Description

The present invention relates to a method of predicting/determining the responsiveness of a cancer disease to treatment with an inhibitor of CD95/CD95L signalling on the basis of methylation levels of specific DNA sequences.

Treatment regimens used for cancers, such as progressive glioblastoma, are of very limited efficacy [1 -3]. For years, alkylating chemotherapy has been the mainstay, although patients already had been exposed to temozolomide in conjunction with radiotherapy after diagnosis and surgery. Recent and ongoing trials often have used lomustine (CCNU) as control arm and tested anti-vascular endothelial growth factor (receptor) strategies [4,5], protein kinase C-beta inhibition with enzastaurin [6], or they embarked on various temozolomide regimens [7,8].

When it became evident that tissue recovery in the brain might be much better than initially thought, options for a second biologically relevant radiotherapy (rRT) were explored in retrospective analyses or uncontrolled trials, and positive clinical outcomes were reported [9,10]. Different concepts of hypofractionation, target delineation and dosing, e.g. 18 x 2 Gy, 15 x 2.33, 6 x 5 Gy, exist, but there is no consensus on one particular regimen.

CD95 (Fas and APO-1 ) is a pleiotrophic receptor that regulates tissue homeostasis. During cancer progression, CD95 is frequently down-regulated or tumour cells are rendered apoptosis resistant. However, evidence exists that cancer cells, regardless of their CD95 apoptosis sensitivity, depend on constitutive activation of CD95 for optimal growth [1 1 ], stimulated by CD95L produced in an autocrine or paracrine manner.

A growth-promoting role of the CD95/CD95L system has recently been described for cancer and in particular for glioblastoma [12,13] where activation of CD95 by CD95L stimulates AKT kinase- and β-catenin- dependent genes [12]. CD95 activation in glioblastoma leads to invasive growth and migration facilitated by increased expression of matrix metalloproteinases (MMP), which are key mediators of glioma invasiveness [13]. In vitro, blocking of CD95 activation was demonstrated to inhibit increased invasiveness of sublethally irradiated glioblastoma cells as an adaptive evasive response to radiation [14,15]. This unwanted effect of radiotherapy is proposed to be mediated by stimulation of PI3K/AKT- dependent MMP-2 and MMP-9 activity [16,17] or alternative mechanisms [18].

Correspondingly, inhibitors of CD95/CD95L signalling and their use in the treatment of cancer diseases are known in the art.

In this context, the provision of a method for predicting/determining if a specific patient suffering from a specific cancer disease will respond to an inhibitor of CD95/CD95L signalling allowing a patient-tailored treatment would be desirable.

Therefore, it was the object of the present invention to provide methods which enable further characterization of a cancer disease and to predict how efficient or successful treatment with an inhibitor of CD95/CD95L signalling would be. According to the present invention this object is achieved by a method of determining the methylation level of a DNA sequence located upstream of and/or in a gene involved in CD95/CD95L signalling in a sample obtained from a patient and classifying the cancer disease according to said methylation level. This classification can then be used to predict/determine the responsiveness of the cancer disease to the treatment described above.

In the present invention it was surprisingly found that the methylation level of specific DNA sequences located upstream of and/or in a gene involved in CD95/CD95L signalling correlates with the clinical outcome of treatment with an inhibitor of CD95/CD95L signalling, optionally in combination with radiotherapy.

DNA methylation is a biochemical process which involves the addition of methyl groups to adenine or cytosine in the DNA. DNA methylation has been shown to play an important role, for example, in developmental processes and in regulation of gene expression. In this regard, methylation of cytosines of CpG sites within so-called CpG islands is especially interesting. The term "CpG island" which is known to the person skilled in the art denotes DNA regions which exhibit a higher frequency of the di-nucleotide sequence CpG (a CpG site) compared to the corresponding frequency over the whole genome. In general, CpG islands are several hundred base pairs long and mostly found in the 5' region of genes.

One aspect of the present invention is a method of predicting/determining the responsiveness of a cancer disease to treatment with an inhibitor of CD95/CD95L signalling comprising the steps of

i) determining the methylation level of a preselected DNA sequence, in particular of a specific CpG site, located upstream of and/or in a gene involved in CD95/CD95L signalling in a sample obtained from a patient; and

ii) predicting/determining the responsiveness of the cancer disease according to said methylation level.

Preferably, the cancer disease is considered to be responsive to said treatment if the methylation at defined CpGs level is < 98%, < 95%, < 90%, < 85%, < 80%, or < 75%. In this context, a methylation level of 100% denotes that in a given sample in all DNA copies the respective CpG sites are methylated.

The methylation level of a DNA sequence may be determined by any method known in the art. For example, the methylation level can be determined by the MassARRAY technique (Sequenom, San Diego, CA, USA). This technique is based on detection of mass shifts introduced through sequence changes following bisulfite treatment. A further aspect of the present invention is a method of treating a cancer disease comprising the steps of

i) determining the methylation level of a DNA sequence located upstream of and/or in a gene involved in CD95/CD95L signalling in a sample obtained from a patient;

ii) classifying the cancer disease according to said methylation level; and

iii) administering to said patient a pharmaceutically effective dose of an inhibitor of CD95/CD95L signalling if the cancer disease is classified to be responsive to treatment with said inhibitor.

The to be classified may be any malignant disease, in particular cancer disease or malignancy derived from tissues from epithelial or other than epithelial tissues known in the art. According to one embodiment the cancer is characterized by increased CD95 and/or CD95L transcription and/or expression. For example, the cancer disease may be an epithelial or hematological cancer. The cancer disease may be a cancer of lymphoid or myeloid origin. It may be any type of cancer, in particular solid tumor tissue. The cancer or other malignant disease can be selected from the group consisting of brain tumors, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, lung cancer, renal cancer, liver cancer or/and metastatic disease thereof. Preferably, the malignant disease is glioma, in particular glioblastoma. The cancer can be newly diagnosed and/or progressive glioblastoma.

Responsiveness of a cancer disease to a specific treatment may be evaluated by any method known in the art. A non-limiting prognostic factor to be considered when evaluating responsiveness of a cancer disease is tumor size. Objective radiological responses, increased progression-free survival or overall survival, set intervals thereof and objective clinical improvement are indicative of responsiveness of a cancer. Further definitions can be found in the examples section. The samples to be obtained from a patient can be obtained by any method known in the art such as initial surgery and/or biopsy and also samples from blood or cerebrospinal fluid. The samples are obtained from tumor tissue. Preferably, the inventive method is an in vitro method.

