Single-cell atlas of hepatic T cells reveals expansion of liver-resident naive-like CD4+ T cells in primary sclerosing cholangitis.

BACKGROUND & AIMS: Little is known about the composition of intrahepatic immune cells and their contribution to the pathogenesis of primary sclerosing cholangitis (PSC). Herein, we aimed to create an atlas of intrahepatic T cells and thereby perform an in-depth characterization of T cells in inflamed human liver. METHODS: Different single-cell RNA sequencing methods were combined with in silico analyses on intrahepatic and peripheral T cells from patients with PSC (n = 11) and healthy donors (HDs, n = 4). Multi-parameter flow cytometry and functional in vitro experiments were conducted on samples from patients with PSC (n = 24), controls with other liver diseases and HDs. RESULTS: We identified a population of intrahepatic naive-like CD4+ T cells, which was present in all liver diseases tested, but particularly expanded in PSC. This population had a transcriptome and T cell receptor repertoire similar to circulating naive T cells but expressed a set of genes associated with tissue residency. Their periductal location supported the concept of tissue-resident naive-like T cells in livers of patients with PSC. Trajectory inference suggested that these cells had the developmental propensity to acquire a T helper 17 (TH17) polarization state. Functional and chromatin accessibility experiments revealed that circulating naive T cells in patients with PSC were predisposed to polarize towards TH17 cells. CONCLUSION: We report the first atlas of intrahepatic T cells in PSC, which led to the identification of a previously unrecognized population of tissue-resident naive-like T cells in the inflamed human liver and to the finding that naive CD4+ T cells in PSC harbour the propensity to develop into TH17 cells. LAY SUMMARY: The composition of intrahepatic immune cells in primary sclerosing cholangitis (PSC) and their contribution to disease pathogenesis is widely unknown. We analysed intrahepatic T cells and identified a previously uncharacterized population of liver-resident CD4+ T cells which are expanded in the livers of patients with PSC compared to healthy liver tissue and other liver diseases. These cells are likely to contribute to the pathogenesis of PSC and could be targeted in novel therapeutic approaches.

Biliary damage and liver fibrosis are ameliorated in a novel mouse model lacking l-histidine decarboxylase/histamine signaling.

Primary sclerosing cholangitis (PSC) is characterized by biliary damage and fibrosis. Multidrug resistance-2 gene knockout (Mdr2-/-) mice and PSC patients have increased histamine (HA) levels (synthesized by l-histidine decarboxylase, HDC) and HA receptor (HR) expression. Cholestatic HDC-/- mice display ameliorated biliary damage and hepatic fibrosis. The current study evaluated the effects of knockout of HDC-/- in Mdr2-/- mice (DKO) on biliary damage and hepatic fibrosis. WT, Mdr2-/- mice, and homozygous DKO mice were used. Selected DKO mice were treated with HA. We evaluated liver damage along with HDC expression and HA serum levels. Changes in ductular reaction were evaluated along with liver fibrosis, inflammation and bile acid signaling pathways. The expression of H1HR/PKC-α/TGF-ß1 and H2HR/pERK/VEGF-C was determined. In vitro, cholangiocyte lines were treated with HA with/without H1/H2 inhibitors before measuring: H1/H2HR, TGF-ß1, and VEGF-C expression. Knockout of HDC ameliorates hepatic damage, ductular reaction, fibrosis, inflammation, bile acid signaling and H1HR/PKC-α/TGF-ß1 and H2HR/pERK/VEGF-C signaling. Reactivation of the HDC/HA axis increased these parameters. In vitro, stimulation with HA increased HR expression and PKC-α, TGF-ß1, and VEGF-C expression, which was reduced with HR inhibitors. Our data demonstrate the key role for the HDC/HA axis in the management of PSC progression.

Short-term prognostic factors for primary sclerosing cholangitis.

