The present invention relates to a new-generation imrnunochernical fecal occult blood test for detection of proximal and distal colon neoplasia in subjects screened for colorectal cancer (CRC). More precisely, the present invention concerns a method and a kit for determining the presence of occult human blood in a stool sample of a patient using sensitive immunochemical detection of the hemogiobm/haptoglobin (Hb/Hp) complex in said sample.

BACKGROUND

Colorectal cancer (CRC) is a mal ignant tumor arising from the mucosa of the large intestine, including the segments of the 1) caecum, 2) ascending colon, 3) transverse colon, 4) descending colon, 5) sigmoid, and 6) rectum. Tumors in locations 1-3 are called proximal CRC, while those of 4-6 are distal CRC. CRC does not include tumors in the a us or the small intestine. Adenomas are benign epithelial tumors that are considered precancerous lesions. Adenomas can present with different degrees of dysplasia and different histological characteristics (tubular, tubule-vi!lous, and villous) associated with increasing malignant potential. Carcinoma in situ (CIS) refers to adenomas with severe dysplasia, while lesions that invade the muscularis mucosa are considered invasive adenocarcinomas. The concept advanced neoplasia (AN) refers to a composite outcome, including adenocarcinoma, adenomas 10mm or greater in diameter, adenomas with high- grade dysplasia, and those with≥25 % of villous histology.

CRC continues to be a major global disease burden. In 2012, CRC ranked in the third place among the most common malignancies, with over 1.3 million new cases and around 700,000 annual deaths being recorded worldwide [1 ]. In Europe, the corresponding figures are 471.000 and 230.000, making CRC the single most frequent malignancy and second only to lung cancer in annual cancer deaths [1]. In the US, CRC ranks the 3 ^ld most common cancer, with 153.000 incident cases and 52.000 cancer deaths (1,2). Similarly, in Asia, CRC holds the 3 ^rd position among most frequent cancers, with over 500.000 new cases and 267.000 annual deaths (1). In Finland, CRC is the second most common cancer among women (1240 new cases), and third most common among males (1246 cases)(3). The global age-adjusted incidence and mortality rates of CRC are 17.2/100.000 and 8.2/100.000, respectively (1). There is good evidence e.g. from the US, that increasing age, male sex, and Black race are associated with an increased incidence of CRC (2). Accordingly, age-adjusted incidence rates for CRC are higher in men than women, and Blacks have the highest incidence of CRC among the racial/ethnic subgroups in the US. Blacks also have a disproportionately high disease-specific mortality (4). Interestingly, age, sex, and race/ethnicity also appear to influence on the anatomic distribution of CRC (5). Data from the NCI's Surveillance,

Epidemiology, and End Results Program ( SEER)(2) demonstrate a proximal migration of CRC over the past two decades, which is attributed to a decrease in incidence of distal CRCs, and an aging population in which proximal lesions are more common (5). This proximal migration appears in both men and women, and in Whites and Blacks. This difference between the two races was not evident during the 1970's.

Risk factors

Most cases of CRC are sporadic, with 75% of cases developing in average-risk persons, and only some 20% of cases are found in persons with some type of family history (6,7). The remainder of cases develop in persons who have predisposing inflammatory bo wel disease (e.g. ulcerative colitis) or a known genetic mutation, including familial adenomatous polyposis (FAR) and hereditary non-polyposis colorectal cancer (HNPCC). Case-control and cohort studies indicate an approximately two-fold increase in CRC risk for persons with a first-degree relative (e.g., parent, sibling, or child) with CRC. This increased risk is also applicable to first-degree relatives of individuals with colorectal adenomas (8). CRC may be associated with non-genetic risk factors, such as smoking or obesity, although evidence is limited to case-control and cross-sectional data (6-8). There has been substantial progress in understanding the molecular genetics of CRC, and these scientific advances underpin the efforts to develop D A testing (fecal or plasma) for CRC detection.

Natural history

It is estimated that at least 95% of all CRC cases arise f om pre-existing polypoid or flat adenomas ( 9). The notion of an adenoma-carcinoma sequence stems from observations of a markedly elevated CRC risk among patients with hereditary polyposis syndromes, and from observational studies showing an estimated 60 to 90% reduction in CRC incidence after polypectomy during colonoscopy or FS (flexible sigmoidoscopy)(10). The most convincing evidence from FS studies comes from well-designed case-control studies that have demonstrated a decrease in CRC mortality, and in some cases, in CRC incidence. The landmark case-control study by Selby and colleagues (11) found a 60% reduction in mortality from distal CRC over 10 years in persons who underwent sigmoidoscopy with polypectomy, compared to matched contemporaneous controls. Despite some uncertainty around the magnitude of this benefi t, these studies provide the best available estimates for the impact of polypectomy on CRC incidence (10,11),