According to the method of the present invention treatment of the cancer disease is effected by administration of an inhibitor of CD95/CD95L signalling, alone or in combination with other treatments and/or chemotherapeutic agents. Other treatments may be any treatments known in the art, such as radiotherapy and/or surgery. Examples for preferred chemotherapeutic agents are alkylating agents such as temozolomide and/or nitrosoureas. Other treatments are targeted based therapies like kinase inhibitors or antibody therapy, e.g. based on bevacizumab. Conventionally a combination of radiotherapy and temozolomide is used. In a method according to the present invention, treatment is preferably effected by the administration of an inhibitor of CD95/CD95L signalling in combination with radiotherapy.

Radiotherapy increases the permeability of the blood-brain-barrier (BBB), which may lead to oedema and potentially worsening of neurological symptoms. At the same time, however, an increased BBB-permeability may facilitate agents entering the tumour stroma and the brain parenchyma surrounding the tumour, specifically the invasive front.

As used throughout the specification, the terms CD95, CD95R and CD95 receptor may be used interchangeably. Further synonyms are Apo-1 or Fas which may be used interchangeably herein. Further, the terms CD95L, CD95 ligand and the corresponding synonyms FasL, Apo-1 L, CD178 or TNF-SF6 may be used interchangeably.

It is to be understood that the term "CD95/CD95L signalling" is known to a person skilled in the art. It refers to the CD95/CD95L signalling pathway and comprises any component or interaction of this signalling pathway. The components are not restricted to a specific class of molecules. For example, such a component may be a protein but it may also be a nucleic acid or a small molecule. Non-limiting examples include the proteins CD95, CD95 ligand, Yes, FADD, PI3K, GSK-3 , β-Catenin, JNK, ERK1/2, AKT, NFKB or MMPs or the respective nucleic acid sequences.

"Genes involved in CD95/CD95L signalling" according to the present invention are, e.g., genes which encode components of the CD95/CD95L signalling pathway as specified above.

The "DNA sequence located upstream of and/or in a gene involved in CD95/CD95L signalling" may be any type of DNA sequence. In this respect, "upstream of a gene" refers to the 5' region of a gene. According to the present invention this DNA sequence may be part of a regulatory sequence and/or a CpG island, or it may comprise a regulatory sequence and/or a CpG island, as well as flanking regions. For example, the DNA sequence may comprise or be comprised by a regulatory sequence or the DNA sequence may comprise or be comprised by a CpG island. The length of the DNA sequence may depend on the specific type of cancer disease and/or the specific gene involved in CD95/CD95L signalling. For example, the DNA sequence may be > 100 nucleotides long, preferably > 50 nucleotides or > 10 nucleotides. The DNA sequence can also be from 1 -10 nucleotides in length. In the most preferred embodiment the DNA sequence to be methylated consists of one nucleotide. In this embodiment the DNA sequence is C at position 135 in SEQ ID NO: 2, denoted as CpG1 , and/or C at position 180 of SEQ ID NO: 2, denoted as CpG2 (based on Human Feb. 2009 (GRCh37/hg19) Assembly), ranging from chrl :172,628,000- 172,628,120 (reference genome GrCh37).

The terms "inhibitor of CD95/CD95L signalling" or "inhibitor of the CD95/CD95L signalling pathway" in terms of the present invention may be used interchangeably and may be any compound which interferes or blocks at least partially the CD95/CD95L signalling pathway. According to a preferred embodiment an "inhibitor of CD95/CD95L signalling" blocks the CD95/CD95L signalling pathway. Methods for determining and/or assessing CD95/CD95L signalling pathway activity are known to the person skilled in the art and are, for example, described by Lavrik et. al. (Cell Death Differ. 2012 Jan;19(1 ):36-41 Regulation of CD95/Fas signalling at the DISC).

An inhibitor used in a method according to the invention may act on the protein level and/or the nucleic acid level. Inhibitors acting on the protein level may be selected from antibodies, proteins and/or small molecules. Inhibitors acting on the nucleic acid level are for example antisense molecules, RNAi molecules and/or ribozymes.

According to a preferred embodiment the inhibitor binds to the CD95 receptor (CD95) and/or the CD95 ligand (CD95L). For example, the CD95/CD95L interaction may be inhibited.

In one preferred embodiment, the inhibitor used in a method according to the invention is an antibody or a functional fragment thereof. The inhibitor being an antibody may bind to CD95, but, of course, also to CD95L. An example for an antibody binding CD95L is Nok-1 .

The antibody may be, for example, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a human antibody, a chimeric antibody, a multi-specific antibody, or an antibody fragment thereof (e.g., a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a diabody, or a single chain antibody molecule). The antibody can be of the lgG1 -, lgG2-, lgG3- or lgG4-type.

The antibody may be used with or without modification, and may be labelled, either covalently or non-covalently, with, for example, a reporter group or an effector group.

An "antibody fragment" used in a method according to the invention presents essentially the same epitope binding site as the corresponding antibody does and/or has substantially the same CD95 and/or CD95L inhibiting activity as the corresponding antibody has.

Methods for producing antibodies of the invention are known to the person skilled in the art.

One kind of inhibitor encompassed by the present invention may be a CD95 ligand inhibitor. For example, CD95 ligand inhibitors may be selected from (a) an inhibitory anti-CD95 ligand-antibody or a fragment thereof as outlined above; (b) a soluble CD95 receptor molecule or a CD95 ligand-binding portion thereof; and (c) a CD95 ligand inhibitor selected from FLINT, DcR3 or fragments thereof.

Soluble CD95 receptor molecules, e.g. a soluble CD95 receptor molecule without transmembrane domain are described in EP-A-0 595 659 and EP-A- 0 965 637 or CD95 receptor peptides as described in WO 99/65935

The Fas ligand inhibitor FLINT or DcR3 or a fragment, e.g. soluble fragment thereof, for example the extracellular domain optionally fused to a heterologous polypeptide, particularly a Fc immunoglobulin molecule is described in WO 99/14330 or WO 99/50413. FLINT and DcR3 are proteins which are capable of binding the CD95 ligand.

In a further embodiment the inhibitor is a fusion protein, in particular a fusion protein that binds to a CD95L.

In a preferred embodiment the inhibitor of CD95/CD95L signalling comprises a fusion protein comprising at least an extracellular CD95 domain or a functional fragment thereof and at least a Fc domain or a functional fragment thereof, an anti-CD95L specific antibody or a CD95L recognising fragment thereof, an anti-CD95 specific antibody or a CD95 recognising fragment thereof, a small molecule and/or combinations thereof. In a preferred embodiment the fusion protein is selected from APG101 as set forth in SEQ ID NO: 1 , polypeptides having at least 70% sequence identity to APG101 and/or functional fragments thereof.

