FIELD OF THE INVENTION:

The present invention relates to methods for the diagnosis of patients suffering from liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma. The present invention also relates to methods and pharmaceutical compositions for the treatment of liver cancer, in particular intrahepatic cholangiocarcinoma or hepatoblastoma.

BACKGROUND OF THE INVENTION:

Liver cancer is an important public health issue. In adult life, the two major primary liver cancers are hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CC). In addition mixed forms of HCC and CC are described (Roskams 2006). In child life, the major primary liver cancers is hepatoblastoma.

Liver which normally is a silent organ, but harbors an enormous regenerative capacity after injuries like partial hepatectomy or toxic injury. In liver several cell types have longevity: hepatocytes, cholangiocytes, and also bipotential progenitor cells residing in the most terminal branches of the bilary tree, the ductules and/or canals of Hering. Expression of keratin 19 a marker of cholangiocytes, hepatic progenitor cells and early hepatoblasts has been linked with a poor prognosis for patients diagnosed with hepatocellular carcinoma (Roskams, 2006).

When the mature epithelial cell compartments of the liver, hepatocytes and/or cholangiocytes are damaged of inhibited in their proliferation, a reserve cell compartment is activated (Roskams 2003): human progenitor cell compartment or oval cell compartment in rodents. The activation of oval cells or in human liver called“ductal reaction” comprises expansion of a transit amplifying cell compartment of small biliary cells, which can differentiate into at least bilary epithelial cells and hepatocytes.

HCC expressing CK19 and CK7 have a lower tumor free survival rate after curative resection and CK19, CK7, EpCAM, CD133, and CD44 expression were found as independent predictors of postoperative recurrence (Uenishi et a , 2003).

The diversity of liver cancer etiologies does not explain by itself its invasiveness. Less than 10 percents liver cancers are classed as invasive harboring anatomo-pathological transformation such as formation of unfiltered ducts in hepatic parenchyma (intrahepatic cholangiocarcinoma: ICC).

The inventors investigated the invasive tissue transformation associated to abnormal expression in liver of cholangio/hepato bipotent progenitor markers. SUMMARY OF THE INVENTION:

The present invention relates to methods for the diagnosis of patients suffering from liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma. The present invention also relates to methods and pharmaceutical compositions for the treatment of liver cancer, in particular intrahepatic cholangiocarcinoma or hepatoblastoma.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors investigated the invasiveness of liver cancer and the formation of unfiltered ducts in hepatic parenchyma (intrahepatic cholangiocarcinoma: ICC, hepatoblastoma). The inventors also investigated the invasive tissue transformation associated to abnormal expression in liver of cholangio/hepato bipotent progenitor markers. PIK3CD expression was found significantly correlated to progenitor markers such as CD44, KRT19, PROM1 and THY1 in liver cancers and mutated in some liver cancer cases (2.2%), especially in PI3Ka domain suggested to be implicate in substrate presentation. PIK3CD mutations are significantly associated with a lower overall survival (log rank test p-value=0.043). PIK3CD play an important role in apico-basal membrane organization from epithelial cells. The inventors performed overexpression of PIK3CD in HUH7 3D cell culture and its influenced lumen and tubular formation of this cell model. Transcriptome analysis in triplicate was performed on this cell model with WT and PIK3CD knock-in conditions. After transfection the inventors verified expression of PIK3CD: PIK3CD was found overexpressed 53X in Knock-in condition as compared to control (p-value=4.58E-5). Palvidis template matching algorithm allowed us to found an expression profile of 660 affymetrix probes correlated in absolute value to PIK3CD (312 positive correlation and 348 negative correlation). Totality of this expression profile allowed to significantly discriminated ductal infiltrated liver cancer from the others in dataset GSE15765 (p-value=2.33E-l2). By learning machine, 85 genes from HUH7 KI- PIK3CD expression profile were found predictive for ICC and cholangiocarcinoma (CC) subclass of liver cancer. By functional enrichment network analysis on gene ontology biological process database, SRC linker overexpression was highlighted as crosstalk between regulation of cell adhesion and epithelial cell transformation in functional HUH7 3D model which predict in ICC and CC liver cancer subclass; FOXC2 transcription factor was also found overexpressed and as a major crosstalk between epithelial tissue morphogenesis and extracellular communication. In hepatic 3D cell culture model and in ICC and CC liver cancer, the inventors connected the interplay of PIK3CD between regulation of extracellular communication and epithelial tissue morphogenesis through major cross talk SRC linker and FOXC2 transcription factor.

A first object of the invention relates to a method of identifying a patient having or at risk of having or developing liver cancer, comprising a step of determining the expression level of PIK3CD in a biological sample obtained from the patient.

In a particular embodiment, the invention relates to a method of identifying a patient having or at risk of having or developing intrahepatic cholangiocarcinoma (ICC), comprising a step of determining the expression level of PIK3CD in a biological sample obtained from the patient.

In a particular embodiment, the invention relates to a method of identifying a patient having or at risk of having or developing hepatoblastoma, comprising a step of determining the expression level of PIK3CD in a biological sample obtained from the patient.

The method of the invention may further comprise a step consisting of comparing the expression level of PIK3CD in the biological sample with a reference value, wherein detecting differential in the expression level of PIK3CD between the biological sample and the reference value is indicative of patient having or at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma.

As used herein, the term“patient” denotes a mammal. Typically, a patient according to the invention refers to any patient (preferably human) afflicted with liver cancer. The term “patient” also refers to any patient afflicted with resectable liver cancer, early stage of hepatocellular carcinoma (HCC), or HCC. The term“patient” also refers to any patient afflicted with cholangiocarcinoma (CC or CCA). The term“patient” also refers to any patient afflicted with intrahepatic cholangiocarcinoma (ICC). The term“patient” also refers to any patient afflicted with hepatoblastoma.

The term“liver cancer” has its general meaning in the art and refers to malignant neoplasm of liver and intrahepatic bile ducts such as revised in the World Health Organisation Classification ICD10 C22.

The term“cholangiocarcinoma” or“CCA” has its general meaning in the art and refers to a group of cancers developed from the bile duct epithelium. The term“cholangiocarcinoma” also refers to bile duct cancer of three classes: intrahepatic cholangiocarcinoma (ICC or iCCA), extrahepatic cholangiocarcinoma (such as perihilar cholangiocarcinoma or Klatskin) and distal cholangiocarcinoma.

The term“intrahepatic cholangiocarcinoma” or“ICC” or“iCCA” has its general meaning in the art and refers to Malignant neoplasm of intrahepatic bile ducts, Adenocarcinoma of intra-hepatic bile ducts such as revised in the World Health Organisation Classification ICD10 C22.1 (Squadroni et al., 2017; Kennedy et al., 2017). The intrahepatic cholangiocarcinoma (ICC) is subdivided to different subgroups such as described in Aishima et al. 2007; Sempoux et al, 2011; Nakanuma et al. 2010; Komuta et al, 2012; and Liau et al, 2014.

The term“hepatoblastoma” has its general meaning in the art and refers to an uncommon malignant liver neoplasm occurring in infants and children (1% of pediatric cancers, 0.02% of all cancers and around 3,500 new cases by year worldwide) with a lO-year survival of 61% (Allan B et al, HPB 2013, 15:741-46).

In a further aspect, the method of the invention allow differential diagnosis of liver cancer, in particular cholangiocarcinoma (CCA) or hepatoblastoma.

The term“biological sample” refers to any biological sample derived from the patient such as blood sample, plasma sample, serum sample, biopsy sample, or liver cancer sample.

As used herein, the term“PIK3CD” has its general meaning in the art and refers to Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Delta, also known as phosphoinositide 3-kinase (PI3K) delta isoform or pl 105.