BACKGROUND: It was recently reported that alkaline phosphatase (ALP) levels below 1.5 upper limit of normal (ULN) predicted better prognosis in primary sclerosing cholangitis (PSC). We evaluated whether ALP as well as other laboratory values were useful for the short-term prognosis of PSC in a Japanese cohort. METHODS: In 78 patients with PSC (41 males and 37 females, mean onset age 41.9 years), the relationship between nine parameters (albumin, bilirubin, international normalized ratio of prothrombin time [PT-INR], ALP, aspartate aminotransferase [AST], alanine aminotransferase [ALT], γ-glutamyl transpeptidase [γ-GTP], platelet, and calculated Model for End-Stage Liver Disease [MELD] score), and liver related clinical endpoints (death due to liver failure, variceal bleeding, liver transplantation, and biliary carcinoma) were retrospectively examined. Using receiver operating characteristic (ROC) analysis, we investigated which parameter was useful for predicting the short-term prognosis. RESULTS: Average follow-up period was 8.6 years. The endpoints were evaluated in 40 patients. Seven patients died of liver failure, three patients developed variceal bleeding, nine patients received liver transplantation from a living donor, 13 patients received certified brain-dead liver transplantation, and eight patients developed biliary carcinoma. The parameters with an area under the curve (AUC) of more than 0.8 were albumin, bilirubin, PT-INR, ALP, and MELD score. AUC for ALP was 0.85. The optimal cutoff value was 2.3 ULN. Despite the use or non-use of ursodeoxycholic acid, short-term prognosis of patients with an ALP level below 2.3 ULN was good. CONCLUSIONS: We confirmed that keeping ALP low is associated with better short-term prognosis in a Japanese cohort. In addition, Alb, Bil, PT-INR, and MELD score were good predictors.

Liver alkaline phosphatase: a missing link between choleresis and biliary inflammation.

Several lines of evidence show that serum alkaline phosphatase (AP) is not only a signpost of cholestasis but also a surrogate marker of the severity of primary biliary cirrhosis and primary sclerosing cholangitis. In the present opinion article, we review and discuss the putative role of liver AP in health and in cholestatic diseases. In inflammatory cholestatic conditions, loss of activity of liver AP (resulting from its relocation from canaliculi and the acidic milieu) might promote hyper-adenosine triphosphate-bilia, lipopolysaccharide overload, and subsequent exacerbation and perpetuation of inflammation. Drugs that can restore the polarity of hepatocytes and canalicular export of bile acids or act as bile alkalinity modifiers are predicted to exert anti-inflammatory effects and to benefit both primary biliary cirrhosis and primary sclerosing cholangitis. Oral administration of intestinal AP could be a valid therapeutic intervention that deserves further study under experimental conditions as well as in human diseases. Overall, the key role of the liver microenvironment that might shape the different facets of the inflammatory processes in fibrosing cholangiopathies is highlighted.

Gain of Function Mutations of PIK3CD as a Cause of Primary Sclerosing Cholangitis.

Gain of function (GOF) mutation in the p110δ catalytic subunit of the phosphatidylinositol-3-OH kinase (PIK3CD) is the cause of a primary immunodeficiency (PID) characterized by recurrent sinopulmonary infections and lymphoproliferation. We describe a family of two adults and three children with GOF mutation in PIK3CD, all with recurrent sinopulmonary infections and varied infectious and non-infectious complications. The two adults have Primary Sclerosing Cholangitis (PSC) without evidence of Cryptosporidium parvum infection and have required liver transplantation. PSC is a novel phenotype of GOF mutation in PIK3CD.

Reduction in alkaline phosphatase is associated with longer survival in primary sclerosing cholangitis, independent of dominant stenosis.

BACKGROUND: Alkaline phosphatase (ALP) is an important serum marker in primary sclerosing cholangitis (PSC). Patients with obstruction of the large bile ducts due to dominant strictures (DS) are a special, clinically important phenotype. AIM: To determine the impact of ALP reduction on liver transplantation-free survival in PSC patients with DS. METHODS: Prospective cohort study in 215 PSC patients. We performed subgroup analysis for patients without DS (no DS, n = 84), DS at first presentation (DS early, n = 72) and development of DS during the course of the study (DS late, n = 59). We evaluated two scores of ALP reduction. ALP reduction 1 was defined as ALP normalisation, 50% reduction compared with baseline values, or reduction below 1.5 times of upper limit of normal (ULN) within 6 months. ALP reduction 2 was defined as ALP reduction below 1.5 times of ULN within 12 months. RESULTS: Of the patients, 59.5% reached an ALP reduction 1 and 56.7% according to ALP reduction 2. Achievement of each score was associated with longer transplantation-free survival in all three groups (ALP reduction 1: no DS P = 0.001; DS early P < 0.001; DS late P = 0.022; ALP reduction 2: no DS P = 0.014; DS early P = 0.001; DS late P = 0.002). Cox-regression analysis revealed each score as an independent predictor for improved transplantation-free survival (ALP reduction 1 and 2 P < 0.001 each). We further analysed previously published scores of ALP improvement in PSC showing also improved survival in patients with ALP normalisation or a reduction below 1.5 times of ULN (P = 0.003, P = 0.001, respectively), whereas the score determined by 40% reduction did not show significant differences in survival (P = 0.55). CONCLUSIONS: Reduction in alkaline phosphatase values within the first year is associated with improved transplantation-free survival in patients with primary sclerosing cholangitis independent of the presence of dominant strictures. Alkaline phosphatase might be an adequate surrogate marker for outcome assessment in clinical studies both for patients with and without dominant strictures.