Fusion proteins comprising the extracellular domain of the death receptor CD95 (Apo-1 ; Fas) fused to an immunoglobulin Fc domain are described in PCT/EP04/03239, the disclosure of which is included herein by reference. "Fusion protein", as used herein, includes a mixture of fusion protein isoforms, each fusion protein comprising at least an extracellular CD95 domain (APO-1 ; Fas) or a functional fragment thereof and at least a second domain being an Fc domain or a functional fragment thereof distributing within a pi range of about 4.0 to about 8.5. Accordingly, the extracellular CD95 domain as used herein may be also called "first domain", while the Fc domain may be called "second domain".

The mixture of fusion proteins can be provided in a composition.

The first protein domain is an extracellular CD95 domain, preferably a mammalian extracellular domain, in particular a human protein, i.e. a human extracellular CD95 domain. The first domain, i.e. the extracellular CD95 domain, of the fusion protein preferably comprises the amino acid sequence up to amino acid 170, 171 , 172 or 173 of human CD95 (SEQ ID NO. 1 ). A signal peptide (e.g. position 1 -25 of SEQ ID NO: 1 ) may be present or not. Particularly for therapeutic purposes the use of a human protein is preferred.

The fusion protein can comprise one or more first domains which may be the same or different. One first domain, i.e. one extracellular CD95 domain is preferred to be present in the fusion protein.

According to a preferred embodiment, the Fc domain or functional fragment thereof, i.e. the second domain of the fusion protein used in method according to the invention, comprises the CH2 and/or CH3 domain, and optionally at least a part of the hinge region, or a modified immunoglobulin domain derived therefrom. The immunoglobulin domain may be an IgG, IgM, IgD, or IgE immunoglobulin domain or a modified immunoglobulin domain derived, therefrom. Preferably, the second domain comprises at least a portion of a constant IgG immunoglobulin domain. The IgG immunoglobulin domain may be selected from lgG1 , lgG2, lgG3 or lgG4 domains or from modified domains therefrom. Preferably, the second domain is a human Fc domain, such as a IgG Fc domain, e.g. a human lgG1 Fc domain.

The fusion protein can comprise one or more second domains which may be the same or different. One second domain, i.e. one Fc domain is preferred to be present in the fusion protein.

Further, both the first and second domains are preferably from the same species.

The first domain, i.e. the extracellular CD95 domain or the functional fragment thereof may be located at the N- or C-terminus. The second domain, i.e. the Fc domain or functional fragment may also be located at the C- or N-terminus of the fusion protein. However, the extracellular CD95 domain at the N-terminus of the fusion protein is preferred.

According to a further preferred embodiment, the fusion protein is APG101 (CD95-FC, position 26-400 in SEQ ID NO: 1 ).

APG101 is a CD95L-binding protein consisting of the extracellular domain of human CD95 fused to the Fc region of human lgG1 . It interferes with CD95- dependent signalling by binding to CD95L, thereby blocking subsequent CD95-dependent activation [19].

As defined by SEQ ID NO: 1 APG101 can be a fusion protein comprising a human extracellular CD95 domain (amino acids 26-172) and a human lgG1 Fc domain (amino acids 172-400), further optionally comprising an N- terminal signal sequence (e.g. amino acids 1 -25 of SEQ ID NO: 1 ). The presence of the signal peptide indicates the immature form of APG101 . During maturation, the signal peptide is cleaved off. According to an especially preferred embodiment the signal sequence is cleaved off. APG101 with the signal sequence being cleaved off is also comprised by the term "unmodified APG101 ". In a further embodiment the fusion protein is a polypeptide having at least 70% identity, more preferably 75% identity, 80% identity, 85% identity, 90% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity with APG101 . According to the present application the term "identity" relates to the extent to which two amino acid sequences being compared are invariant, in other words share the same amino acids in the same position.

The term "APG101 " includes a fusion protein of position 26-400 of SEQ ID NO: 1 , with and without a signal peptide. The term "APG101 " also includes fusion proteins containing N-terminally truncated forms of the CD95 extracellular domain.

In another preferred embodiment the fusion protein used in a method according to the invention is a functional fragment of APG101 . As used herein, the term "fragment" generally designates a "functional fragment", i.e. a fragment or portion of a wild-type or full-length protein which has essentially the same biological activity and/or properties as the corresponding wild-type or full-length protein has.

A person skilled in the art is aware of methods to design and produce fusion proteins used in a method according to the present invention. The mixture of fusion protein isoforms, in particular APG101 isoforms, however, can be obtained by a method described, e.g., in PCT/EP04/03239, the disclosure of which is included herein by reference. According to a preferred embodiment designing a fusion protein of the present invention comprises a selection of the terminal amino acid(s) of the first domain and of the second domain in order to create at least one amino acid overlap between both domains. The overlap between the first and the second domain or between the two first domains has a length of preferably 1 , 2 or 3 amino acids. More preferably, the overlap has a length of one amino acid. Examples for overlapping amino acids are S, E, K, H, T, P, and D.

As indicated above, "fusion protein", as used herein, includes a mixture of isoforms. The term "isoform" as used herein designates different forms of the same protein, such as different forms of APG101 , in particular APG101 without signal sequence. Such isoforms can differ, for example, by protein length, by amino acid, i.e. substitution and/or deletion, and/or post- translational modification when compared to the corresponding unmodified protein, i.e. the protein which is translated and expressed from a given coding sequence without any modification. Different isoforms can be distinguished, for example, by electrophoresis, such as SDS- electrophoresis, and/or isoelectric focussing which is preferred according to the present invention.

Isoforms differing in protein length can be, for example, N- terminally and/or C-terminally extended and/or shortened when compared with the corresponding unmodified protein. For example, a mixture of APG101 isoforms used in a method according to the invention can comprise APG101 in unmodified form as well as N- terminally and/or C-terminally extended and/or shortened variants thereof. Thus, according to a preferred embodiment, the mixture used in a method according to the invention comprises N-terminally and/or C-terminally shortened variants of APG101 . In particular preferred is a mixture of fusion protein isoforms comprising N- terminally shortened fusion proteins. Such N-terminally shortened fusion proteins may comprise -1 , -2, -3, -4, -5, -6, -7, -8, -9, -10, -1 1 , -12, -13, -14, -15, -16, -17, -18, -19, -20, -21 , -22, -23, -24, -25, -26, -27, -28, -29, -30, -35, -40, -45 and/or -50 N-terminally shortened variants of unmodified APG101 . Particularly preferred are -17, -21 and/or -26 N-terminally shortened variants. The numbering refers to the APG101 protein including signal sequence according to SEQ ID NO: 1 . In other words, the shortened fusion proteins can comprise a sequence SEQ ID NO: 1 N-terminally truncated by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 and/or 50 amino acids. Preferred shortened fusion proteins have SEQ ID NO: 1 N-terminally truncated by 16, 20, or 25 amino acids.