Significance of the polyp size

While there is general agreement that the risk of local cancer, or progression to cancer, for polyps 10 mm or larger is sufficient to require immediate removal, the necessity and benefit of removing small polyps is not clear (12,13), Sensitivity estimates for optical methods (e.g., CTC, FS, and colonoscopy) depend on the threshold for the size of polyp considered clinically meaningful. The threshold for polyp size also determines the number of colonoscopy referrals th at wil l result from primary CT Co tonography (CTC' ) and other visualization-only screening methods. No large observationai studies are available to determine the consequences of untreated adenomas. Similarly, the natural history of smaller adenomas, particularly those of different sizes (e.g., 5 mm or under, 6 to 9 mm), is practical!)' unknown. The tendency towards net growth or regression may vary by polyp size and histology, as well as by other characteristics such as patient age, tumor location, and number of lesions (14). Some colonoscopy database studies suggest that the prevalence of CRC in lesions <6 mm in diameter ranges from zero to 0.8%, and in those with. 6-9 mm, from 0.4% to .1.1% (15). While advanced neoplasia (AN) seems to be somewhat more common than CRC in small polyps, the clinical significance of AN remains poorly understood.

Significance of advanced neoplasia (AN)

Several studies have estimated the accuracy of optical screening methods not only for CRC, but also for AN. By definition, AN is a composite endpoint for three conditions: an adenoma 10mm. or larger in size, ii) a smaller adenoma with at least 25% of villous histology, or iii) adenomas containing high-grade dysplasia or invasive carcinoma. It is important to understand both the impact of polypectomy of ANs on the risk of future CRC, and conversely, the impact of leaving an AN lesion intact on the risk of future CRC. In a long-term follow-up study after polypectomy (n=1618 patients), the risk of subsequent CRC was increased at least 3-fold in patients with tubule- villous, villous, or large (>10 mm) adenomas (i.e., AN), as compared with other types of recto-sigmoid adenomas (16). Similarly, the frequency of invasive carcinomas in polyps with surface villous histology ranged from 10 to 40%, as compared with 6 to 23% among polyps with surface tubule- villous histology, 2 to 5% in polyps with surface tubular histology, as well as 34% among polyps with high-grade dysplasia (17). Importantly, however, there are no prospective studies on the natural histoiy of AN, and no longitudinal studies have validated the clinical benefi t of targeting AN in screening populations. The prevalence of flat and depressed (non-polypoid) adenomas in screening populations is largely unknown, as is the prevalence of dysplasia in these lesions, and the risk of CRC associated with these lesions is currently under dispute.

Screening of colorectal cancer

Among few human cancers, CRC meets the criteria of a screening condition; it is a highly prevalent disease, wit well-established preclinical period during which the majority of CRC develops from precursor lesions, i.e., adenomatous polyps of AN. Thus, screening for CR ' can impact both i) the primary prevention (finding precancerous polyps that could later become malignant) and ii) the secondary prevention (detecting early cancers that can be more effectively treated). Based on evidence from randomized controlled trials (RCT), a screening program using simple, reasonably acceptable, guaiac-based fecal occult blood (FOB) screening tests reduces CRC mortality when used with repeated application over time and endoscopic follow-up of positive results (18). Also other screening approaches are recommended, based on extrapolation from the evidence of RCTs (19). However, no current CRC ¹ screening tests are without flaws, including potential harms, limited accessibility, or imperfect acceptability to patients. Ongoing research aims to make more accurate screening tests available to further improve CRC screening programs (20). While there is general consensus that CRC screening reduces disease-specific mortality, newer screening tests have created uncertainty about the optimal methods for CRC screening in the general population (19,20). Methods available for C C screesiisig

CRC screening tests in common use include FOBT (fecal occult blood test), FS (flexible sigmoidoscopy), and colonoscopy (19,20,21). In many countries, the US included the use of colonoscopy for CRC screening has increased recently while the use of FS has decreased, in part due to the changed policies of coverage by health insurances (22). Public perceptions of accuracy also play an important role in this issue. Significant variation in community CRC screening practices, which may impact effectiveness of screening, has also been reported. Similarly, there appears to be variation in practice for follow-up of positive FOBT (e.g., using FS instead of colonoscopy)(23). Lastly, there remains significant variation in operator characteristics for endoscopies, both FS and colonoscopy, which may affect test characteristics for screening and confirmatory endoscopy.