Changes of activity and isoforms of alkaline sphingomyelinase (nucleotide pyrophosphatase phosphodiesterase 7) in bile from patients undergoing endoscopic retrograde cholangiopancreatography.

BACKGROUND: Alkaline sphingomyelinase (NPP7) is an ecto-enzyme expressed in intestinal mucosa, which hydrolyses sphingomyelin (SM) to ceramide and inactivates platelet activating factor. It is also expressed in human liver and released in the bile. The enzyme may have anti-tumour and anti-inflammatory effects in colon and its levels are decreased in patients with colon cancer and ulcerative colitis. Active NPP7 is translated from a transcript of 1.4 kb, whereas an inactive form from a 1.2 kb mRNA was found in colon and liver cancer cell lines. While the roles of NPP7 in colon cancer have been intensively studied, less is known about the function and implications of NPP7 in the bile. The present study examines the changes of NPP7 in bile of patients with various hepatobiliary diseases. METHODS: Bile samples were obtained at endoscopic retrograde cholangiopancreatography (ERCP) in 59 patients with gallstone, other benign disease, tumour, and primary sclerosing cholangitis (PSC). The NPP7 activity was determined. The appearance of the 1.4 and 1.2 kb products in the bile was examined by Western blot. The results were correlated to the diseases and also plasma bilirubin and alkaline phosphatase. RESULTS: NPP7 activity in the tumour group was significantly lower than in the gallstone group (p < 0.05). The activity in the tumour plus PSC group was also lower than in gallstone plus other benign disease group (p < 0.05). Within the tumour group NPP7 activity was lowest in cholangiocarcinoma patients, being only 19% of that in gallstone patients. Bilirubin correlated inversely to NPP7 and was higher in the tumour than in the gallstone group. Western blot identified both the 1.4 kb and the 1.2 kb products in most bile samples. The density ratio for the 1.4/1.2 kb products correlated to NPP7 activity significantly. Two patients (one PSC and one cholangiocarcinoma) lacking NPP7 activity had only the 1.2 kb form in bile. CONCLUSION: NPP7 activity and the ratio of 1.4/1.2 kb products in bile are significantly decreased in malignancy, particularly in cholangiocarcinoma. The implications of the finding in diagnosis of cholangiocarcinoma and 1.2 kb product in hepatobiliary diseases require further investigation.

Liver enzyme elevations within 3 months of diagnosis of inflammatory bowel disease and likelihood of liver disease.

BACKGROUND: Inflammatory bowel disease-associated liver diseases (IBD-LDs) include autoimmune hepatitis (AIH), primary sclerosing cholangitis (PSC), and an overlap syndrome. Prospective unbiased multicenter data regarding the frequency of IBD-LD in patients with pediatric inflammatory bowel disease (IBD) are lacking. We examined early alanine aminotransferase (ALT) and γ-glutamyl transpeptidase (GGT) elevations in children diagnosed as having IBD and assessed the likelihood of IBD-LD. METHODS: Data collected from the prospective observational Pediatric Inflammatory Bowel Disease Collaborative Research Group Registry enrolling children of age <16 years within 30 days of diagnosis. AIH, PSC, and overlap syndrome were diagnosed using local institutional criteria. RESULTS: A total of 1569 subjects had liver enzymes available. Of the total, 757 had both ALT and GGT, 800 had ALT only (no GGT), and 12 had GGT only (no ALT). Overall, 29 of 1569 patients (1.8%) had IBD-LD. IBD-LD was diagnosed in 1 of 661 (0.15%) of patients with both ALT and GGT ≤ 50 IU/L compared with 21 of 42 (50%) of patients with both ALT and GGT > 50 (odds ratio 660, P < 0.0001). Of the 29 patients with IBD-LD, 21 had PSC, 2 had AIH, and 6 had overlap syndrome. IBD-LD was more common in patients with ulcerative colitis and IBD-unclassified (indeterminate colitis) than in those with Crohn disease (4% vs 0.8%, respectively, P < 0.001). CONCLUSIONS: Elevation of both ALT and GGT within 90 days after the diagnosis of IBD is associated with a markedly increased likelihood of IBD-LD. Both ALT and GGT levels should be measured in all of the pediatric patients newly diagnosed as having IBD.