An example for a C-terminal shortening of APG101 isoforms is C-terminal Lys-clipping.

According to a preferred embodiment of the present invention the mixture of fusion proteins used in a method according to the present invention preferably comprises 50 mol-% unmodified APG101 in relation to modified isoforms, more preferably 40 mol-% unmodified APG101 , more preferably 30 mol-% unmodified APG101 , more preferably 20, more preferably 10 mol- % unmodified APG101 , more preferably 5 mol-% unmodified APG101 and even more preferably 3 mol-% unmodified APG101 and most preferably 1 mol-% and/or less unmodified APG101 . Most preferred is an embodiment comprising a mixture of fusion protein isoforms that does not comprise any unmodified APG101 .

As outlined above, isoforms can also differ by amino acid substitution, amino acid deletion and/or addition of amino acids. Such a substitution and/or deletion may comprise one or more amino acids. However, the substitution of a single amino acid is preferred according to this embodiment.

Isoforms used in a method according to the invention can also differ with regard to post-translational modification. Post-translational modification according to the present invention may involve, without being limited thereto, the addition of hydrophobic groups, in particular for membrane localisation such as myristoylation, palmitoylation, isoprenylation or glypiation, the addition of cofactors for enhanced enzymatic activity such as lipoyation, the addition of smaller chemical groups such as acylation, formylation, alkylation, methylation, amidation at the C-terminus, amino acid addition, γ- carboxylation, glycosylation, hydroxylation, oxidation, glycilation, biotinylation and/or pegylation.

According to the present invention the addition of sialic acids, Fc-based glycosylation, in particular Fc-based N-terminal glycosylation, and/or pyro- Glu-modification are preferred embodiments of post-translational modification.

Beside the first and second domain as defined herein, the fusion proteins used in a method according to the invention may comprise further domains such as further targeting domains, e.g. single chain antibodies or fragments thereof and/or signal domains. According to a further embodiment, the fusion protein used in a method according to the invention may comprise an N- terminal signal sequence, which allows secretion from a host cell after recombinant expression. The signal sequence may be a signal sequence which is homologous to the first domain of the fusion protein. Alternatively, the signal sequence may also be a heterologous signal sequence. In a different embodiment the fusion protein is free from an additional N-terminal sequence, such as a signal peptide.

The fusion protein as described herein may be an N-terminally blocked fusion protein, which provides a higher stability with regard to N-terminal degradation by proteases, as well as a fusion protein having a free N- terminus, which provides a higher stability with regard to N-terminal degradation by proteases.

Modifications blocking the N-terminus of protein are known to a person skilled in the art. However, a preferred post-translational modification according to the present invention blocking the N-terminus is the pyro-Glu- modification. Pyro-Glu is also termed pyrrolidone carboxylic acid. Pyro-Glu- modification according to the present invention relates to the modification of an N-terminal glutamine by cyclisation of the glutamine via condensation of the a-amino group with a side chain carboxyl group. Modified proteins show an increased half-life. Such a modification can also occur at a glutamate residue. Particularly preferred is a pyro-Glu-modification, i.e. a pyrrolidone carboxylic acid, with regard to the N-terminally shortened fusion protein -26.

A mixture as described herein may comprise 80-99 mol-% N-terminally blocked fusion proteins and/or 1 -20 mol-% fusion proteins having a free N- terminus.

According to a further preferred embodiment the mixture as described herein comprises 0.0 to 5.0 mol-%, more preferably 0.0 to 3.0 mol-% and even more preferably 0.0 to 1 .0 mol-%, of fusion protein high molecular weight forms such as aggregates. In a preferred embodiment the mixture does not comprise any aggregates of fusion protein isoforms, in particular no dimers or aggregates of APG101 . Dimers or aggregates are generally undesired because they have a negative effect on solubility.

In still a further embodiment of the present invention the inhibitor is a nucleic acid effector molecule. The nucleic acid effector molecule may be DNA; RNA, PNA or an DNA-RNA-hybrid. It may be single stranded or double stranded. Expression vectors derived from retroviruses, adenovirus, herpes or vaccina viruses or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system.

The nucleic acid effector molecule may be in particular selected from antisense molecules, RNAi molecules and ribozymes which are preferably capable of inhibiting the expression of the CD95R and/or CD95L gene. An inhibitor of the CD95 signalling pathway used in a method according to the present invention course may be provided as a pharmaceutical composition. This composition may comprise pharmaceutically acceptable carriers, diluents and/or adjuvants, etc.

Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal means.

Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient, for example a nucleic acid or a protein of the invention or an antibody, which is sufficient for treating a specific condition. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment.

Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. A person skilled in the art is aware of further methods to provide sufficient levels of the active moiety and/or to maintain the desired effect. Factors, which may be taken into account, include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. In a preferred embodiment the total amount of the inhibitor of the CD95 signalling pathway according to the present invention to be administered for a patient suffering from MDS is from about 50 to about 600 mg/week, preferably from about 50 to about 400 mg/week and more preferably about 100 to about 200 mg/week. The preferred weekly dose can be administered as a single dose or as several doses. Especially preferred is a single dose particularly from 100 to 200 mg/week which is administered intravenously as a single dose.

The treatment can last for several weeks. In each individual case, the duration of the treatment is determined by the supervising doctor and is e.g. based on the success of the treatment, the occurrence of side effects etc.

A further aspect of the present invention is a CD95L inhibitor for use in the treatment of a cancer disease in a subject selected from the group consisting of patients exhibiting a methylation level of a DNA sequence located upstream of and/or in a gene involved in CD95/CD95L signalling in a sample obtained from the patient of < 98%, < 95%, < 90%, < 85%, < 80%, or < 75%.

A still further aspect of the present invention is a method of selecting a treatment for a cancer disease, comprising the steps of

i) determining the methylation level of a DNA sequence located upstream of and/or in a gene involved in CD95/CD95L signalling in a sample obtained from a patient;

ii) classifying the cancer disease according to said methylation level; and iii) selecting a treatment suitable for treating the cancer disease according to its classification.

Preferably, a treatment with an inhibitor of CD95/CD95L signalling is selected, if the methylation level is < 98%, < 95%, < 90%, < 85%, < 80%, or < 75%.