As used herein, the“reference value” refers to a threshold value or a cut-off value. The setting of a single“reference value” thus allows discrimination between a poor and a good prognosis with respect to the overall survival (OS) for a patient. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the expression level (obtained according to the method of the invention) with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the expression level (or ratio, or score) determined in a biological sample derived from one or more patients having liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma. Furthermore, retrospective measurement of the expression level (or ratio, or scores) in properly banked historical patient samples may be used in establishing these threshold values. Predetermined reference values used for comparison may comprise “cut-off’ or “threshold” values that may be determined as described herein. Each reference (“cut-off’) value for the biomarker of interest may be predetermined by carrying out a method comprising the steps of

a) providing a collection of samples from patients suffering of liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma;

b) determining the expression level of the biomarker for each sample contained in the collection provided at step a);

c) ranking the tumor tissue samples according to said expression level;

d) classifying said samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their expression level,

e) providing, for each sample provided at step a), information relating to the responsiveness of the patient or the actual clinical outcome for the corresponding cancer patient (i.e. the duration of the event-free survival (EFS), metastasis-free survival (MFS) or the overall survival (OS) or both);

f) for each pair of subsets of samples, obtaining a Kaplan Meier percentage of survival curve;

g) for each pair of subsets of samples calculating the statistical significance (p value) between both subsets;

h) selecting as reference value for the expression level, the value of expression level for which the p value is the smallest.

For example the expression level of a biomarker has been assessed for 100 cancer samples of 100 patients. The 100 samples are ranked according to their expression level. Sample 1 has the best expression level and sample 100 has the worst expression level. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding cancer patient, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.

The reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of expression levels.

In routine work, the reference value (cut-off value) may be used in the present method to discriminate cancer samples and therefore the corresponding patients.

Kaplan-Meier curves of percentage of survival as a function of time are commonly to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art.

The man skilled in the art also understands that the same technique of assessment of the expression level of a biomarker should of course be used for obtaining the reference value and thereafter for assessment of the expression level of a biomarker of a patient subjected to the method of the invention.

In one embodiment, the reference value may correspond to the expression level of PIK3CD determined in a biological sample associated with a patient not afflicted with liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma. Accordingly, a higher expression level of PIK3CD than the reference value is indicative of a patient having or at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma, and a lower or equal expression level of PIK3CD than the reference value is indicative of a patient not having or not at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma.

In another embodiment, the reference value may correspond to the expression level of PIK3CD determined in a biological sample associated with a patient afflicted with liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma. Accordingly, a higher or equal expression level of PIK3CD than the reference value is indicative of a patient having or at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma, and a lower expression level of PIK3CD than the reference value is indicative of a patient not having or not at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma.

In some embodiments, the invention relates to a method of identifying a patient having or at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma, comprising the steps of: i) determining the expression level of PIK3CD in a biological sample obtained from the patient, ii) comparing the expression level determined at step i) with a predetermined reference value, and iii) concluding that the patient is having or at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma when the level determined at step i) is higher than the predetermined reference value, or concluding that the patient is not having or at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma when the level determined at step i) is lower than the predetermined reference value.

In some embodiments, the method of the invention further comprises determining the expression level of at least one biomarker selected from the group consisting of: ZNF385D, ZDHHC8P1, WWP2, WT1-AS, TUBB6, TRIM31, TNN, TNFAIP2, TCTN1, SVIL, SRPX2, SRC, SPINK5, SPEG, SMAD7, SLN, RS1, RNF40, RBFA, RASL12, PTOV1, PRSS2, PRB3, PPP1R13L, POMT1, PLEKHJ1, PLEKHF1, PLEKHA8P1, PLD2, PIM2, PHKA1, PFKFB3, PDYN, PCDHB11, OCA2, NPHS1, MYOZ1, MY05C, FRRC37A2, FMNB2, FDB1, KFHF21, ISYNA1, IRF1, IP6K1, IGSF3, HYAF2, GUCY1B3, GRHF2, GOFGA8A, GADD45B, FZD7, FYB, FOXD2, FOXC2, FOS, FGF3, FARP2, FAM111A, FAIM2, EYA1, ERICH 1, EFF3, EGR1, DMPK, DENND6B, DBN1, CXCF3, CSDC2, CNNM2, CENPB, Cl9orf60, BTG2, BRD2, BNIP1, BEST2, B3GAFT1, ATP8B3, ART3, ANXA3, ANKRD1, AFDH3B1, ADM, and ADAMTSF4, wherein higher expression level of at least one biomarker is indicative of a patient having or at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma, and lower expression level is indicative of a patient not having or at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma.

In some embodiments, the method of the invention further comprises determining the expression level of at least one biomarker selected from the group consisting of: ZNF510, ZNF407, ZNF140, ZG16, TTC13, TSHZ2, TRMT5, TPK1, TMPRSS6, TAP2, TAOK3, SUFT1E1, SRD5A1, RHCG, PRF1, POFR3K, PFA2G7, PFA2G12A, PIGO, PIBF1, PEG3, PAGE1, MORC3, MNAT1, MINA, METTF16, MEP1A, FCP2, KDEFC1, ITGB2, IRF9, IGFBP1, HFA-DRB4, GUCY2C, GABPA, FOXOl, ENOPH1, CYP4F8, CYP2D6, CSTF2T, CRYAA, CCF13, C6orfl20, Cl2orf29, Cl0orf88, BTN2A1, BTG3, BHMT, ATRN, AGTPBP1, and ACSF5, wherein lower expression level of at least one biomarker is indicative of a patient having or at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma, and higher expression level is indicative of a patient not having or at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma.

In some embodiments, the method of the invention further comprises determining the expression level of at least one biomarker selected from the group consisting of: ZNF385D, ZDHHC8P1, WWP2, WT1-AS, TUBB6, TRIM31, TNN, TNFAIP2, TCTN1, SVIF, SRPX2, SRC, SPINK5, SPEG, SMAD7, SLN, RS1, RNF40, RBFA, RASF12, PTOV1, PRSS2, PRB3, PPP1R13F, POMT1, PFEKHJ1, PFEKHF1, PFEKHA8P1, PFD2, PIM2, PHKA1, PFKFB3, PDYN, PCDHB11, OCA2, NPHS1, MYOZ1, MY05C, FRRC37A2, FMNB2, FDB1, KFHF21, ISYNA1, IRF1, IP6K1, IGSF3, HYAF2, GUCY1B3, GRHF2, GOFGA8A, GADD45B, FZD7, FYB, FOXD2, FOXC2, FOS, FGF3, FARP2, FAM111A, FAIM2, EYA1, ERICH 1, EFF3, EGR1, DMPK, DENND6B, DBN1, CXCF3, CSDC2, CNNM2, CENPB, Cl9orf60, BTG2, BRD2, BNIP1, BEST2, B3GAFT1, ATP8B3, ART3, ANXA3, ANKRD1, AFDH3B1, ADM, ADAMTSF4, ZNF510, ZNF407, ZNF140, ZG16, TTC13, TSHZ2, TRMT5, TPK1, TMPRSS6, TAP2, TAOK3, SUFT1E1, SRD5A1, RHCG, PRF1, POFR3K, PFA2G7, PFA2G12A, PIGO, PIBF1, PEG3, PAGE1, MORC3, MNAT1, MINA, METTF16, MEP1A, FCP2, KDEFC1, ITGB2, IRF9, IGFBP1, HFA-DRB4, GUCY2C, GABPA, FOXOl, ENOPH1, CYP4F8, CYP2D6, CSTF2T, CRYAA, CCF13, C6orfl20, Cl2orf29, Cl0orf88, BTN2A1, BTG3, BHMT, ATRN, AGTPBP1, and ACSF5.

In some embodiment, the method of the invention comprises determining the expression level of at least one biomarker selected from the group consisting of PIK3CD, CXCF3, PRSS2, ANXA3, FOXC2, FZD7, SMAD7, PPP1R13F, GRHF2, and SRC.

In some embodiment, the method of the invention comprises determining the expression level of PIK3CD, CXCF3, PRSS2, ANXA3, FOXC2, FZD7, SMAD7, PPP1R13F, GRHF2, and SRC.