Performance of the different CRC screening tests

Flexible sigmoidoscopy (FS) and colonoscopy

The most comprehensive treatise on the performance of different methods used for CRC screening is to be found in the systematic review of Whitlock et al. 2008, published in two separate reports (19,20). According to this comprehensive review, in community settings, FS (with or without biopsy to determine colonoscopy referral) has an estimated sensitivity of 58% to 75% for CRC in the entire colon and an estimated sensitivity of 72% to 86%o for AN. Variations in these estimates are likely due to differences in examiner skills and the patient's risks for proximal lesions in the unexamined colon (19,20). While colonoscopy remains the most accurate screening test for CRC at a single application, recent studies have confirmed that colonoscopy misses polyps and may also miss CRC. Colonoscopy also presents a higher risk for harms than other tests. Serious harms from community endoscopies are about 10 times more common with colonoscopy (3.1 per 1,000

procedures) than with FS (3.4 per 10,000 procedures) (19,20).

Quaiac-based fecal occult blood tests (FOBT)

During the past, different quaiac-based FOBTs have been very popular methods in CRC screening in many countries (19,20). These tests detect fecal occult blood based on peroxidase activity of hemoglobin (Hb)-derived heme groups, but, unfortunately, are not specific for human blood. In addition to human blood, these quaiac-based tests can also trace animal blood derived from food, and in addition, peroxidases derived from some raw vegetables. This can lead to false positive results and unnecessary referrals to colonoscopy. In addition, these tests are usually not highly sensitive, which can also lead to false negative results.

Not unexpectedly, the data on the efficacy of FOBT screening are controversial (19,20). While two studies with long-term follow-up of biennial FOBT screening suggest CRC mortality reduction by 13 to 21 %, after 8 to 13 years of screening, another two trials did not show mortality benefit until after 15-18 years of screening. In a recent meta-analysis from the Cochrane Collaboration, the overall estimate of CRC mortality reduction by biennial FOBT screening was 15%, using either random- or fixed-effect models (RR=0.85, 95%CI: 0.78-0.92)(18). However, this analysis did not incorporate the recently reported data from one of these trials, suggesting that CRC mortality benefit is no longer statistically significant at 17 years when deaths due to CRC treatment are included •RR 0.89. 95%CI: 0.78-1.01 )(24). When critically assessed in the recent systematic review (19,20), a meta-analysis of ail four FOBT screening trials indicated no benefit for all-cause mortality ( RR 1 .00, 95%CI: 0.99-1.03)(18,24). This made the authors to suggest that CRC screening is not be expected to reduce all-cause mortality in these ongoing FOBT trials (19,20).

Fecal immunochemical tests (FIT)

In japan, the pioneering country of CRC screening, different fecal immunochemical tests (FITs) have been the principal screening method since the early 1990's (25). In the recent systematic revie w (19,20), altogether 12 types of FITs were identified in the literature, representing 20 different proprietary names. Due to the major differences in test methodology, the authors were unable to assess all FITs as a class, however, but had to satisfy with a sub-group analysis ( 19,20).

In their systematic assessment, four individual FITs (Magstrearn/HemeSelect; FlexSure OBT/Hemoccult ICT; OC-Hemodia; Monohaem) showed higher sensitivity for CRC (61% to 91% percent) than estimates for (quaiac-based) non-rehydrated Hemoccult II (25% to 38%), with somewhat reduced specificity (91% to 97%, respectively). Sensitivity for ANs or large adenomas is less commonly reported, but ranges between 20% and 67% for FITs, which is comparable or superior to the sensitivity for non-rehydrated Hemoccult II (19,20). Fewer good-quality studies are available on another FIT (HemoccultSENSA), and although it appears to improve sensitivity for CRC (64% to 80%), it seems to have a lower specificity (87% to 90%). The authors concluded that in the l ack of test accuracy results indicating clearly superior test sensitivity with comparable specificity, determining the trade-offs between sensitivity and specificity of these newer fecal tests in organized CRC screening programs requires further studies (19,20).