In a yet further aspect the methylation level of CpGs contained in SEQ ID NO: 2 located at chrl :172,628,020 (CpG2) and chrl :172,628,065 (CpG1 ) (GRCh37/hg19) are used as a biomarker for the prediction/determination of diseases. Exemplary diseases are cancer diseases such as epithelial or hematological cancer. The cancer disease may be a cancer of lymphoid or myeloid origin. It may be any type of cancer, in particular solid tumor tissue. The cancer disease can be selected from the group consisting of brain cancer, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, lung cancer, renal cancer, liver cancer or/and metastatic disease thereof. Preferably, the cancer disease is brain tumor such as but not limited to glioblastoma, glioma, astrocytoma, oligodendroglioma, ependymoma, in particular glioblastoma. The cancer can be newly diagnosed and/or progressive glioblastoma.

FIGURE LEGENDS

Figure 1 : Trial design and CONSORT flow chart. Patients were

randomized 1 :2 to receive rRT or rRT+APG101 (Abbreviations: intention-to- treat population, ITT; reirradiation, rRT).

Figure 2: Kaplan-Meier survival estimates. Data of PFS (panel a) or OS (panel b) were analyzed by treatment arm.

Figure 3: Supplementary Figure 1. Overview of the genomic position of cg10161121 , cg06983746 and CD95 ligand.

Figure 4: Supplementary Figure 2. Survival by treatment and

cg06983746 methylation. Kaplan-Meier curves for overall survival in patients with lower (a) and higher (b) than median methylation, by treatment group.

Figure 5: Supplementary Figure 3. Kaplan-Meier survival estimates.

Data of PFS (panel a,b) or OS (panel c,d) were analyzed by treatment arm and CD95L expression in the tumor tissue.

Figure 6: Supplementary Figure 4. Cox regression for cg10161121 and cg06983746 and OS in a NOA-8 biomarker cohort, (a) OS in NOA-08 (n=40) by cg10161 121 or cg06983746. (b) OS in NOA-08 by treatment (21 RT / 19 TMZ) by cg10161 121 or cg06983746.

Examples

Example 1 METHODS Patients

Adult patients with first or second progression of a histologically confirmed glioblastoma, either not being eligible for tumour resection or having macroscopic residual tumour after tumour resection documented by contrast- enhanced magnetic resonance imaging (MRI) with the largest diameter measuring 1 to 4 cm and a Karnofsky performance score (KPS) > 60, were eligible. No more than two prior therapy regimens including one or two resections, one or two chemotherapies of which one is temozolomide- containing, and one radiotherapy (completed >8 months prior to enrolment) were allowed. All patients were required to give signed informed consent prior to enrolment.

Radiation Therapy

rRT at 36 Gy in 2 Gy single fractions was required to be performed as highly precise treatment, either as stereotactic radiotherapy, or as image-guided radiotherapy. To assess the quality of each participating centre, a dedicated dummy run evaluating technical equipment, quality assurance as well as treatment planning was performed (see Supplementary Methods).

APG101

APG 101 was given at 400 mg weekly as a 30-minute i.v. infusion. Neuropathological Methods

Archived tumour tissue was available from 81 patients. This tumour tissue was used to examine the neuropathological markers IDH1 , MGMT, CD95 and CD95L, as well as genome-wide methylation levels in a discovery set of 20 patients. CD95L promoter methylation at probes interrogating two CpGs located at chrl 172.628,020 (CpG2) and chrl :172,628,065 (CpG1 ) (GRCh37/hg19) was then examined in a validation set of 40 patients by MassARRAY. For clinical data evaluations, a median based cut-off for each CpG was used (see Supplementary Methods and Tables S1-4).

Statistical analysis

The primary endpoint was the proportion of patients free of progression based on the central assessment and alive at six months (PFS-6), calculated in days from randomisation.

The sample size of the study was planned according to the optimal two- stage design of Simon ²⁴ for the rRT+APG101 arm with a PFS-6 target rate of 30%, a non-interesting rate of 15%, first-type error rate of 0.05 and a power of 80%. A control arm of patients treated with rRT alone was added to the Simon design to calibrate the PFS-6 rate. The sample size of the control arm was defined as 50% of the investigational treatment arm. The Simon design required the recruitment of 55 patients for the rRT+APG101 arm (19 patients in Stage 1 and 36 patients in Stage 2). With the addition of 28 patients in the control arm, the study was hence planned with a total sample size of 83 patients.

According to the Simon design, the study was considered positive if 13 PFS- 6 responses were observed among the 55 patients treated with rRT+APG101 , based on the assumption that five PFS-6 responses would be seen in the control arm.

Secondary efficacy endpoints were: objective response rates, OS, PFS, quality-of-life as determined by EORTC PAL QLQ-C15 and BN-20, and cognitive function determined by MMSE. Safety and tolerability of APG101 were assessed by adverse events with intensity mild/moderate/severe. PFS was defined as time from randomisation to next progression for patients with progression or, respectively, as time to death of any cause for patients without progression described with Kaplan-Meier estimates. Patients without progression or death were censored at the day of the last assessment of tumour response. The significance level for remarkable findings was set to 0.05 for all tests in this study. All analyses of the primary and secondary efficacy endpoints were based on the intention-to-treat population, which included all randomised patients except patients who did not receive any dose of trial medication or rRT after randomisation. The per-protocol analyses were limited to patients without major protocol violations (Fig. 1 ).

The safety analyses were done on the entire documentation of adverse events (details in Supplementary Methods).

Analyses were performed with SAS ^® 9.1 .3 (SAS Institute, Cary, NC). During the study, the data were documented into the Oracle Clinical ^® data management system of Premier Research (Darmstadt, Germany). Premier Research monitored the data quality.

RESULTS

Patients

The trial enrolled and randomised 91 patients. The ITT population included 84 patients who were randomised and received at least one dose of APG101 or rRT. The per-protocol population consisted of 72 patients (Fig. 1 ). As of the data cut-off date, median follow-up was 1 1 .4 months in both treatment arms. Baseline patient and disease characteristics were well balanced (Table 1 ).

Tolerability and toxicity

Most patients tolerated both treatments well. Toxicities are listed in Table 2. There were three patients in the rRT arm who discontinued rRT due to disease progression and one patient receiving 20 fractions. All other patients (22/26 = 84.6%) received the planned 18 x 2 Gy. In the rRT+APG101 arm, all patients except of one received the planned RT (57/58=98.3%). The median duration of APG101 treatment was 3.6 months [range: 0.1 -24 months]. Discontinuations from the study occurred due to disease progression (67/84, 79.8%), withdrawal of consent (2/84, 2.4%), investigator judgment (7/84, 8.3%), withdrawal from treatment (4/84, 4.8%) and other reasons (4/84, 4.8%). Efficacy outcomes

At a minimal follow-up of 6 months (median 1 1 .4 months [range: 2 - 36+ months]) after the last patient had been randomised, 84 patients were evaluable for the primary endpoint. In the control arm, rRT resulted in a PFS- 6 rate of 3.8% (95%-CI: 0.1 - 19.6), i.e. one patient was free of progression, whereas PFS-6 in the rRT+APG101 arm was 20.7% (95%-CI: 1 1 .2 - 33.4, p=0.04), i.e. 12 patients were free of progression. These data were confirmed in the central review.