Analyzing the biomarker expression level may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed nucleic acid or translated protein.

In one embodiment, the biomarker expression level is assessed by analyzing the expression of the protein translated from said gene. Said analysis can be assessed using an antibody (e.g., a radio-labeled, chromophore- labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin- streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from the gene encoding for the biomarker.

Methods for measuring the expression level of a biomarker in a sample may be assessed by any of a wide variety of well-known methods from one of skill in the art for detecting expression of a protein including, but not limited to, direct methods like mass spectrometry- based quantification methods, protein microarray methods, enzyme immunoassay (EIA), radioimmunoassay (RIA), Immunohistochemistry (IHC), Western blot analysis, EFISA, Luminex, ELISPOT and enzyme linked immunoabsorbant assay and undirect methods based on detecting expression of corresponding messenger ribonucleic acids (mRNAs). The mRNA expression profile may be determined by any technology known by a man skilled in the art. In particular, each mRNA expression level may be measured using any technology known by a man skilled in the art, including nucleic microarrays, quantitative Polymerase Chain Reaction (qPCR), next generation sequencing and hybridization with a labelled probe.

Said direct analysis can be assessed by contacting the sample with a binding partner capable of selectively interacting with the biomarker present in the sample. The binding partner may be an antibody that may be polyclonal or monoclonal, preferably monoclonal (e.g., a isotope-label, element-label, radio-labeled, chromophore- labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from the gene encoding for the biomarker of the invention. In another embodiment, the binding partner may be an aptamer.

The binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as an isotope, an element, a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

As used herein, the term "labelled", with regard to the antibody, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as an isotope, an element, a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be produced with a specific isotope or a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited to radioactive atom for scintigraphic studies such as 1123, 1124, Inl l l, Rel86, Rel88, specific isotopes include but are not limited to 13C, 15N, 1261, 79Br, 8lBr.

The afore mentioned assays generally involve the binding of the binding partner (ie. antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, silicon wafers. In a particular embodiment, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize said biomarker. A sample containing or suspected of containing said biomarker is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art such as Singulex, Quanterix, MSD, Bioscale, Cytof.

In one embodiment, an Enzyme-linked immunospot (ELISpot) method may be used. Typically, the sample is transferred to a plate which has been coated with the desired anti biomarker capture antibodies. Revelation is carried out with biotinylated secondary Abs and standard colorimetric or fluorimetric detection methods such as streptavidin-alkaline phosphatase and NBT-BCIP and the spots counted.

In one embodiment, when multi-biomarker expression measurement is required, use of beads bearing binding partners of interest may be preferred. In a particular embodiment, the bead may be a cytometric bead for use in flow cytometry. Such beads may for example correspond to BD™ Cytometric Beads commercialized by BD Biosciences (San Jose, California). Typically cytometric beads may be suitable for preparing a multiplexed bead assay. A multiplexed bead assay, such as, for example, the BD(TM) Cytometric Bead Array, is a series of spectrally discrete beads that can be used to capture and quantify soluble antigens. Typically, beads are labelled with one or more spectrally distinct fluorescent dyes, and detection is carried out using a multiplicity of photodetectors, one for each distinct dye to be detected. A number of methods of making and using sets of distinguishable beads have been described in the literature. These include beads distinguishable by size, wherein each size bead is coated with a different target- specific antibody (see e.g. Fulwyler and McHugh, 1990, Methods in Cell Biology 33:613-629), beads with two or more fluorescent dyes at varying concentrations, wherein the beads are identified by the levels of fluorescence dyes (see e.g. European Patent No. 0 126,450), and beads distinguishably labelled with two different dyes, wherein the beads are identified by separately measuring the fluorescence intensity of each of the dyes (see e.g. U.S. patent Nos. 4,499,052 and 4,717,655). Both one-dimensional and two-dimensional arrays for the simultaneous analysis of multiple antigens by flow cytometry are available commercially. Examples of one-dimensional arrays of singly dyed beads distinguishable by the level of fluorescence intensity include the BD(TM) Cytometric Bead Array (CBA) (BD Biosciences, San Jose, Calif.) and Cyto-Plex(TM) Flow Cytometry microspheres (Duke Scientific, Palo Alto, Calif.). An example of a two-dimensional array of beads distinguishable by a combination of fluorescence intensity (five levels) and size (two sizes) is the QuantumPlex(TM) microspheres (Bangs Laboratories, Fisher, Ind.). An example of a two- dimensional array of doubly-dyed beads distinguishable by the levels of fluorescence of each of the two dyes is described in Fulton et al. (1997, Clinical Chemistry 43(9): 1749- 1756). The beads may be labelled with any fluorescent compound known in the art such as e.g. FITC (FL1), PE (FL2), fluorophores for use in the blue laser (e.g. PerCP, PE-Cy7, PE-Cy5, FL3 and APC or Cy5, FL4), fluorophores for use in the red, violet or UV laser (e.g. Pacific blue, pacific orange). In another particular embodiment, bead is a magnetic bead for use in magnetic separation. Magnetic beads are known to those of skill in the art. Typically, the magnetic bead is preferably made of a magnetic material selected from the group consisting of metals (e.g. ferrum, cobalt and nickel), an alloy thereof and an oxide thereof. In another particular embodiment, bead is bead that is dyed and magnetized.

In one embodiment, protein microarray methods may be used. Typically, at least one antibody or aptamer directed against the biomarker is immobilized or grafted to an array(s), a solid or semi-solid surface(s). A sample containing or suspected of containing the biomarker is then labelled with at least one isotope or one element or one fluorophore or one colorimetric tag that are not naturally contained in the tested sample. After a period of incubation of said sample with the array sufficient to allow the formation of antibody-antigen complexes, the array is then washed and dried. After all, quantifying said biomarker may be achieved using any appropriate microarray scanner like fluorescence scanner, colorimetric scanner, SIMS (secondary ions mass spectrometry) scanner, maldi scanner, electromagnetic scanner or any technique allowing to quantify said labels.

In another embodiment, the antibody or aptamer grafted on the array is labelled.

In another embodiment, reverse phase arrays may be used. Typically, at least one sample is immobilized or grafted to an array(s), a solid or semi-solid surface(s). An antibody or aptamer against the suspected biomarker is then labelled with at least one isotope or one element or one fluorophore or one colorimetric tag that are not naturally contained in the tested sample. After a period of incubation of said antibody or aptamer with the array sufficient to allow the formation of antibody-antigen complexes, the array is then washed and dried. After all, detecting quantifying and counting by D-SIMS said biomarker containing said isotope or group of isotopes, and a reference natural element, and then calculating the isotopic ratio between the biomarker and the reference natural element may be achieve using any appropriate microarray scanner like fluorescence scanner, colorimetric scanner, SIMS (secondary ions mass spectrometry) scanner, maldi scanner, electromagnetic scanner or any technique allowing to quantify said labels.

In one embodiment, said direct analysis can also be assessed by mass Spectrometry. Mass spectrometry-based quantification methods may be performed using either labelled or unlabelled approaches (DeSouza and Siu, 2012). Mass spectrometry-based quantification methods may be performed using chemical labeling, metabolic labelingor proteolytic labeling. Mass spectrometry-based quantification methods may be performed using mass spectrometry label free quantification, LTQ Orbitrap Velos, LTQ-MS/MS, a quantification based on extracted ion chromatogram EIC (progenesis LC-MS, Liquid chromatography-mass spectrometry) and then profile alignment to determine differential expression of the biomarker.

In another embodiment, the biomarker expression level is assessed by analyzing the expression of mRNA transcript or mRNA precursors, such as nascent RNA, of biomarker gene. Said analysis can be assessed by preparing mRNA/cDNA from cells in a sample from a patient, and hybridizing the mRNA/cDNA with a reference polynucleotide. The prepared mRNA/cDNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses, such as quantitative PCR (TaqMan), and probes arrays such as GeneChip(TM) DNA Arrays (AFFYMETRIX) .