Studies comparing FOBT and FIT tests

During the recent past, a number of studies have been published where the performance of guaiac-based FOBT has been compared with the fecal immunochemical tests (FIT) in detecting different study endpoints of CRC screening (26-32). In their study of 8,104 patients, Allison et al. (26) compared two guaiac tests (Hemoccult II, Hemoccult II

SENSA) with HemeSelect (a FIT). They found that the sensitivity of Hemoccult II (37.1%; 95%CI, 19.7-54.6), was clearly inferior to that of HemeSelect (68.8%; 95%CI, 51.1-86.4), and the same was tme with test specificity as well: 86.7% and 94.4%, respectively (26). In an earlier study, Frommer et al. (27) concluded that immunological detection of occult blood in fecal samples seems to show more adenomas and carcinomas (particularly early lesions) than the Hemoccult II kit and has a rate of false positive results that is acceptably low (27). In another recent study, comparing a new bedside immunological test strip device (Prevent ID CC) with a sensitive guaiac-based test (Hemoccult), and an established immunochemical test (Human Hb ELISA)(28), the sensitivity and specificity of the beside immunochemical strip test for detection of adenomas and CRC were 60% and 95%, which favorably competes with an established immunochemical fecal occult blood test and exceeds the performance of the guaiac-based test (28). In contrast, Rozen et al. found a guaiac-based test (HemoccultSENSA) to be more sensitive but less specific than a FIT (FlexSure) in detecting any colorectal neoplasms (29).

R esults in alignment of these were recently reported by van Rossum et al. (2008), who compared a guaiac-based FOBT and a FIT in the first population-based study on a random sample of 20,623 individuals, randomized to either Hemoccult-II or OC-Sensor (31). Of the 10,993 tests returned, 117 FOBTs (2.4%) were positive vs. 339 (5.5%) of FITs. Cancer and advanced adenomas were found, respectively, in 11 and 48 of FOBTs and in 24 and 121 of FITs. Differences in PPV for cancer and advanced adenomas were, respectively, 2.1% (P=0.4) and -3.6% (P=0.5). Differences in specificities favor FOBT and were, respectively, 2.3% (P<G.01) and -1.3% (P<G.01). Differences in intention-to-screen detection rates favor FIT and were, respectively, 0.1% (P<0.05) and 0.9% (P<0.01). Taken together, the number-to-scope to find 1 cancer was comparable between the two tests. However, participation and detection rates for advanced adenomas and cancer were significantly higher for FIT than FOBT, the latter significantly underestimates the prevalence of advanced adenomas and cancer in the screening population compared with FIT (31).

Finally, Wong et a!. (2003) compared the performance characteristics of a guaiac-based FOBT (Hemoccult SENSA) and a FIT (FlexSure OBT) in a Chinese population referred for colonoscopy (32). The sensitivity, specificity and positive predictive value (PPV) for the detection of significant colorectal neoplasia were 91%, 70% and 18% for Hemoccult SENSA and 82%, 94% and 47% for FlexSure OBT. The specificity and PPV were significantly higher for FlexSure OBT than for Hemoccult SENSA (P<0.001 and P=0.016, respectively). The authors concluded that PPV of the FIT for detection of colorectal neoplasia was 29% better than that of the sensitive FOBT, and the poor specificity of the guaiac-based test (without dietary restriction), makes it less useful for CRC screening in a Chinese population (32).

Taken together, the studies comparing the performance of FOBT and FIT tests show in part contradictory results (19,20,26-32). The data suggest, however, that the sensitivity of FIT assays is substantially better than that of FOBT. In some studies, the specificity of the two assays is comparable, whereas in some others, also the specificity of FITs seems to be clearly superior to that of FOBTs. In the only RCT conducted so far, the evidence suggests that the performance of FITs is clearly superior to FOBTs in detecting any type of colorectal neoplasia (31).

Clearly, more studies are mandatory to evaluate the performance of the new-generation FITs in comparison to the most sensitive guaiac-based FOBTs on the market, in both a clinical setting and particularly as screening tools for CRC. Considerations on the clinical performance of CRC screening tests

It is important to realize that occult blood in the stools can be derived from several different sources (diseases and conditions) that are unrelated to the targets, i.e. polyps, adenomas and carcinomas, of an organized CRC screening. A FIT test with high analytical sensitivity is capable of detecting FOB of this non-neoplastic origin as well. The list of such conditions include, but is not complete, e.g. vascular malformations (arterio -venous fistulas), angiectasia lesions, diverticulitis, diverticulosis, and importantly, inflammatory bowel disease (IBD). The two most important conditions of IBD are Ulcerative Colitis (UC) and Crohn's disease. Because of the fact that these non-neoplastic causes of FOB comprise a substantial proportion of the conditions detected by FIT in a systematic population-based CRC screening, the specificity of any FIT test cannot be very high, if adenoma/carcinoma is used as an endpoint, without correcting for these non-neoplastic confounders. In a screening setting, high analytical sensitivity of the FIT test is an advantage, because all FOB is an abnormal condition and warrants a clinical examination to disclose the cause. A screening test with high analytical sensitivity has a high specificity for adenoma/carcinoma endpoint only when all these non-neoplastic confounders are excluded from the analysis. In contrast, a screening test with low analytical sensitivity (i.e., FOBT)(missing most of the non-neoplastic FOB cases), can have a higher specificity for the adenoma/carcinoma endpoint, because of the fact that bleeding from these neoplastic lesions is usually (but not always) profuse enough to be detected by these low-sensitivity tests.