Efficacy of rRT+APG101 was also suggested by the analysis of the per- protocol population (Figure 1 ) for PFS-6 with 4.8% (95%-CI: 0.1 - 23.8) versus 21 .6% (95%-CI: 1 1 .3 - 35.3, p=0.0469).

Median PFS was 2.5 months (95%-CI: 2.3 - 3.8) versus 4.5 months (95%-CI: 3.7 - 5.4, p=0.0162) (Fig. 2a).

In the univariate analysis, median overall survival (OS) was 1 1 .5 (95% CI: 6.5 - 15.4) months in the rRT+APG101 and 1 1 .5 (95% CI: 8.8 - 16.2) months in the rRT arm (Fig. 2b). After correcting for tumour size, the HR for the secondary endpoint of OS was 0.60 (95% CI: 0.36 - 1 .01 , p=0.0559) (Table 3a).

In both arms, all patients experienced a progression in the observation interval of the study. Pseudoprogression [21 ] was reported in 19/26 (73.1 %) patients in the rRT and in 43/58 (74.1 %) patients in the rRT+APG101 arm. It was confirmed in 8/19 and 22/43 patients, respectively (see Supplementary Table S1 ). Patients in both arms had a similar type and frequency of salvage therapies. These post-progression treatments are listed in the Supplementary Table S2.

Prognostic and Predictive Factors

Tumour size at rRT was a prognostic factor for OS with HR=0.45 (95% CI: 0.27 - 0.75], p=0.0022). Tumour size as a prognostic factor for PFS conferred a HR=0.61 (95% CI: 0.35 - 1 .05, p=0.0744) (Table 3b). Tumour tissue was analysed for IDH1 R132H mutation (7/84, 8.4%), MGMT promoter methylation (57/84, 67.9%), expression of the APG101 target pathway, CD95 and CD95L, as well as CpG methylation analysis upstream of CD95L (Supplementary Figure 1). Lower methylation at CpG1 and CpG2 located at chrf :172,628,02ο (CpG2) and chrl :172,628,065 (CpG1 ) (GRCh37/hg19) in the CD95L promoter was a positive prognostic factor for OS in the rRT+APG101 arm (Supplementary Table 4 and Supplementary Figure 2).

DISCUSSION

The present trial evaluated a novel therapeutic approach for recurrent glioblastoma aiming to block the CD95/CD95L system previously shown to trigger invasive growth. CD95L is the target of APG101 . The addition of APG101 to rRT produces a relevant number of patients with first or second progression of a glioblastoma without tumour progression at six months after randomisation. There is a beneficial PFS signal and also promising OS data after correction for tumour size. Patients with expression of CD95L in the tumour, as signified by low methylation at probes cg10161 121 and cg06983746 upstream of CD95L, have a greater benefit from APG101 treatment (Supplementary Figure 2). Thus, CD95L promoter methylation can be used as a selection marker. Expression of CD95L seems to be associated with impaired prognosis in other malignancies as well [24].

The main reason for a poor outcome in glioblastoma is the therapy resistance, the highly invasive behaviour of the tumour as well as the local immunosuppression. While for patients with newly diagnosed glioblastoma the current standard of care is radiochemotherapy with temozolomide, no such standard exists for progressive disease. Therapeutic options at recurrence depend on the individual disease situation and include reoperation, rRT, alkylating chemotherapy with temozolomide or nitrosoureas, bevacizumab and experimental agents within clinical trials. By targeting the invasive growth, APG101 addresses a pathological hallmark of glioblastoma different from all previous approaches.

In uncontrolled series of rRT with fractionated stereotactic rRT [9,10] or stereotactic radiosurgery [28], rRT appeared as a relatively safe and effective approach in well-selected patient groups as one option for salvage therapy. In the current trial, the PFS-6 rate (12/56 patients) achieved by the addition of APG101 to rRT is remarkable given the prespecified target (13/56 patients) corrected for the performance of the rRT arm which was lower than assumed, but well within the range for (negative) recurrent therapy trials. Patients received similar post-progression treatments in both study arms (Supplementary Table S1). The positive PFS signal (Fig. 2a) also translates into a meaningful OS benefit in the rRT +APG101 arm (Fig. 2b). The macroscopic diameter of the tumour as determined by MRI is strongly prognostic. Hence, a neurosurgical reduction of the tumour size is an adequate measure enabling patients to achieve a greater benefit from the combined treatment with APG101 + rRT.

RRT+APG101 was well tolerated (Table 2). There were no serious AEs causally related to APG101 , and APG101 did not impair tolerability of rRT resulting in a favourable risk/benefit assessment.

The present study was designed to identify a therapeutic effect of APG101 when combined with rRT in the treatment of progressive glioblastoma. Thus, the study population was selected with regard to tumour size, prior first-line therapy, and the long time frame between first-line therapy and first or second progression, as needed for a second RT. The latter is documented by the high number of patients with MGMT promoter methylation (Table 1 ). There were insignificant imbalances in favour of the rRT+APG101 arm with more /DH-mutated tumours and in favour of the rRT arm with smaller tumours (Table 1 ). Every effort was made to ensure high quality and comparability of therapeutic and diagnostic measures applied during the study. MRI and RT dummy runs had to be completed by all sites, and all sites had to obtain central approval prior to participation in the study. MRIs were assessed centrally in a blinded fashion and a strict algorithm to identify pseudoprogression was used. All measures ensured the accuracy of the observed therapeutic effects, however, the effect of rRT on PFS is at the lower end of what was reported in scientific publications.

The examination of tumour tissue for the expression of CD95 and CD95L had been specified in the study protocol. Methylation analyses were also carried out. Both tests were carried out on identical archived tumour samples obtained during surgery at the time of diagnosis, and provided quantitative data with an easy to reproduce PCR-based assay. Patients with low methylation levels at two CpG upstream of CD95L showed the best response to treatment with APG101 .

The present data show clinical efficacy of APG101 in combination with rRT. Given the very limited options at progression of glioblastoma, rRT+APG101 represents a therapeutic chance, especially for the subset of patients with the option for second RT. CD95/CD95L inhibition may also be exploited in newly diagnosed glioblastoma patients.