Advantageously, the analysis of the expression level of mRNA transcribed from the gene encoding for biomarkers involves the process of nucleic acid amplification, e. g., by RT- PCR (the experimental embodiment set forth in U. S. Patent No. 4,683, 202), ligase chain reaction (Barany, 1991), self sustained sequence replication (Guatelli et a , 1990), transcriptional amplification system (Kwoh et a , 1989), Q-Beta Replicase (Lizardi et a , 1988), rolling circle replication (U. S. Patent No. 5,854, 033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. A further object of the invention relates to a method for predicting the survival time of a patient suffering from liver cancer and/or intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma comprising the steps of: i) determining the expression level of PIK3CD and/or detecting at least one mutation of PIK3CD in a biological sample obtained from the patient, ii) comparing the expression level determined at step i) with a reference value, and iii) concluding that the patient will have a short survival time when the expression level of PIK3CD is higher than the reference value and/or detecting said at least one mutation of PIK3CD.

The term“mutation” has its general meaning in the art and refers to a coding mutations affecting PIK3CD. The term“mutation” of the invention also refers to mutation in the PIK3CD coding region, nonsynonymous mutation, and missense mutations. The term“mutation” of the invention also refers to mutation affecting in domain PI3Ka (Phosphoinositide 3-kinase family, accessory domain (PIK domain)), PI3K_C2 (Phosphoinositide 3-kinase C2) domain and PI3K_p85B domain (PI3-kinase family, p85-binding domain).

The step of detecting if at least one mutation of PIK3CD is present in a biological sample may be performed by any method well-known by the skilled person. More particularly, said step of detecting the presence or not of at least one mutation of PIK3CD may comprise:

sequencing the PIK3CD gene from the DNA present in the biological sample or sequencing the PIK3CD cDNA corresponding to the mRNA present in the biological sample, and

comparing the obtained sequence to a reference sequence encoding a functional PIK3CD protein or comparing the amino acid sequence encoded by the obtained sequence to a reference sequence of a functional PIK3CD protein.

A reference sequence encoding a functional PIK3CD protein is for example sequence EGAS00001000604, NG_023434, NC_00000l. l l and NC_0l89l2.2 when comparing the PIK3CD gene sequence or sequence NM_001350234.1, NM_00l350235.l and NM_005026.4 when comparing the PIK3CD cDNA sequence.

A reference sequence of a functional PIK3CD protein is for example sequence 000329, NP_001337163.1, NP_001337164.1 and NP_0050l7.3.

The sequence comparison may be performed by any method well-known by the skilled person such as sequence alignment.

The method of the present invention is particularly suitable for predicting the duration of the overall survival (OS), progression-free survival (PFS) and/or the disease-free survival (DFS) of the cancer patient. Those of skill in the art will recognize that OS survival time is generally based on and expressed as the percentage of people who survive a certain type of cancer for a specific amount of time. In general, OS rates do not specify whether cancer survivors are still undergoing treatment at five years or if they've become cancer- free (achieved remission). DFS gives more specific information and is the number of people with a particular cancer who achieve remission. Also, progression-free survival (PFS) rates (the number of people who still have cancer, but their disease does not progress) includes people who may have had some success with treatment, but the cancer has not disappeared completely. As used herein, the expression“short survival time” indicates that the patient will have a survival time that will be lower than the median (or mean) observed in the general population of patients suffering from said cancer. When the patient will have a short survival time, it is meant that the patient will have a“poor prognosis”. Inversely, the expression“long survival time” indicates that the patient will have a survival time that will be higher than the median (or mean) observed in the general population of patients suffering from said cancer. When the patient will have a long survival time, it is meant that the patient will have a“good prognosis”.

In some embodiment, the method of the invention in performed for predicting the overall survival (OS), progression-free survival (PFS) and/or the disease-free survival (DFS) of a patient suffering from resectable liver cancer (early stage of hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma).

In some embodiments, the present invention relates to a method for predicting the overall survival (OS) of a patient suffering from liver cancer and/or intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma comprising the steps of: i) determining the expression level of PIK3CD and/or detecting at least one mutation of PIK3CD in a biological sample obtained from the patient, ii) comparing the expression level determined at step i) with a reference value, and iii) concluding that the patient will have a short survival time when the expression level of PIK3CD is higher than the reference value and/or detecting said at least one mutation of PIK3CD.

A further object of the invention relates to a PIK3CD inhibitor for use in the treatment of liver cancer in a patient in need thereof.

A further object of the invention relates to a PIK3CD inhibitor for use in the treatment of intrahepatic cholangiocarcinoma (ICC) in a patient in need thereof.

A further object of the invention relates to a PIK3CD inhibitor for use in the treatment of hepatoblastoma in a patient in need thereof.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patients at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

The term“PIK3CD inhibitor” has its general meaning in the art and refers to a compound that selectively blocks or inactivates the PIK3CD. The term“PIK3CD inhibitor” also refers to a compound that selectively blocks the binding of PIK3CD to its substrate phosphatidylinositol 4,5 bisphosphate [Ptdlns(4,5) P2 ] or the regulatory subunit p85 and then inhibiting the generation of phosphatidylinositol(3,4,5)-triphosphate (PtdIns(3,4,5)P3) lipid). The term“PIK3CD inhibitor” also refers to a compound able to prevent the action of PIK3CD for example by inhibiting the PIK3CD controls of downstream effectors such as inhibiting the activation of the PI3K/PTEN/Akt/mTOR pathway signaling. As used herein, the term “selectively blocks or inactivates” refers to a compound that preferentially binds to and blocks or inactivates PIK3CD with a greater affinity and potency, respectively, than its interaction with the other sub-types of the PI3K family. Compounds that block or inactivate PIK3CD, but that may also block or inactivate other PIK3CD sub-types, as partial or full inhibitors, are contemplated. The term“PIK3CD inhibitor” also refers to a compound that inhibits PIK3CD expression. Typically, a PIK3CD inhibitor is a small organic molecule, a polypeptide, an aptamer, an antibody, an oligonucleotide or a ribozyme.

Tests and assays for determining whether a compound is a PIK3CD inhibitor are well known by the skilled person in the art such as described in Lannutti et a , 2011; WO2011005119; Denny, 2013; Puri and Gold, 2012; Norman, 2011; Martini et a , 2014; Fruman and Rommel, 2011; Bartholomeusz and Gonzalez-Angulo, 2012, Takashima and Faller, 2013.

The term“PIK3CD inhibitor” has its general meaning in the art and refers to compounds such as CAL-101 (idelalisib; GS-1101; 5-Fluoro-3-phenyl-2-[(S)-l-(9H-purin-6-ylamino)- propyl]-3H-quinazolin-4-one), AMG-319, CAL-263, XL-499, RP-5090, RP-5237, KAR-4141, X-339, IC87114 and compounds described in Lannutti et a , 2011; WO2011005119; Denny, 2013; Puri and Gold, 2012; Norman, 2011; Martini et a , 2014; Fruman and Rommel, 2011; Bartholomeusz and Gonzalez-Angulo, 2012, Takashima and Faller, 2013.

In some embodiments, the term“PIK3CD inhibitor” also refers to dual PI3K alpha/delta inhibitors such as (benzimidazole- 1, 3, 5-triazinyl)morpholines, ETP-00046321, ETP- 00047022, Pictrelisib, GDC-0941; dual PI3K gamma/delta inhibitors such as IPI-145, TG- 100115; dual PI3K beta/delta inbitors such as RP-5002, KAR-4139, CAL-120, AZD-6482; and pan-PI3K inhibitors such as LY294002, Buparlisib (B KM 120); and compounds described in Lannutti et a , 2011; WO2011005119; Denny, 2013; Puri and Gold, 2012; Norman, 2011; Martini et a , 2014; Fruman and Rommel, 2011; Bartholomeusz and Gonzalez-Angulo, 2012, Takashima and Faller, 2013.

