[Show abstract][Hide abstract] ABSTRACT: Background Every colorectal cancer (CRC) patient should be tested for microsatellite instability (MSI, a marker for defective DNA mismatch repair) as a first screen for Lynch syndrome (LS). In this study, we investigated whether it may be possible to improve the detection of MSI in CRC. We examined whether the HT17 DNA repeat (critical for correct splicing of the chaperone HSP110) might constitute a superior marker for diagnosis of the MSI phenotype in patients with CRC compared with the standard panel of markers (pentaplex).
Methods The HT17 polymorphism was analysed in germline DNA from 1037 multi-ethnic individuals. We assessed its sensitivity and specificity for detecting MSI in a multicentre, population-based cohort of 685 patients with CRC and an additional series of 70 patients with CRC considered to be at-risk of LS. All cases were screened earlier for MSI using pentaplex markers. Cases showing discordant HT17/pentaplex results were further examined for the expression of mismatch repair proteins.
Results HT17 status was analysed independently and blinded to previous results from pentaplex genotyping. HT17 showed no germline allelic variation outside a very narrow range. Compared with the pentaplex panel, HT17 showed better sensitivity (0.984 (95% CI 0.968 to 0.995) vs 0.951 (95% CI 0.925 to 0.972)) and similar specificity (0.997 (95% CI 0.989 to 1.000) for both) for the detection of MSI. Furthermore, HT17 alone correctly classified samples judged to be uncertain with the pentaplex panel and showed excellent ability to detect MSI in patients with LS.
Conclusions HT17 simplifies and improves the current standard molecular methods for detecting MSI in CRC.
Full-text · Article · Feb 2016 · Journal of Medical Genetics
[Show abstract][Hide abstract] ABSTRACT: To compare characteristics and outcomes of resected and nonresected main-duct and mixed intraductal papillary mucinous neoplasms of the pancreas (IPMN).
Over a 14-year period, 50 patients who did not undergo surgery for resectable main-duct or mixed IPMN, for reasons of precluding comorbidities, age and/or refusal, were compared with 74 patients who underwent resection to assess differences in rates of survival, recurrence/occurrence of malignancy, and prognostic factors. All study participants had dilatation of the main pancreatic duct by ≥ 5 mm, with or without dilatation of the branch ducts. Some of the nonsurgical patients showed evidence of mucus upon perendoscopic retrograde cholangiopancreatography or endoscopic ultrasound and/or after fine needle aspiration. For the surgical patients, pathologic analysis of resected specimens confirmed a diagnosis of IPMN with involvement of the main pancreatic duct or of both branch ducts as well as the main pancreatic duct. Clinical and biologic follow-ups were conducted for all patients at least annually, through hospitalization or consultation every six months during the first year of follow-up, together with abdominal imaging analysis (magnetic resonance cholangiopancreatography or computed tomography) and, if necessary, endoscopic ultrasound with or without fine needle aspiration.
The overall five-year survival rate of patients who underwent resection was significantly greater than that for the nonsurgical patients (74% vs 58%; P = 0.019). The parameters of age (< 70 years) and absence of a nodule were associated with better survival (P < 0.05); however, the parameters of main pancreatic duct diameter > 10 mm, branch duct diameter > 30 mm, or presence of extra pancreatic cancers did not significantly influence the prognosis. In the nonsurgical patients, pancreatic malignancy occurred in 36% of cases within a mean time of 33 mo (median: 29 mo; range: 8-141 mo). Comparison of the nonsurgical patients who experienced disease progression with those who did not progress showed no significant differences in age, sex, symptoms, subtype of IPMN, or follow-up period; only the size of the main pancreatic duct was significantly different between these two sub-groups, with the nonsurgical patients who experienced progression showing a greater diameter at the time of diagnosis (> 10 mm).
Patients unfit for surgery have a 36% greater risk of developing pancreatic malignancy of the main-duct or mixed IPMN within a median of 2.5 years.
Full-text · Article · Mar 2015 · World Journal of Gastroenterology
[Show abstract][Hide abstract] ABSTRACT: The three biomarkers commonly used in the management of colorectal cancers (CRCs) are: microsatellite instability (MSI), RAS and BRAF mutations. Colorectal tumors exhibiting microsatellite instability (MSI-High) form a distinct subgroup of CRCs (15%). MSI-High tumors have a better prognosis than microsatellite stable tumors, especially in located stages. Adjuvant therapy is therefore not recommended in stage II MSI-High CRC. Moreover, the determination of MSI status is commonly used as a screening tool for Lynch syndrome families since the MSI-High phenotype is supposed constant in Lynch spectrum's tumors. A RAS mutation (exon 2, 3 or 4 of KRAS and NRAS genes) is present in 50 to 60% of CRCs. It is a well admitted biological marker predictive of resistance to anti-EGFR therapy (cetuximab and panitumumab). These therapies are restricted to wild-type metastatic CRCs. The BRAF mutation is found in 5 to 10% of the CRCs. It has a strong negative prognostic impact, especially in metastatic CRCs. These patients should be quickly included in clinical studies, whenever available, testing aggressive strategies or new anti-BRAF therapies. Testing MSI, RAF and RAS is routinely realised by one of the 28 genetic research laboratories mandated by INCA, using formalin fixed paraffin embedded (FFPE) tumor tissue samples. A standardised prescription is transmitted from the initial pathological laboratory to the genetic laboratory, with the FFPE tissue sample. Many new biomarkers are emerging (PI3KCA, c-MET, genomic signature, etc.) and these developments might ensure a better management of patients having a colorectal cancers in terms of personalised medicine.