US patent 4,092,120 describes a method and an apparatus for storing samples for the purpose of later analyzing such samples to establish the presence of fecal occult blood. Additionally, US patent 4,427,769 describes a very first immunoassay method for the detection of human hemoglobin or decomposition products of human hemoglobin in feces. This development has its origin in Finland since the 1970's, when Labsystems Oyj established by Osmo Suovaniemi developed Fecatwin/EIA test for screening and diagnosis of colorectal cancer (39, www.biohithealthcare.com/AboutUs/History:Aggressive innovation and patenting strategy). The test is thus specific for human blood, and it was patented e.g. in USA (US patent 4,427,769). SUMMARY OF THE INVENTION

It is an aim of the present invention to provide a novel method for collecting stool samples and measuring their content of human fecal occult blood (FOB). Particularly, it is an aim of the present invention to provide a simpler and more efficient method and kit for extracting human blood from a stool sample, compared to known methods.

These and other objects, together with the advantages thereof over known methods and kits, are achieved by the present invention, as hereinafter described and claimed.

It has been attempted to alleviate the above mentioned problems related to the prior art by limiting the amount of sampling tubes to one. Into this sampling tube, stool samples are collected during two or more days. The method ensures the detection of even minor amounts of human blood, in amounts barely above the detection limit, even with several combined samples.

Thus, the present invention helps in ensuring that the subject can be guided to a colonoscopy and with eventual detection of CRC, provides the potential for subsequent treatment at an early stage.

The present invention concerns a method of determining the presence of human blood in a stool sample of a human patient, including sample collection and measurement of the content of human blood of the sample. More specifically, the method of the present invention is characterized by what is stated in Claims 1 and 14.

Further, the kit of the present invention is characterized by what is stated in Claim 8, and the method for detecting colorectal neoplasia is characterized by what is stated in Claim 15.

Considerable advantages are obtained by means of the invention. The present invention provides a method, by which fecal occult blood (FOB) due to intestinal or gastric bleeding (of whatever cause) can be detected at an even earlier stage than before. Thus, the method can be utilized, for example in CRC screening, thereby increasing patient safety. The method and kit also significantly reduce the amounts of costs, biological waste and environmental damage that are related to the known procedures and kits. In addition, because the present method is based on a measurement of only one component, which is Hb/Hp complex, the test is cheaper to manufacture and simpler to cany out than any other test currently available.

Next the present technology is described with reference to embodiments below.

Colon View Hb/Hp Fecal Occult Blood Test is a visual (or automated) test, using immunochromatography for quick and qualitati ve detection of human

hemoglobin/haptoglobin complex (Hb/Hp) in stool samples. Hb molecule consists of 2 pairs of peptide (a- and β-globins) chains and 4 heme groups, each with one atom of iron. Free Hb may separate into α-β molecules, which are bound to a protein called haptoglobin (Hp). Hb/Hp complex plays an important role in the retrieval of Hb from lysed

erythrocytes and is relatively stable against acid and proteolytic degradation. This means that the Hb/Hp complex can be detected even after longer passage along the bowel, increasing the chance that also the blood derived from intestinal polyps (adenomas) and carcinomas of the proximal colon can be detected .

The present ColonView Hb/Hp Test is based on an immunochromatographic method, in which Hb/Hp complexes are specifically recognized through specific antibody reactions. When concerning a kit, the test cassette strip is pre-coated with anti-human Hb and anti- human Hp antibodies on the test region (T) and goat anti-mouse antibodies on the control region (C). An anti-human Hb/Hp complex antibody-colloidal gold conjugate pad is placed at the end of the membrane. When human Hb/Hp complexes are present in the stool sample, dissolved in buffered saline (preservation liquid), the mixture of colloidal gold conjugate and extracted sample moves along the membrane chromato graphically by capillary action.

In the case of a positive result, the Hb and/or Hb/Hp molecules in the stool sample loaded with gold-marked antibodies attach to the test band (T) and become visible by means of a pink/red coloration. In the case of a negative result, there are no Hb and/or Hb/Hp molecules that can attach to the test band (T) as complexes and therefore, there can also be no coloration of the test band (T). If the control strip (C) turns red/pink in color, this shows that the sample has been correctly taken and has migrated properly, indicating that the test is technically valid.