In one embodiment of the invention, PIK3CD inhibitors include but are not limited to miRNAs such as miR-7 (Fang et a , 2012).

In another embodiment, the PIK3CD inhibitor of the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Then after raising aptamers directed against PIK3CD of the invention as above described, the skilled man in the art can easily select those blocking or inactivating PIK3CD.

In another embodiment, the PIK3CD inhibitor of the invention is an antibody (the term including“antibody portion”) directed against PIK3CD.

In one embodiment of the antibodies or portions thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.

As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of PIK3CD. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.

Briefly, the antigen may be provided as synthetic peptides corresponding to antigenic regions of interest in PIK3CD. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). Following culture of the hybridomas, cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.

Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modem Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDRS). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.

It is now well-established in the art that the non CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or hetero specific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody.

This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization.

In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et a , /. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.

In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non human sequences. The present invention also includes so-called single chain antibodies.

The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. In a preferred embodiment, the PIK3CD inhibitor of the invention is a Human IgG4.

In another embodiment, the antibody according to the invention is a single domain antibody. The term“single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called“nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term“VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term“complementarity determining region” or“CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.

The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.

VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen- specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the“Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The“Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

In one embodiment, the PIK3CD inhibitor of the invention is a PIK3CD expression inhibitor.

The term“expression” when used in the context of expression of a gene or nucleic acid refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include messenger RNAs, which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins (e.g., PIK3CD) modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation.

An“inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.

PIK3CD expression inhibitors for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of PIK3CD mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of PIK3CD proteins, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding PIK3CD can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as PIK3CD expression inhibitors for use in the present invention. PIK3CD gene expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that PIK3CD expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as PIK3CD expression inhibitors for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of PIK3CD mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful a PIK3CD inhibitors can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing PIK3CD. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in KRIEGLER (A Laboratory Manual," W.H. Freeman C.O., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild- type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et a , "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUCl8, pUQ9, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

In another embodiment, the present invention relates to a method of treating liver cancer in a patient in need thereof, comprising the step of administering to said patient a therapeutically effective amount of PI3KCD inhibitor wherein the patient is identified as having or at risk of having or developing liver cancer. In a particular embodiment, the patient is identified as having or at risk of having or developing intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma. Typically the inhibitors according to the invention as described above are administered to the patient in a therapeutically effective amount.

By a "therapeutically effective amount" of the inhibitor of the present invention as above described is meant a sufficient amount of the inhibitor for treating liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the inhibitors and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific inhibitor employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific inhibitor employed; the duration of the treatment; drugs used in combination or coincidential with the specific inhibitor employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the inhibitor at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the inhibitor of the present invention for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the inhibitor of the present invention, preferably from 1 mg to about 100 mg of the inhibitor of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

In some embodiments, the PIK3CD inhibitor of the present invention is administered to the patient in combination with a PBK/PTEN/Akt/mTOR pathway signaling inhibitor. The term“PBK/PTEN/Akt/mTOR pathway signaling inhibitor” has its general meaning in the art and refers to but not limited to compounds selected from the group consisting of PI3K inhibitor such as GDC-0032 (RG-7604), GDC-0349 (RG-7603), GDC-0941 (RG-7321), LY-290042, pictrelisib, GSK-1059615, BKM120, PX-866, BAY80-6946, XL147, ZSTK474, CH5132799; mTORC inhibitor such as Rapamycin, temsirolimus (42-[2,2-bis (hydroxymethyl)] rapamycin, also known as CCI779), everolimus (42-0-(2-hydroxyethyl) rapamycin, also known as RAD001), and ridaforolimus (macrolide dimethylphophinic acid rapamycin-40-O-yl ester derivative of sirolimus, also known as AP23573 and deforolimus), AZD8055, OSI027, INK- 128 and compounds described in Mohindra et al., 2014; Nelson et al., 2013; Pal and Quinn, 2013; S6K inhibitor such as selective S6K1 or S6K2 inhibitors, and inhibitors of S6K phosphorylation such as metformin; PF-4708671 (2-((4-(5-Ethylpyrimidin-4-yl)piperazin-l- yl)methyl)-5-(trifluoromethyl)-lH-benzo[d]imidazole), LYS6K2, XL418, Lefinnomide (also be known as SU101 or ARAVA), active metabolite of leflunomide such as All 1726 and SU0020, AZD5363; and compounds described in Fenton and Gout, 2011 and US 2014/121235; PDK/mTOR inhibitor such as intellikine, PIK-294, INK-1197, GDC-0980 (RG-7422), BEZ235, BGT226, PF-04691502, PKI-587, PF-05212384, XL765, SAR245409,

GSK2126458, DS-7423, PWT33597-101, SF1126 (LY294002); AKT inhibitor such as Perifosine, MK2206, GSK2110183, GDC-0068, AZD5363, ARQ092, GSK2141795, GSK690693; and compounds described in Lannutti et al., 2011; WO2011005119; Denny, 2013; Puri and Gold, 2012; Norman, 2011; Martini et al., 2014; Fruman and Rommel, 2011; Bartholomeusz and Gonzalez-Angulo, 2012, Takashima and Faller, 2013.

In some embodiments, the PIK3CD inhibitor of the present invention is administered to the patient in combination with anti-liver cancer treatment, in particular anti-intrahepatic cholangiocarcinoma (ICC) treatment or anti-hepatoblastoma treatment. The terms“anti-liver cancer treatment”, “anti-intrahepatic cholangiocarcinoma (ICC) treatment” or “anti hepatoblastoma treatment” have their general meaning in the art and refer to any type of liver cancer therapy undergone by the liver cancer subjects including surgical resection of liver cancer, external-beam radiation therapy (EBRT), transarterial chemoembolization (TACE), Radioembolization using yttrium- 90 microspheres, and any type of agent conventional for the treatment of liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma.

In some embodiments, the PIK3CD inhibitor of the present invention is administered to the patient in combination with at least one compound selected from the group consisting of g- Secretase-inhibitor such as DAPT (N-[N-(3,5-difluoro-phenacetyl)-F-alanyl]-S-phenylglycine t-butyl ester), GSI IX; Anti-Notchl compound such as Brontictuzumab; Anti-Notch2/3 compound such as Tarextumab; Demcizumab; Anti-miR-2l; MO-T1144, MO-T1150, and MO-T1151; Corilagin; Curcumin; FGFR inhibitor such as BGJ398, Erdafitinib, ARQ 087, AZD4547, TAS120, CH5183284/Debio 1347, ponatinib, FPA144, derazantinib,

NVP-BGJ398; CDK4/6 inhibitor such as ribociclib, palbociclib; anti-mesothelin compound such as anetumab ravtansine; MET inhibitor such as tivantinib, cabozantinib; IDH inhibitor compound such as AG- 120, AG-221; Her-2/neu (ERBB2) inhibitor such as erlotinib, lapatinib, nertatinib, neratinib, afatinib, dacomatinib; BH3 mimetics; EZH2, HDAC, and DNMT inhibitors; immune checkpoints inhibitor; Anti-CTLA-4 compound such as ipilimumab, tremelimumab; PD-l inhibitor such as pembrolizumab, nivolumab; PD-L1 inhibitor such as durvalumab; and SRC inhibitor such as Dasatinib; Bosutinib; KX2-391; Saracatinib; and Quercetin and compounds described in Cigliano et al., 2017; Mertens et al., 2017; Rizvi et al., 2017; Yang et al., 2017; Squadroni et al., 2017

In some embodiments, the PIK3CD inhibitor of the present invention is administered to the patient in combination with at least one compound selected from the group consisting of gemcitabine, fluorouracil, FOLFIRINOX (fluorouracil, irinotecan, oxaliplatin, and leucovorin), nab-paclitaxel, inhibitors of programmed death 1 (PD-l), PD-l ligand PD-L1, anti-CTLA4 antibodies, EGFR inhibitors such as erlotinib, chemoradiotherapy, inhibitors of PARP, inhibitors of Sonic Hedgehog, gene therapy and radiotherapy.