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28. Tsao MN, Mehta MP, Whelan TJ, et al. (2009) The American Society for Therapeutic Radiology and Oncology (ASTRO) evidence-based review of the role of radiosurgery for malignant glioma. Int J Radiat Oncol Biol Phys 63:47-55. Table 1. Baseline patient and disease characteristics

rRT + APG101

Characteristic rRT (n=26) (n=58)

Median age, y (range) 59 (25-79) 57 (20-73)

Sex, %

Male 46.2 67.2

Karnofsky performance status, N

(%) 8 (30.8) 17 (29.3)

60-80 18 (69.2) 41 (70.7) 90-100

MGMT status, n (%)

Methylated 15 (57.7) 41 (70.6) Non-methylated 8 (30.8) 14 (24.1 ) Missing 3 (1 1 .5) 3 (5.2)

IDH status, n (%)

Mutated 0 6 (10.3) Wild type 25 (96.2) 49 (84.5) Missing 1 (3.8) 3 (5.2)

Recurrence status, n (%)

First 19 (73.1 ) 41 (70.7) Second 6 (23.1 ) 15 (25.9) Third 1 (3.8) 2 (3.4)

Mean time since first diagnosis, 20.3 (1 1.7) 23.9 (14.8) months (standard deviation)

Tumour diameter, n (%)

< 2.5 cm 20 (76.9) 29 (50) > 2.5 cm 6 (23.1 ) 29 (50)

CD95L status, n

Positive 16 (61 .5) 39 (67.2) Negative 8 (30.8) 16 (27.6) Missing 2 (7.7) 3 (5.2)

CD95L, CD95 ligand; IDH, isocitrate dehydrogenase; MGMT, O6- methylguanine-DNA methyltransferase; Table 2. Adverse events ^*

rRT rRT +APG101 n = 26 n = 58

Grade Mild Moderate Severe Mild Moderat e

Haematological toxicity, n

0 0 0 3 2

Neutropenia

1 0 0 1 2

Lymphocytopenia

Thrombocytopenia 0 0 0 4 2

Liver enzyme elevation, n 1 0 0 1 0

Infection, n 1 6 0 18 8

Thrombembolic event, n 0 0 0 0 1

Asthenia / Fatigue, n 3 0 0 12 1

Nausea / Vomiting, n 3 0 0 7 4

Metabolic disorders, n 1 1 0 4 3

Neurologic symptoms 2 5 6 3 19

Seizures 2 3 4 8 13

Cutaneous AE (dermatitis, 2 2 0 12 5 allergic rash, alopecia), n

^* Distinct events as recorded in the AE documentation. Each patient was counted only once within each AE category. If a patient experiences more than one AE within a category, only the AE with the maximum intensity was included. Patients in the rRT+APG101 arm have been seen weekly during the post-RT phase until progression whereas patients in the rRT arm were seen six-weekly.

^* exploratory analyses due to the non-comparative design of the trial

Example 2

Supplementary Methods

Trial Design and Conduct

Subsequent data safety monitoring board meetings were held after 25 patients completed the reirradiation (rRT) and at the end of stage 1 of the Simon Two-Stage Design (after 28 patients reached the primary endpoint) combined with a safety evaluation after the first 49 patients completed rRT.

Radiation Therapy

Only centres fulfilling all requirements were eligible to recruit patients into the trial. To ensure protocol -conform treatment planning, two test patients were distributed to each centre and target volume definition as well as treatment planning and dose distributions carried out by the site were evaluated as a dummy run (Medical Centre Heidelberg).

As the basic inclusion criterion was an indication for rRT, any recurrence in- or outside the radiation field that occurred >8 months out of the initial RT was principally eligible. However, since >90% of the lesions occur inside the radiation volume, all except one patient, who had a recurrence at the 20% isodose had a pre-RT at a similar region. For treatment planning, CT as well as contrast-enhanced MRI were mandatory. Target volume delineation was defined to include the gross tumour volume (GTV) defined as the contrast- enhancing lesion on MRI, adding 1 cm safety margin for potential microscopic spread (clinical target volume, CTV). The recommended total dose was 36 Gy in 2 Gy single fractions. After treatment, treatment plans of study patients were reviewed centrally in Heidelberg.

Evaluations

Importantly, an apparent increase in tumour size considering the largest cross-sectional area or contrast-enhancement in the radiation field of >25% in the first or second scan post RT was called pseudoprogression and not deemed a progression until further confirmation on follow up. Further progression resulted in backdating to the scan of the initial suspicion of a progression and stable disease on follow-up in retrospective rating as stable. Stable or decreasing contrast enhancement resulted in a continuation of trial treatment and/or follow up.

Neuropathological Methods

Expression of mutated IDH1 R132H protein was determined by immunohistochemistry [1 ]. The MGMT promoter methylation status was analysed after bisulfite treatment by methylation-specific PCR [2]. Expression of CD95 and CD95L was determined by immunohistochemistry. All CD95- and CD95L-stained slides were evaluated slide-by-slide in a single session by a board-certified neuropathologist (C.H.). Vital tumour tissue of each slide was evaluated regarding the CD95 and CD95L staining intensities 'high', 'moderate', 'low' and 'absent'. CD95 and CD95L calibration figures were used to standardize the evaluation. Because most tumors showed different staining intensities in separate areas, the percentage of these summed areas were counted. Each tumour was assigned to a specific value in percent representing the area showing 'high', 'moderate', 'low' and 'absent' CD95 and CD95L staining intensities. Biomarker identification

To identify epigenetic differences distinguishing responders from non- responders, we performed genome-wide assessment of DNA methylation using the HumanMethylation450 BeadChip (lllumina, San Diego, CA, USA) of 20 patients who received APG101 plus radiotherapy (10 patients with a PFS > 5 months and 10 patients with a PFS < 2 months, discovery cohort) at the Genomics and Proteomics Core Facility of the German Cancer Research Center (Heidelberg, Germany). Data normalization was performed following the manufacturer's recommendations. Unsupervised hierarchical clustering was performed after removing probes (i) targeting the X and Y chromosomes, (ii) containing a single nucleotide polymorphism within 5 base pairs of and including the CpG site and (iii) not mapping uniquely to the human reference genome (hg19), allowing for one mismatch. Student's t test assuming unequal variances was used to detect probes with significantly different mean methylation between the two groups.