According to one embodiment, the present invention comprises a method of determining the presence of occult human blood in a stool sample, said method including sample collection and measurement of the content of a hemoglobin/haptoglobin (Hb/Hp) complex of the sample, which method contains the steps of

- collecting a stool sample of said patient during two or more days,

- ^■ mixing the stool samples of each collection day and dissolving it into a single unit of preservation liquid,

-- shaking the sample in the preservation liquid and allowing the

hemoglobin/haptoglobin complex to dissolve into the preservation liquid, whereby an extract is obtained, and

- carrying out said measurement on the obtained extract by using an immunoassay method. According to one embodiment, the stool samples of said patient are collected during three or more days, preferably three or four days.

A ccording to another embodiment, the measurement of the content of a

hemoglobin/haptoglobin (Hb/Hp) complex of the sample is carried out on the obtained extract by using one single immunochemical test. Preferably, the measurement is carried out by using by using an automated immunoassay method, selected from enzyme immunoassay, such as enzyme-linked immunosorbent (ELISA) assay, radioimmunoassay, fluoro immunoassay, or luminescence immunoassay, preferably ELISA. According to an embodiment, the hemoglobin/haptoglobin complex is dissolved after each sampling by shaking the sample and the preservation liquid. Thus, the method of determining the presence of occult human blood in a stool sample of a patient consists of sample collection and measurement of oniy one component, which is the Hb Hp complex, by the steps described above. The subjects to be tested are preferably selected among those to be screened for lower gastrointestinal pathologies, including colorectal cancers, bleeding polyps and adenomas, as well as other potential causes of occult intestinal (and even gastric) bleeding.

However, the measurements for determining the presence of fecal occult human blood in a stool sample can also be conducted via a quick test consisting of oniy one component (Hb/Hp complex).

According to an embodiment, the present invention comprises a kit for the multiple-day determination of the presence of occult human blood in the stools, which kit contains one or more stool collectors as well as sampling and immunochromatographic measurement devices, which include

- one sample tube for holding preservation liquid and, optionally, one or more

samples,

- one sample tip for collecting samples, which is suitable to be fitted into the sample tube,

- at least one cap suitable for fitting tightly onto the sample tube, and

- means for measuring the content of hemoglobiri/haptoglobm complex of an extract obtained from the sample. It is preferred that the sample tip is attached to the cap via a stick and the tip of the cap is shaped into a detachable part, which can be easily broken to leave an opening defining an applicator region.

According to another preferred embodiment, the kit comprises means for measuring the content of hemoglobin/haptoglobin complex, which means does contain one measurement cassette having one or more immunochromatography strips and one sample drop region. The measurement cassette strip is pre-coated with anti-human Hb and anti-human Hp antibodies on a test region (T) and goat anti-mouse antibodies on a control region (C), and further comprises an anti-human fib/Hp complex antibody-colloidal gold conjugate pad at the same end of the membrane than the sample drop region.

The kit further contains instructions for use (IFU).

A method for determining the presence of occult human blood (of whatever cause) in a stool sample, including sample collection and measurement of the content of

hemogiobin/haptogiobin complex of the sample, which method is carried out by using a kit described above belongs also to the scope of the present invention.

According to one embodiment, the present invention comprises a method for detection of proximal and distal colorectal neoplasia among screened subjects, wherein positive results obtained by the Hb/Hp measurements as described in the methods above indicate an increased risk of colorectal neoplasia (benign or malignant). FOB due to any reason is an abnormal situation, which requires clinical assessment.

According to further embodiment, the present invention comprises a method for detecting colorectal cancer (or its precursor) of a human patient by using the methods above described, further comprising carrying out a colonoscopy on patients with positive results from said methods, and making a diagnosis based on the combined results of said methods and the colonoscopy.

The present method thus provides tools for detection of proximal and distal colorectal neoplasia in patients subjected to organized (or opportunistic) screening for colorectal cancer. Herein organized refers to e.g. population-based screening and opportunistic refers to non-organized screening.

According to one embodiment, the present FIT test with high analytical sensitivity is capable of detecting FOB of non-neoplastic origin as well. The list of such conditions comprise e.g. vascular malformations (arterio -venous fistulas), angiectasia lesions, diverticulitis, diverticulosis, and most importantly, inflammatory bowel disease (IBD). The two most important conditions of IBD are Ulcerative Colitis (UC) and Crohn's disease. Therefore, the present test can be u tilized for tracking a state of treatment of such IBD by combining the results obtained from an IBD test and the FIT test. Thus, according to one embodiment the present invention comprise a method for detection of a status of an IBD, wherein negative results from both, the FIT test and the IBD test, indicate a balanced treatment. In other words, this is the case when the FIT test shows no fecal occult blood and the IBD test shows no inflammation.