In a further aspect, the present invention relates to a method of screening a candidate compound for use as a drug for treating liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma in a patient in need thereof, wherein the method comprises the steps of:

providing a PIK3CD, providing a cell, tissue sample or organism expressing a

PIK3CD,

providing a candidate compound such as a small organic molecule, a polypeptide, an aptamer, an antibody or an intra-antibody,

measuring the PIK3CD activity,

and selecting positively candidate compounds that inhibit PIK3CD activity.

Methods for measuring PIK3CD activity are well known in the art (Lannutti et al., 2011; WO2011005119; Denny, 2013; Puri and Gold, 2012; Norman, 2011; Martini et al., 2014; Fruman and Rommel, 2011; Bartholomeusz and Gonzalez-Angulo, 2012, Takashima and Faller, 2013). For example, measuring the PIK3CD activity involves determining a Ki on the PIK3CD cloned and transfected in a stable manner into a CHO cell line, measuring cancer cell migration and invasion abilities, measuring hepatic progenitor marker, and measuring PI3K/PTEN/Akt/mTOR pathway signaling in the present or absence of the candidate compound.

Tests and assays for screening and determining whether a candidate compound is a PIK3CD inhibitor are well known in the art (Fannutti et al., 2011; WO2011005119; Denny, 2013; Puri and Gold, 2012; Norman, 2011; Martini et al., 2014; Fruman and Rommel, 2011; Bartholomeusz and Gonzalez-Angulo, 2012, Takashima and Faller, 2013). In vitro and in vivo assays may be used to assess the potency and selectivity of the candidate compounds to inhibit PIK3CD activity.

Activities of the candidate compounds, their ability to bind PIK3CD and their ability to inhibit PIK3CD activity may be tested using isolated cancer cell or CHO cell line cloned and transfected in a stable manner by the human PIK3CD, hepatic progenitor cell, HUH7 3D cell.

Activities of the candidate compounds and their ability to bind to the PIK3CD may be assessed by the determination of a Ki on the PIK3CD cloned and transfected in a stable manner into a CHO cell line, measuring cancer cell migration and invasion abilities, measuring hepatic progenitor marker, and measuring PI3K/PTEN/Akt/mTOR pathway signaling in the present or absence of the candidate compound.

Cells expressing another kinase than PIK3CD may be used to assess selectivity of the candidate compounds.

The inhibitors of the invention may be used or prepared in a pharmaceutical composition.

In one embodiment, the invention relates to a pharmaceutical composition comprising the inhibitor of the invention and a pharmaceutical acceptable carrier for use in the treatment of liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma in a patient of need thereof.

Typically, the inhibitor of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral, sublingual, intramuscular, intravenous, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, intraperitoneal, intramuscular, intravenous and intranasal administration forms and rectal administration forms.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze- dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions comprising inhibitors of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The inhibitor of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active inhibitors in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the patient being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual patient.

In addition to the inhibitors of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.

Pharmaceutical compositions of the invention may include any further compound inhibiting the PI3K/PTEN/Akt/mTOR pathway signaling such as PI3K/PTEN/Akt/mTOR pathway signaling inhibitor.

Pharmaceutical compositions of the invention may include any further compound which is used in the treatment of liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma. In one embodiment, said additional active compounds may be contained in the same composition or administrated separately.

In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma in a patient in need thereof.

The invention also provides kits comprising the inhibitor of the invention. Kits containing the inhibitor of the invention find use in therapeutic methods.

In a further aspect, the present invention relates to a method of treating liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma in a patient in need thereof, comprising the step of administering to said patient a therapeutically effective amount of PI3KCD inhibitor.

In a further aspect, the present invention relates to a method of treating patient at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma comprising the steps of:

i) determining whether the patient is a risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma by performing the method according to the invention, and

ii) administering a PI3KCD inhibitor if said patient was being classified as at risk of having or developing liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma.

In a further aspect, the present invention relates to a method of treating liver cancer, in particular intrahepatic cholangiocarcinoma (ICC) or hepatoblastoma in a patient identified as having a short survival time comprising the steps of:

i) determining whether the patient will have a short survival time by performing the method according to the invention, and

ii) administering a PI3KCD inhibitor if said patient was being classified as having a short survival time.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Gene expression profile of PIK3CD gain in HUH7 3D cell culture: A /

Boxplot of PIK3CD overexpression after transfection in HUH7 3D cell culture model (p-value was calculated unpaired 2 sided student test), B / unsupervised principal component analysis performed on gene expression profile regulated by PIK3CD gain transfection in HUH7 3D cell culture model (p-value was calculated with group correlation to the first principal axis)

Figure 2: Gene expression profile up regulated by PIK3CD gain in HUH7 3D cell culture allowed to predict intrahepatic cholangiocarcinoma subclass of liver cancer: A/ Unsupervised principal component analysis performed on transcriptome liver cancer samples (GSE15765) with gene expression profile regulated by PIK3CD (p-value was calculated with group correlation to the first principal axis); B / Venn diagram between overexpressed genes by PIK3CD gain in HUH7 3D culture and with genes with positive predictive score for intrahepatic cholangiocarcinoma subclass by learning machine

Figure 3: Mutations affecting PIK3CD coding gene in resectable liver cancer are associated to lower overall survival: Overall Survival analysis of liver cancer patients affected (dashed line) or not (solid line) by PIK3CD mutations (logrank test p-value on overall survival).

Figure 4: Prediction of intrahepatic cholangiocarcinoma liver cancer subclass with PIK3CD up regulated expression profile by machine learning: A/ misclassification error plot by class obtained by learning machine (leave one out cross validation) with gene overexpressed by PIK3CD in HUH7 3D cell model on transcriptome dataset of liver cancer GSE15765; B / table of misclassified liver cancer samples (GSE15765) by machine learning with minimal error threshold introduced on PIK3CD regulated genes in HUH7.

Figure 5: Determination of the minimal predictive signature of ductal invasive tumors in all liver tumors: After integration of the PIK3CD overexpression cell model transcriptome in the HUH7 cell line with human tumor transcriptome a signature of 85 biomarkers overexpressed in PIK3CD dependence in ductal invasion liver tumors (intrahepatic cholangiocarcinoma) was determined by machine learning. The predictive staging within the two sets of transcriptome allowed to determine a minimal signature common to the cell model PIK3CD dependent and tumor ICC. Based on this signature restricted to the ten best markers (PIK3CD, CXCL3, PRSS2, ANXA3, FOXC2, FZD7, SMAD7, PPP1R13L, GRHL2, SRC) the Random Forest algorithm built on a mathematical learning of 500 trees made it possible to determine that the misclassification error rate was less than 10% (specifically 7.78% error in overall misclassification of tumors). The multiple combination of expression quantification of these ten markers predicts ICC within liver tumors with an area under the 0.98 curve, a 95% sensitivity, and a 97.5% specificity.

EXAMPLE: Material & Methods

Transcrip tomic dataset

Transcriptome gene expression analysis performed on liver cancer samples (Woo et al., 2010) with technology Affymetrix Human Genome U133A 2.0 Array was downloaded on Geo Omnibus Database (https://www.ncbi.nlm.nih.gov/geo/). RMA Normalized Gene expression matrix from dataset GSE 15765 was annotated with GEO plateform file GPL571 by SQL query.

Next generation sequencing dataset

Next generation sequencing (whole exome with Agilent SureSelect capture and paired end sequencing on Illumina HiSeq 2000 platform) performed on resectable liver cancer cohort constituted of 231 patients (Ahn et al., 2014) was query through web tool application Cbioportal (Gao et al., 2013) in order to check alterations of PIK3CD gene (European Genome-Phenome Archive (accession number: EGAS00001000604)).