Biomarker validation

Two CpGs upstream of the CD95 ligand were screened in an independent validation cohort comprising all patients for whom sufficient DNA was available and which were not part of the discovery cohort (n = 40 patients) using the MassARRAY technique (Sequenom, San Diego, CA, USA). This technology relies on detection of mass shifts, which are introduced through sequence changes following bisulfite treatment. In short, 500 ng genomic DNA was bisulfite-converted using the Epitect Bisulfite Kit (Qiagen, Hilden, Germany). For PCR amplification, the following primers were used:

aggaagagagTTATTTTGTAGTTGAAGTTGAGAAG (forward)

cagtaatacgactcactatagggagaaggctACTAACCTACTCTACAAAATCCC

(reverse)

Next, DNA methylation analysis was performed on a Sequenom mass spectrometer and the results were analyzed by the Epityper software (Version 1 .05, Sequenom, San Diego, CA, USA). For statistical analysis, both CpGs were dichotomized using their median methylation level and Cox regression analysis using a model including tumor size (the main prognostic factor in the analysis of the trial data) and treatment (APG101 + radiotherapy vs. radiotherapy alone) was performed.

Randomization and treatment

• Eligible patients were randomized (2:1 ) to receive APG-101 in addition to rRT or rRT alone, and central stratification by tumour diameter (< vs. > 2.5 cm) and number of recurrences (first vs. second)

• Description of treatment

o APG101 was given i.v. once per week at 400 mg as an 30 min infusion

o Radiotherapy (18 x 2 Gy)

Statistical Analyses

Exploratory objectives included: biomarker analysis, amount of lymphocytes in total blood, proportion of T-cells and B-cells within lymphocyte population, ratio of T-cells to B-cells, determination of T- cell subpopulations (CD3+, CD4+/CD8+, CD4+/CD8-, CD4-/CD8+, CD4-/CD8-, CD45RA, CD45R0), determination of activation marker (HLA-DR, IL-2R).

Within the framework of prospectively planned descriptive analyses

• exact 95%-confidence intervals (CI) according to Clopper-Pearson were calculated for the rates within treatment groups and asymptotic 95%-CI were presented for the difference of rates between treatment groups. Descriptive treatment comparisons of rates were done by use of a Fisher test.

• Kaplan-Meier estimates [3] were used to describe PFS and OS and derive median survival times together with 95%-CI. Cox regression models including prognostic factors as covariates were fitted to PFS and OS data to obtain estimates of treatment hazard ratios and corresponding 95%-CI.

Changes in Quality of Life scores with respect to baseline were classified as improved, unchanged and worsened. Asymptotic 95%-CI were calculated for the differences in improvement rates between treatment groups by visit.

Supplementary Results

Biomarker data

Unsupervised hierarchical clustering of the discovery cohort (n = 20 patients) identified 4 of the 10 responders to carry a hypermethylator phenotype (G-CIMP) [4]. However, the remaining 16 patients did not cluster distinctively depending on treatment response. As G-CIMP positive tumors most likely represent an epigenetically distinct entity [5], we excluded these 4 patients from further analysis. Next, we performed a supervised detection of differentially methylated probes employing Student's t test. For identification of possible markers, we focused on significantly differentially methylated probes which are associated with genes putatively involved in CD95 signaling (including CD95, CD95 ligand, Yes, FADD and others) [6] or with a methylation difference > 0.4 between responders and non- responders. Using these criteria, we identified 2 probes (cg10161 121 and cg06983746), both upstream of the CD95 ligand (Supplementary Figure 1 ), which exhibited significantly lower methylation in responders.

To validate these findings in a larger independent cohort, we performed MassARRAY analysis of these two probes in all patients for whom sufficient DNA was available and which were not part of the discovery cohort (n = 40 patients). Methylation levels were generally comparable to those seen in the 450k array. Cox regression analysis identified a relevant interaction between methylation levels of the cg06983746 and treatment efficacy (Supplementary Table 4), where lower methylation is associated with a significantly longer progression-free survival in patients receiving both APG101 and radiotherapy (Supplementary Figure 2A). For overall survival, this substantiated (patients with lower methylation levels benefit from combination therapy, while those with higher methylation levels tend to have shortened overall survival when receiving both APG101 and radiotherapy as opposed to radiotherapy alone, Supplementary Figure 2C-D). Importantly, methylation levels of cg06983746 had no direct, treatment-independent effect on either progression-free or overall survival, underlining the specific predictive value of cg06983746 methylation for benefit from addition of APG101 to radiotherapy. Also, analyses of a prognostic role for CD95L promoter methylation in the temozolomide- and also radiotherapy-treated patients of the NOA-08 trial revealed no such an effect (Supplementary Figure 4).

Quality-of-Life Assessments

HRQOL [20] data were available from 92% of all patients. No clinically meaningful or statistically remarkable differences between the two groups over time in any of the scales or cohorts were observed.

Supplementary Tables

Table S1. Pseudoprogression.

Pseudoprogression, namely stable disease on follow up, was confirmed on follow-up MRI in 8/19 and 22/43 patients in the rRT and rRT+APG101 arms, respectively, while 5/19 and 12/43 were retrospectively rated as progressive disease.

^* Confirmatory scans were usually not done in patients with a relevant clinical progression, e.g. inability to return to the scheduled study visit. Table S2. Postprogression management.

Table S3: Reduction criteria for CD95/CD95L expression intensities.

Quality Criteria

Positive A tumor that shows any 'high' CD95L labeling independent of the size of the area

Intermediate A tumor with only 'moderate' and/or 'low' CD95L labeling Negative A tumor without any CD95L labeling

Table S4: Hazard Rations for treatment response dependent on cg06983746 methylation

a) Progression-free survival

cg06983746 methylation HR for R alone 95% CI (dichotomized at median) vs. APG101 + RT

< 0.85 7.87 1.91 -32.5

> 0.85 1 .18 0.33-4.19 b) Overall survival

cgO ^' 6983746 methylation HR for RT alone 95% CI

(dichotomized at median) vs. APG101 + RT

< 0.85 5.4 1 .73-16.9

> 0.85 0.38 0.12-1.19

HR, hazard ratio; RT, radiotherapy; CI, confidence interval

Supplementary References

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2. Christians A, Hartmann C, Benner A, et al. (2012) Prognostic value of three different methods of MGMT promoter methylation analysis in a prospective trial on newly diagnosed glioblastoma. PLoS One 7:e33449.

3. Kaplan E, Meier P. (1958) Non-parametric estimation from incomplete observations. J Am Stat Assoc 53:457-481 .

4. Noushmehr H, Weisenberger DJ, Diefes K, et al. Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma. Cancer cell 2010;17:510-522.

5. Lai A, Kharbanda S, Pope WB, et al. Evidence for sequenced molecular evolution of IDH1 mutant glioblastoma from a distinct cell of origin. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 201 1 ;29:4482-4490.

6. Kleber S, Sancho-Martinez I, Wiestler B, et al. Yes and PI3K bind CD95 to signal invasion of glioblastoma. Cancer cell 2008;13:235-248.