PNPLA3 I148M polymorphism and progressive liver disease.

The 148 Isoleucine to Methionine protein variant (I148M) of patatin-like phospholipase domain-containing 3 (PNPLA3), a protein is expressed in the liver and is involved in lipid metabolism, has recently been identified as a major determinant of liver fat content. Several studies confirmed that the I148M variant predisposes towards the full spectrum of liver damage associated with fatty liver: from simple steatosis to steatohepatitis and progressive fibrosis. Furthermore, the I148M variant represents a major determinant of progression of alcohol related steatohepatitis to cirrhosis, and to influence fibrogenesis and related clinical outcomes in chronic hepatitis C virus hepatitis, and possibly chronic hepatitis B virus hepatitis, hereditary hemochromatosis and primary sclerosing cholangitis. All in all, studies suggest that the I148M polymorphism may represent a general modifier of fibrogenesis in liver diseases. Remarkably, the effect of the I148M variant on fibrosis was independent of that on hepatic steatosis and inflammation, suggesting that it may affect both the quantity and quality of hepatic lipids and the biology of non-parenchymal liver cells besides hepatocytes, directly promoting fibrogenesis. Therefore, PNPLA3 is a key player in liver disease progression. Assessment of the I148M polymorphism will possibly inform clinical practice in the future, whereas the determination of the effect of the 148M variant will reveal mechanisms involved in hepatic fibrogenesis.

Relevance of the inner mitochondrial membrane enzyme F1F0-ATPase as an autoantigen in autoimmune liver disorders.

BACKGROUND AND AIMS: Recently, a non-M2-related mitochondrial 60 kDa protein found to be recognized by antimitochondrial antibody (AMA) negative sera from patients with primary biliary cirrhosis (PBC) has been shown to contain parts of the five F(1)-ATPase subunits α, ß, γ, δ and ε. In this study, we examined whether this enzyme is, indeed, a target antigen in PBC. METHODS: Analysed were 60 AMA-positive/anti-M2-negative and 103 anti-M2-positive PBC patients, 46 patients with autoimmune hepatitis (AIH), 35 patients with primary sclerosing cholangitis (PSC), 110 patients with viral hepatitis, 40 patients with inflammatory bowel diseases (IBD), 33 patients with connective tissue diseases (systemic lupus erythematosus, mixed connective tissue disease, Sjögren disease, systemic sclerosis) and 25 blood donors. The F(1)-ATPase-subunits α-δ were recombinantly expressed in Escherichia coli, purified and applied to ELISA and Western blotting. RESULTS: In all, 40 of the 60 AMA-positive/anti-M2-negative (67%) and 44 (43%) of the 103 anti-M2-positive PBC-sera reacted with at least one of the F(1)-subunits α-δ. The ß- and γ-subunits were preferentially recognized. However, also up to 57% of patients with AIH and 34% of patients with PSC had anti-ß- or γ-antibodies, while patients with viral hepatitis had these antibodies in up to 13%. Patients with IBD had anti-ß and anti-γ-antibodies in up to 20 and 5% respectively. None of the patients with connective tissue diseases had antibodies to the ß- and only 6% to the γ-subunit. Sera from healthy blood donors were negative. CONCLUSIONS: Antibodies to the ß- and γ-subunits of F(1)-ATPase are further AMAs in PBC but occur also in other autoimmune liver disorders; they may be, therefore, indicators for a general autoimmune process of the liver.