RNA preparation and Transcriptome analysis

Total RNA extraction from HUH7 3D cell culture was prepared by using preparation kit and by following manufacturer recommendations. Quantification of RNA material was done by using Nanodrop technology and quality of nucleic acid was check by using Bioanalyser (Agilent technologies). Total RNA in triplicate condition which passed quality control was used to synthetize amplified RNA (aRNA) microarray probe by using linear T7 RNA polymerase amplification protocol (Affymetrix). Labeled aRNA probes were hybridized on human Affymetrix Microarray ST2.0. Microarray were scanned by using Affymetrix plateform and normalized by RMA algorithm included in Affymetrix expression console.

Bioinformatics analysis of transcriptome

Bioinformatics analysis were performed in R software environement version 3.0.2. RMA normalized matrix of HUH7 transcriptome was used with genefilter R Bioconductor package in order to remove invariable genes. Pavlidis template matching algorithm was used to found co regulated genes to PIK3CD which was transfected in 3D cell culture HUH7 (Formolo et al., 2011): threshold of correlation was fixed at R>=0.80 in absolute value. Learning machine analysis (leave one out with cross validation) performed with PIK3CD regulated expression profile on liver cancer samples was realized with Pamr R-package (Tibshirani et al., 2002). Microarray expression heatmaps were performed with MADE4 R- package (Culhane et al., 2005). Unsupervised principal component analysis on gene expression matrix was perform with FactoMineR R-package (Le et al., 2008). Functional enrichment on Gene ontology biological process for microarray analysis was performed with the website tools of Enrichr (Kuleshov et al., 2016). Network analysis representation realized on functional enrichment results was investigated in Cytoscape standalone software version 3.2.1 64bit (Cline et al., 2007). Survival analysis was performed with survival R-pakage.

Results

PIK3CD expression is correlated to hepatic progenitor markers and its mutations are associate to a lower overall survival in liver cancer

We performed transcriptome analysis of primary liver cancer dataset GSE15765 composed in majority of hepatocellular carcinoma (n=70), and mixed form of intrahepatic cholangiocarcinoma (n=7) and cholangiocarcinoma (n=l3). Analysis of PIK3CD expression correlation to hepatic progenitor markers revealed a significant correlation with expression of CD44 (Pearson correlation coefficient: r = 0.39, p-value = 0.0001), also a significant correlation with expression of KRT19 (Pearson correlation coefficient: r = 0.23, p-value = 0.027), also a significant correlation with expression of PROM1 (Pearson correlation coefficient: r = 0.23, p- value = 0.028), and a significant correlation with expression of THY1 (Pearson correlation coefficient: r = 0.35, p-value = 0.0007).

Whole exome data analysis from 231 cases of resectable liver cancer cases (early stage hepatocellular carcinoma) showed that 5 patients (2.2% of the cases) were found mutated in PIK3CD coding region (nonsynonymous, missense mutations) affecting in majority domain PI3Ka (Phosphoinositide 3-kinase family, accessory domain (PIK domain)) and in minority PI3K_C2 (Phosphoinositide 3-kinase C2) and PI3K_p85B domains (PI3-kinase family, p85- binding domain). In this cohort, mutations affecting PI3_PI4_kinase (Phosphatidylinositol 3- and 4-kinase), PI3K_rbd (PI3-kinase family, ras-binding domain) domains were not found (data not shown). Presence of these coding mutations affecting PIK3CD were found significantly associated with a lower overall survival (log rank test, p=0.043, Figure 3). In this cohort mutations of IDH were also screen: no mutations was found in IDH2 and one patient was found mutated for IDH 1 (R132L) but this mutation was not found co-associated in patients presenting PIK3CD mutations (data not shown).

Gained of PIK3CD expression in HUH7 3D culture cell model regulates large expression profile

After transfection of PIK3CD in HUH7 culture cell model, transcriptome analysis was performed in triplicate on mRNA of these cells as compared to WT control. PIK3CD expressed was verified and effectively its expression was found significantly over-expressed after tranfection with a fold change of FC=+53 as compared to Wide type (WT) control condition (unpaired 2 sided Student ttest p-value= 4.59E-5, Figure 1A). Transcriptome RMA normalized matrix was filtered with genefilter R Bioconductor package to remove invariable genes. PIK3CD Affymetrix probe was used as quantitative predictor for Palvidis Template Matching algorithm applied to HUH7 dataset allowed to found 660 probes co-regulated genes to PIK3CD with correlation coefficient R>=0.80 in absolute value: 312 positive correlation and 348 negative correlation (data not shown). Unsupervised classification performed with this expression profile allowed to discriminate the experimental groups on heatmap representation (data not shown). And this discrimination was confirmed as significant by unsupervised principal component analysis (p-value = 0.00017, calculated with groups correlation to the first principal axis, Figure IB). These results showed that transfecting PIK3CD in a hepatic HUH7 3 dimension cell culture model allowed a large transcriptional regulation in these cells.

Genes up regulated by PIK3CD gain in HUH7 3D cell culture allowed to predict intrahepatic cholangiocarcinoma subclass of liver cancer

PIK3CD have already been reported has playing important role in epithelial cell polarity especially in the organization of the basolateral plasma membrane of these cells (Gassama- Diagne et al., 2006). HUH7 cell culture in 3 dimensions allowed to observed duct formation with lumens. It seems to reproduce tissue modifications observed in intrahepatic cholangiocarcinoma. Transfection of PIK3CD in HUH7 3D cell culture model allowed to observed deregulation of duct-lumens structural formations. Transcriptome dataset GSE15765 of liver cancer samples performed by Affymetrix technology is interesting because it contains a subclass of liver cancer samples which have intrahepatic cholangiocarcinoma characteristic: effectively these data already predict cholangiocarcinoma like markers in the past (Woo et a , 2010). Using totality of the expression profile 3D-HUH7-PIK3CD dependent in this dataset of liver cancer allowed to have a significant discrimination of the cholangiocarcinoma-like samples from the hepatocellular carcinoma samples (p-value = 2.34E- 12, calculated by the group correlation on the first principal axis, Figure 2A). With 3D-HUH7-PIK3CD expression profile, a supervised learning machine algorithm (leave one out with cross validation) was applied to the dataset GSE15765 between the two classes: hepatocellular carcinoma (HCC) and cholangiocarcinoma-like =“mixed” original class and“CC” original class (data not shown). This analysis allowed to perform a good prediction of the liver cancer subclasses with an important reduction of misclassification error by class during the learning process (Figure 4A). 3D-HUH7-PIK3CD expression profile is really efficient to predict invasion in this liver cancer dataset because total error sample misclassification is near 8% (Figure 4B). Crossing the information between genes up regulated in HUH7 and gene which were found up regulated in “cholangiocarcinoma-like” subclass of liver cancer allow to found a list of 85 genes (Figure 2B). These genes up regulations in both transcriptome dataset (3D HUH7 transfected by PIK3CD and liver cancer dataset GSE15765) could be seen on corresponding heatmap (data not shown).

Among the 85 genes, the inventors identified a signature of 10 genes predictive of intrahepatic cholangiocarcinoma (ICC) with 95% of sensibility and 97.5% of specificity

(Figure 5).

PIK3CD invasive expression profile highlight SRC linker as crosstalk between regulation of cell adhesion and epithelial cell morphogenesis

Functional enrichment performed on“Gene Ontology Biological Process” database with PIK3CD predictive signature for“cholangiocarcinoma-like” liver cancer revealed that this expression profile is well enriched on epithelial cell development functionalities (data not shown) such as: regulation of epithelial cell migration, morphogenesis of a branching epithelium, epithelial cell differentiation, branching morphogenesis of an epithelial tube, epithelium development, morphogenesis of an epithelium. This expression signature allowed also to enrich some functionalities implicated in cell polarity such as: cellular protein complex localization and regulation of membrane depolarization. Some PIK3CD specific signaling functions were also found enriched in this expression profile such as: inositol phosphate metabolic process, phosphatidylinositol phosphorylation. This analysis revealed also consequent implication of functionalities in relation with the extracellular compartment such as: regulation of cell adhesion and extracellular matrix compartment. Functional network analysis performed with gene ontology biological process database (data not shown) allowed to highlight some molecules highly connected to these important functionalities such as: FOXC2 transcription factor which seems largely implicated in epithelium morphogenesis functionalities, also SMAD7 which have some connections with cell polarity and BMP&TGFb signaling, finally SRC signaling linker seems to have central role by connecting some functionalities implicated in epithelium morphogenesis and some functionalities implicated in the communication with the extracellular part (regulation of cell adhesion, response to interleukin and TGFb). It is interesting to notify that SRC was classed in the best predictive genes for intrahepatic cholangiocarcinoma subclass (data not shown). Boxplot of SRC expression in 3D-HUH7-PIK3CD cell models also confirm significant over-expression (unpaired 2 sided student ttest p-value=0.024, data not shown).