For carrying out the tests as described herein, e.g. three stool samples need to be tested and the result must be interpreted positive if any of the samples test posi tive. Thus, any sample positive for Hb/Hp complex represents a positive test. The analytical sensitivity for the Hb/Hp complex is 4 ng/ml.

According to one embodiment, all stool samples are collected to one single sample tube. Samples may also be collected to separate sample tubes.

According to a further embodiment, the test requires two or more stool samples, preferably at least three stool samples.

The present invention is illustrated by the following non-limiting examples. It should be understood, however, that the embodiments given in the description above and in the examples below are for illustrative purposes only, and that various changes and modifications are possible within the scope of the appended claims.

EXAMPLES - METHODS

This example describes a procedure and guidelines for a comparative experiment conducted by the inventors of the present invention. All stool samples must be collected before appointment to colonoscopy. Samples from three different stools collected from three different (consecutive) days as follows are needed:

® Day I : The first stool sample: ColonView samples from the same stool.

• Day 2: The second stool sample: ColonView samples from the same stool.

· Day 3 : The third stool sample: ColonView samples from the same stool.

These three days should, if possible, be three consecutive days. In any case, the samples should be coliected within one week time (max).

1. Collecting a random sample of stool in a clean dry container or receptacle, for example the Stool Col lection Paper provided with the Sample Collection Bag. Use the Stool

Collection Paper as follows:

• Peeling off the liner covering the adhesive tape on each end of the collection paper.

• Lifting toilet seat. Unfold collection paper and place on the rim of toilet bowl .

Securing the adhesive tabs on the collection paper to the sides of the toilet rim. · Making bowel movement onto collection paper.

2. Unscrewing the colored cap and remove the Sample Col lection Tube applicator stick. Be careful not to spill or spatter solution from container.

3. Collecting random samples by inserting the applicator stick into the stool specimen. Take samples from various surfaces of the stool specimen.

4. Re-inserting the applicator stick into the tube and screw the cap tightly. Be careful not to break the tip of the Sample Collection Tube (colored end).

• If using the Stool Collection Paper, releasing adhesive tabs and flushing the collection paper with stool.

5. Writing your name and the date of sample collection in the space provided on the Sample Collection Tube.

6. Returning the specimen for testing promptly by mail or in person. The specimen(s) can be stored in the refrigerator (2-8 °C) for no more than 1 1 days, or at room temperature (max. 25 °C) for no more than 5 days. Sample processing in the test laboratory

1 . The Test Cassette and the Sample Collection Tube containing the stool sample is brought to room temperature (20 ... 30°C) at least 10 minutes before testing.

2. Taking the required number of lest Cassettes from the foil packaging only immediately before performing the test. Marking the Test Cassette with the name of the patient or with another form of identification.

3. Carefully shaking the Sample Collection Tube to ensure that the stool sample mixes properly with the saline solution.

4. Taking a paper towel and breaking the seal of the Sample Collection Tube with rotary motion or using a pair of scissors to cut the seal. Holding the collection tube upright and adding 3 drops of the solution into both round sample windows (S) of the lest Cassette.

5. Reading the results after 5 minutes. Strongly positive results may be evaluated even sooner. Evaluating the result within a maximum of 15 minutes. Quality Control or Interna! Procedural Control

The test also contains a procedural control. A colored line that appears in the control region (C) shows that each test is performed correctly. It is not unusual that the background turns slightly yellowish in color during the testing, depending on the color of the stool sample. This is acceptable, as long as evaluation of the test results is not adversely affected.

A clear background in the observation window is considered an internal negative control. However, when the stool samples are tested, the background may appear slightly yellowish due to the original color of the stool samples. This is acceptable as long as it does not interfere with the interpretation of test result. The test is invalid if the background fails to clear and obscures the reading of the result.

Evaluation of the test results

Test result is evaluated as "positive", if two lines appear.

Positive: 2 pink-colored lines appear; one in the control region (C) and one in the test region (T) in the Hb/Hp test. If you are testing strongly positive samples, the intensity of the control line may be reduced. It is not recommended to compare the intensity of the lines.