[No complaints, but constantly elevated cholestatic serum liver tests]. / (Fast) beschwerdefreie Patientin, aber konstant erhöhte Cholestaseparameter.

A 75 year old woman presented with long-lasting pruritus and elevation of serum levels of alkaline phosphatase and gamma-glutamyl transpeptidase (GGT). Potentially hepatotoxic drugs were stopped; an abdominal ultra-sonography was normal. Antimitochondrial antibodies were elevated and are the serologic hallmark of primary biliary cirrhosis. The disease can progress from an asymptomatic inflammation to a cirrhosis. The disease-modifying treatment consists in ursodeoxycholic acid 13-15 mg/kg per day. Concomitant to the primary biliary cirrhosis hypothyroidism, osteoporosis, malabsorption and dyslipidemia can occur.

Alkaline phosphatase normalization is associated with better prognosis in primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis results in elevated but fluctuating serum alkaline phosphatase levels that occasionally return to normal. AIMS: To investigate the frequency of normalization of alkaline phosphatase in newly diagnosed primary sclerosing cholangitis patients and the subsequent clinical outcomes. METHODS: Records of newly diagnosed primary sclerosing cholangitis patients were examined retrospectively for laboratory values and clinical end points (cholangiocarcinoma, liver transplantation and death) within 10 years of diagnosis. Data from a recent prospective ursodeoxycholic acid treatment trial were also studied. RESULTS: Eighty-seven patients met the inclusion criteria. Normalization of alkaline phosphatase was seen in 35 (40%) patients. Five (14%) patients with normalization reached an end point whereas 17 (33%) of the patients with persistent elevation reached an end point (P = 0.02). Ursodeoxycholic acid was used similarly by both groups. When the investigative criteria were applied to a prospective trial, there was again a significant relationship between normalization of alkaline phosphatase and survival in patients receiving ursodeoxycholic acid (P < 0.01) and the placebo group (P = 0.02). CONCLUSIONS: Serum alkaline phosphatase was found to normalize in a high proportion of newly diagnosed primary sclerosing cholangitis patients. This was significantly associated with a better prognosis in a retrospective cohort and when data from a prospective treatment trial was evaluated.

Genetic polymorphisms of matrix metalloproteinase 3 in primary sclerosing cholangitis.

BACKGROUND: The damaging cholestasis inherent to primary sclerosing cholangitis (PSC) results from bile duct stricturing because of progressive fibrosis. The matrix metalloproteinase 3 (MMP3) degrades a wide range of matrix components and is expressed by activated liver stellate cells, and so is a candidate for involvement with the fibrotic processes underlying PSC. Moreover, the MMP3 gene harbours polymorphisms associated with variation in its activity directly impacting clinical phenotypes. AIMS: We aimed to examine the influence of MMP3 polymorphisms on PSC risk and progression. METHODS: Nine single nucleotide polymorphisms (SNPs) tagging the common genetic variation of MMP3 were genotyped in 266 PSC patients and 407 controls. SNPs and inferred haplotypes were assessed for PSC association by logistic regression and score tests. The effect of SNPs on survival to liver transplant or death was analysed using Cox regression, and Kaplan-Meier curves were constructed. RESULTS: No association of PSC with individual SNPs or haplotypes of MMP3 was detected. However, progression to death or liver transplant was significantly associated with homozygosity for minor alleles of rs522616, rs650108 and rs683878, particularly among PSC patients with concurrent ulcerative colitis (UC) (strongest in redundant SNPs rs650108/rs683878, hazard ratio=3.23, 95% confidence interval 1.45-7.25, P=0.004). CONCLUSIONS: Genetic variation in MMP3 influences PSC progression, possibly in the context of coexisting UC. While the functional variants and specific mechanisms remain unknown, this finding implicates the turnover of the extracellular matrix as an important and variable component of PSC pathogenesis. Efforts to understand this process could form the basis for developing effective treatments, which are currently lacking for PSC.

Bezafibrate for the treatment of primary sclerosing cholangitis.