PIK3delta in HCC associates a stem cell phenotype with chromatin repression of genes involved in liver development

In order to understand PIK3CD role in HCC, we investigated heterogeneity study of its expression in adult HCC transcriptome. We analyzed transcriptome dataset GSE14323 (Mas, Maluf et al. 2009), from viral etiology with HCV virus: this dataset contain normal liver samples, cirrhosis tissues and tumor HCC tissues. LIMMA algorithm was applied to tumor tissues with PIK3CD probe as quantitative predictor (data not shown). Unsupervised classification performed with PIK3CD on normal liver and HCC samples revealed that majority of HCC samples were classified near normal liver samples, but one minor subgroup of HCC samples (n=5) were classified far from the normal liver (data not shown). Effectively there is a significant overexpression of PIK3CD in this subgroup of 5 tumors as compared to the other majority of tumors (p-value <0.0001, data not shown). Gene set enrichment analysis allowed to highlight in liver tumor a positive relation between PIK3CD expression with hepatoblast signature (Normalized enrichment score=+2. l4, p <0.0001, data not shown), also enrichment in subtype of liver cancer harboring stemcell profile with H3K27me3 histone mark (Normalized enrichment score=+2.06, p <0.0001, data not shown), and also an enrichment with signature of genes implicated in liver development (Normalized enrichment score=+2.02, p <0.0001, data not shown). Functional network performed with these enrichments allow to see that the major common gene profile was shared with the enrichment performed with tumors which harbor repressive mark H3K27me3. H3K27me3 CHIP- sequencing performed on HEPG2 hepatoblast cell line was analyzed and revealed that majority of the detected peaks were found well conserved around transcription starting sites (TSS) on vertebrae promoter database (data not shown). Overlapping of H3K27Me3 repressive marks and gene expression profile negatively correlated to PIK3CD in HCC transcriptome allowed to discover a repressed program of 166 genes (data not shown), which are well organized all over the human genome but without event on chromosomes 5 and 18 (data not shown). Functional enrichment performed with this HCC repressive program on epigenetic peaks from H3K27me3 allow to discover that this program is enriched in hepatocyte differentiation (Negative loglO p-value=42.07, data not shown), also in targets of FOXA2 (Negative loglO p-value=l9.84), also in genes downregulated in liver development (early fetal liver stage E11.5-E12.5, Negative p-value=l4.20, data not shown), and also in gene implicated in Hepatoblastoma: undifferentiated pediatric liver cancer (Negative p-value=6.23, data not shown). Genes repressed in these processes implicated in liver development and repressed in PIK3CD liver cancers were used to build a functional network highly connected (data not shown). It is interesting to see that genes found to be correlated to PIK3CD were also capable to predict stratification of the invasive subgroup of tumors in an independent cohort of tumor liver transcriptome (Woo HG and al. 2008) (GSE15765, p- value=4.50274le-l l , data not shown). By integrating epigenetic H3K27me3 with 214 transcriptome liver samples from independent cohorts, this work highlighted that PIK3CD is highly expressed in HCC which repressed its liver development program controlled by H3K27me histone mark in patient with invasive liver cancer.

Discussion

In our work, PIK3CD was found correlated to hepatic progenitor markers associated to the worst prognosis in liver cancer. Firstly, a correlation to CD 133 alias PROM1 which has been recognized as tumor initiating cell maker in HCC cell lines, effectively compared with CD133- cells, CD133+ cells isolated from HCC cell lines showed higher expression of CD44 and CD34 (Ma et a , 2007). In regenerative liver tissue the CD117+/CD133+ hepatic progenitors have been recognized as tumor- initiating cells (Craig et a , 2004). Strong expression of CD133 was found increased in cholangiocarcinoma progression especially with nodal metastasis: the CDl33(+) cells had a higher invasive ability compared with CDl33(-) cells (Leelawat et a , 2011). PIK3CD was also found correlated to CD90 marker. CD90 expression was shown to be increased in hepatic tumors as compared to both its paired cirrhotic tissue and normal liver and hepatocellular carcinoma cell line JHH-6 CD90+ present more proliferative properties than CD90- (Sukowati et ah, 2013). CD90+ cells are not present in normal liver, but pathologic ones injected into immune-deficient mice have the ability to created repeated tumors (Ma et ah, 2007). CD45-/CD90+ subpopulation of tumor cells in HCC has also been characterized as tumor-initiating cells (Yang et a , 2008). PIK3CD was also found correlated to CD44. In intrahepatic cholangiocarcinoma CD44 expression showed an association with periductal infiltrative type, poor differentiation, and vascular invasion (Gu and Jang, 2014). Finally PIK3CD was also found correlated to CK19 in liver cancer, HCC expressing highly CK19 have lower tumor free survival after curative resection and CK19 is an independent predictor of postoperative recurrence (Uenishi et a , 2003). Liver cancer which overexpressed CK19 and have poor prognosis harbored a sternness gene expression profile in their transcriptome (Kim et a , 2011). High level of CK19 expression have also been attached to tumors which harbored NOTCH signaling pathway activation and presenting an increase of “Side population” cell compartment (Govaere et a , 2014).

Mutations affecting PIK3 signaling pathway have already been observed HCC cohort (Ahn et a , 2014) but mutations concerning PIK3delta wasn’t been highlighted in the past. In our work, analysis of exome sequencing from early stage liver cancer revealed more frequent mis sense mutations in PIK domain of PIK3CD as compared to other protein domains. PIK domain is conserved in all PI3 and PI4-kinases. Its role is unclear but it has been suggested to be involved in substrate presentation (Flanagan et al., 1993). In our analyzed, we associated presence of PIK3CD missense mutations to a significant lower overall survival in liver cancer suggesting maybe important role of this protein physiopathology of liver cancer.

In our study, we found a FOXC2 regulation dependent to PIK3CD and this regulation was found in a context of polarity transformation of HUH7 hepatic cell lines. FOXC2 (Forkhead Box C2), is a transcription factor which has been found as promoter of epithelia-mesenchymal transition in breast cancer harboring metastasis by induction of ZEB1 (Werden et al., 2016), suggesting is important role in cancer invasive process. FOXC2 transcription factor previously have been attached to poor prognosis of extrahepatic cholangiocarcinoma and its role in invasion have demonstrated by its knock down in cholangiocarcinoma cell lines : resulting an inhibition of cell mobility and invasion, also decreased expression of EMT markers such as N- cadherin and matrix metalloproteinase MMP2 and angiopoietin-2 (Watanabe et al., 2013). Functional interpretation of our polarity hepatic cell model revealed large implication of this transcription factor in epithelial tissue morphogenesis and extracellular matrix organization and this functions predict alterations observed in transcriptome of invasive liver cancer (intrahepatic cholangiocarcinoma) .

In intrahepatic cholangiocarcinoma, SRC dependency have been linked to Isocitrate Dehydrogenase Mutations and confers hypersensitivity to Dasatinib therapy (Saha et al., 2016). Interestingly, in our analysis of liver cancer patients, mutations present in PIK3CD are not concerned by IDH mutations suggesting that some other events than IDH mutations could influence SRC regulation in intrahepatic cholangiocarcinoma.

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