Negative: I pink-colored line appears in the control region (C) only, in the Hb/Hp test. Invalid: If no red line appears in the control (C) region in the Hb/Hp test, this is a sign that the test is not functioning properly, or that the test materials are not correct. In this case, repeat the test with a new Test Cassette or contact the manufacturer for technical support. Colonoscopy and biopsy procedures

In the demonstration study, a cohort of 300 colonoscopy-referral patients were examined by colonoscopy, thus providing the histological confirmation used as the gold standard in calculating the performance indicators of the test. If colonoscopy was completely normal, biopsies were not considered necessary and, in such a case, normal colonoscopy was used as an indicator of a negative result regarding the study endpoints.

Colonoscopy was performed according to the usual practice with a detailed record of all findings in the colonoscopy report. This applies to all study endpoints ((adenomas (A), adenocarcinomas (AC)) and other potential causes of occult blood (confounders), including their number, size and locations. The lesion site (caecum, ascending-, transverse- , descending colon, sigma, recto-sigmoid, rectum) was used as dichotomized variable (proximal and distal colon) in the final analyses.

All colonoscopy biopsies were examined by the expert pathologists. The diagnoses were reported using the standard WHO classification of colorectal neoplasia. In addition to their size, all polypoid lesions were classified as hyperplastic polyps or adenomas, with the latter being further classified according to their histological pattern as tubular, tubule- villous, villous or serrate adenomas. Statistical A alyses

Al l statistical analyses were performed, using the SPSS 22.0.0.1 for Windows (IBM, NY, USA) and STATA/SE 13.1 software (STATA Corp., Texas, USA). The descriptive statistics was conducted according to routine procedures. Performance indicators

(sensitivity, specificity, positive predictive value, PPV, negative predictive value, NPV and their 95%CI) of the two tests were calculated separately for each study endpoint, using the STATA/SE software and the diagti algorithm introduced by Seed et al. (2001 )(38). This algorithm also calculates the area under ROC (Receiver Operating Characteristics) called A.UC [(SE+SP)/2], for each study endpoint. Because the ColonView Hb and Hb/Hp test consists of two components, these performance indicators were calculated separately for lib and b/Hp complex, increasing the flexibility of this assay. Significance of the difference between AUC values is estimated using the roccomb test (STATA) with 95 %CL Before closing the cohort enrolment, careful power analyses will be conducted to estimate the statistical power of this study, it has been estimated that with the 10-15% prevalence of positive FIT or FOBT test among colonoscopy referral patients, we can expect 50 to 75 patients testing positive in a cohort of 500 subjects. With the test sensitivity level of 80%, that would yield some 40 to 60 cases of clinically significant pathologies (study endpoints). This number is more than enough to give 100% power for each test in calculating the difference in effect size in the disease/no disease setting (e.g. in two-sample proportion test). This sample size of 60 (but not 40) would also probably be powered enough (at 80% level) to compare the three tests in detection of clinically significant disease, within the effect size difference of e.g. 0.8 vs. 0.55, i.e. 80% detection (by test 1) vs. 55% detection (by test 2). Any effect size difference smaller than this necessitates more cases to maintain the power of the study at the acceptable 80% level.

EXAMPLES - RESULTS

Tables I-iV below describe the superior results obtained by the Hb/Hp test in a comparative experiment between Hb and Hb/Hp complex conducted by the inventors of the present invention by the methods as earlier described,

Table I. K ey characteristics of the patients and their colorectal disease.

*For the patients included in the final cohort; **Patients who failed to attend colonoscopy Table II. Performance indicators of the Colon View test for different endpoints.

SE, sensitivity; SP, specificity, PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval; AUC, area under ROC curve; *Three consecutive samples from each patient, any single test positive for Hb or Hb/Hp (VR, visual reading); ** Three consecutive samples from each patient, any single test positive for Hb or Hb/Hp (AR, automate reading: n ^::: :1Ί( ^" Differences in AUC values: 0.0001 : /; 0 000 1 :

31

0/180: ⁿ p=0.0001 ; ^{z l} p=0.0Q01 ; ^J1 p=0.71

Table III. Performance indicators of Hb and the Hb/Hp complex detection by ColonView* for different endpoints.

SE, sensitivity; SP, specificity, PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval; AUG, area under ROC curve; *Pooied results for Lot I and Lot2; VR, visual reading; AR, automatic reading; ''^Differences in AUC values: 'ρ^Ο.111 ; p=0A7%; ³ p=HM ; /> 0.444: w (ί.649: ^{: :} /; i).(;55. Table IV, Performance indicators of Colon View* in detection of neoplasia in the proximal and distal colon**.

SE, sensitivity; SP, specificity, PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval; AUC, area under ROC curve: *Pooled results for Lot 1 and Lot2; VR, visual References and related publications:

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Patent literature:

1. US 4,092,120

2. US 4,427,769