BACKGROUND: It is known that bezafibrate decreases serum alkaline phosphatase (ALP) in patients with hyperlipidemia, and the efficacy of this drug for the treatment of primary biliary cirrhosis has been confirmed. However, there has been little evidence of its efficacy for the treatment of primary sclerosing cholangitis (PSC). METHODS: Bezafibrate (400 mg/day) was orally administered to 7 consecutive patients with PSC, and we analyzed their clinical features and the drug efficacy in terms of the effect on hepatobiliary enzymes, including ALP, gamma-glutamyl transpeptidase (gamma-GTP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) after 6 months. The latest hepatobiliary enzyme levels were also evaluated. RESULTS: In 3 patients (effective group), the levels of all hepatobiliary enzymes had decreased after 6 months. Mean ALP had decreased to approximately 40% of the baseline in this group. The efficacy of bezafibrate was observed for a long period (range, 8-27 months) in these 3 patients. There seemed to be no definite association between the efficacy of bezafibrate and the clinical features in the short term. CONCLUSIONS: This study showed that bezafibrate could lower the levels of hepatobiliary enzymes in about half of a cohort of patients with PSC.

The human UGT1A3 enzyme conjugates norursodeoxycholic acid into a C23-ester glucuronide in the liver.

Norursodeoxycholic acid (norUDCA) exhibits efficient anti-cholestatic properties in an animal model of sclerosing cholangitis. norUDCA is eliminated as a C(23)-ester glucuronide (norUDCA-23G) in humans. The present study aimed at identifying the human UDP-glucuronosyltransferase (UGT) enzyme(s) involved in hepatic norUDCA glucuronidation and at evaluating the consequences of single nucleotide polymorphisms in the coding region of UGT genes on norUDCA-23G formation. The effects of norUDCA on the formation of the cholestatic lithocholic acid-glucuronide derivative and of rifampicin on hepatic norUDCA glucuronidation were also explored. In vitro glucuronidation assays were performed with microsomes from human tissues (liver and intestine) and HEK293 cells expressing human UGT enzymes and variant allozymes. UGT1A3 was identified as the major hepatic UGT enzyme catalyzing the formation of norUDCA-23G. Correlation studies using samples from a human liver bank (n = 16) indicated that the level of UGT1A3 protein is a strong determinant of in vitro norUDCA glucuronidation. Analyses of the norUDCA-conjugating activity by 11 UGT1A3 variant allozymes identified three phenotypes with high, low, and intermediate capacity. norUDCA is also identified as a competitive inhibitor for the hepatic formation of the pro-cholestatic lithocholic acid-glucuronide derivative, whereas norUDCA glucuronidation is weakly stimulated by rifampicin. This study identifies human UGT1A3 as the major enzyme for the hepatic norUDCA glucuronidation and supports that some coding polymorphisms affecting the conjugating activity of UGT1A3 in vitro may alter the pharmacokinetic properties of norUDCA in cholestasis treatment.

Catalytically impaired hMYH and NEIL1 mutant proteins identified in patients with primary sclerosing cholangitis and cholangiocarcinoma.

The human hMYH and NEIL1 genes encode DNA glycosylases involved in repair of oxidative base damage and mutations in these genes are associated with certain cancers. Primary sclerosing cholangitis (PSC), a chronic cholestatic liver disease characterized by inflammatory destruction of the biliary tree, is often complicated by the development of cholangiocarcinoma (CCA). Here, we aimed to investigate the influence of genetic variations in the hMYH and NEIL1 genes on risk of CCA in PSC patients. The hMYH and NEIL1 gene loci in addition to the DNA repair genes hOGG1, NTHL1 and NUDT1 were analyzed in 66 PSC patients (37 with CCA and 29 without cancer) by complete genomic sequencing of exons and adjacent intronic regions. Several single-nucleotide polymorphisms and mutations were identified and severe impairment of protein function was observed for three non-synonymous variants. The NEIL1 G83D mutant was dysfunctional for the major oxidation products 7,8-dihydro-8-oxoguanine (8oxoG), thymine glycol and dihydrothymine in duplex DNA, and the ability to perform delta-elimination at abasic sites was significantly reduced. The hMYH R260Q mutant had severe defect in adenine DNA glycosylase activity, whereas hMYH H434D could excise adenines from A:8oxoG pairs but not from A:G mispairs. We found no overall associations between the 18 identified variants and susceptibility to CCA in PSC patients; however, the impaired variants may be of significance for carcinogenesis in general. Our findings demonstrate the importance of complete resequencing of selected candidate genes in order to identify rare genetic variants and their possible contribution to individual susceptibility to cancer development.