[Show abstract][Hide abstract] ABSTRACT: As many other cancers, pancreatic ductal adenocarcinoma (PDAC) progression is associated with a series of hallmark changes for cancer cells to secure their own growth success. Yet, these very changes render cancer cells highly sensitive to viral infection. A promising strategy may rely on and exploit viral replication for tumour destruction, whereby infection of tumour cells by a replication-conditional virus may lead to cell destruction and simultaneous release of progeny particles that can spread and infect adjacent tumour cells, while sparing healthy tissues. In the present study, we used Myb34.5, a second-generation replication-conditional HSV-1 mutant in which ICP6 gene expression is defective and expression of the HSV-1 γ34.5 gene is regulated by the cellular B-myb promoter. We found that B-myb is present in experimental PDAC and tumours, and is over expressed in patients' tumours, as compared to normal adjacent pancreas. Myb34.5 replicates to high level in human PDAC cell lines and is associated with cell death by apoptosis. In experimental models of PDAC, mice receiving intratumoral Myb34.5 injections appeared healthy and tumour progression was inhibited, with evidences of tumour necrosis, haemorrhage, viral replication and cancer cell death by apoptosis. Combining standard-of-care chemotherapy with Myb34.5 successfully led to a very impressive antitumoral effect that is rarely achieved in this experimental model, and resulted in a greater reduction in tumour growth than chemotherapy alone. These promising results warrant further evaluation in early phase clinical trial for patients diagnosed with PDAC for whom no effective treatment is available.
No preview · Article · Nov 2014 · Human Gene Therapy
[Show abstract][Hide abstract] ABSTRACT: Despite tremendous efforts from scientists and clinicians worldwide, pancreatic adenocarcinoma (PDAC) remains a deadly disease due to the lack of early diagnostic tools and reliable therapeutic approaches. Consequently, a majority of patients (80%) display an advanced disease that results in a low resection rate leading to an overall median survival of less than 6 months. Accordingly, robust markers for the early diagnosis and prognosis of pancreatic cancer, or markers indicative of survival and/or metastatic disease are desperately needed to help alleviate the dismal prognosis of this cancer. In addition, the discovery of new therapeutic targets is mandatory to design effective treatments. In this review, we will highlight the translational studies demonstrating that microRNAs may soon translate into clinical applications as long-awaited screening tools and therapeutic targets for PDAC.
No preview · Article · Aug 2014 · World Journal of Gastroenterology
[Show abstract][Hide abstract] ABSTRACT: Sporadic early onset colorectal carcinoma (EOCRC) which has by definition no identified hereditary predisposition is a growing problem that remains poorly understood. Molecular analysis could improve identification of distinct sub-types of colorectal cancers (CRC) with therapeutic implications and thus can help establish that sporadic EOCRC is a distinct entity. From 954 patients resected for CRC at our institution, 98 patients were selected. Patients aged 45-60 years were excluded to help define "young" and "old" groups. Thirty-nine cases of sporadic EOCRC (patients≤45 years with microsatellite stable tumors) were compared to both microsatellite stable tumors from older patients (36 cases, patients>60 years) and to groups of patients with microsatellite instability. Each group was tested for TP53, KRAS, BRAF, PIK3CA mutations and the presence of a methylator phenotype. Gene expression profiles were also used for pathway analysis. Compared to microsatellite stable CRC from old patients, sporadic EOCRC were characterized by distal location, frequent synchronous metastases and infrequent synchronous adenomas but did not have specific morphological characteristics. A familial history of CRC was more common in sporadic EOCRC patients despite a lack of identified hereditary conditions (p = 0.013). Genetic studies also showed the absence of BRAF mutations (p = 0.022) and the methylator phenotype (p = 0.005) in sporadic EOCRC compared to older patients. Gene expression analysis implicated key pathways such as Wnt/beta catenin, MAP Kinase, growth factor signaling (EGFR, HGF, PDGF) and the TNFR1 pathway in sporadic EOCRC. Wnt/beta catenin signaling activation was confirmed by aberrant nuclear beta catenin immunostaining (p = 0.01). This study strongly suggests that sporadic EOCRC is a distinct clinico-molecular entity presenting as a distal and aggressive disease associated with chromosome instability. Furthermore, several signaling pathways including the TNFR1 pathway have been identified as potential biomarkers for both the diagnosis and treatment of this disease.
[Show abstract][Hide abstract] ABSTRACT: Mutation of the KRAS oncogene is present in 75% to 95% of pancreatic cancer tissues. This study aimed to evaluate whether endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), combined with analysis of the KRAS mutation, improves the diagnosis of pancreatic cancer in cases of inconclusive or doubtful cytopathologic analysis.
We prospectively included 186 patients with a pancreatic mass (103 men; mean age: 62 y). Cytopathology and KRAS mutations, using TaqMan MGB allelic discrimination, were performed on EUS-FNA material. A final diagnosis was obtained from EUS-FNA analysis and/or a subsequent biopsy if necessary, and/or surgery, and follow-up: these were pancreatic adenocarcinoma (n=104), other malignant pancreatic tumors (n=22), and benign lesions (n=60, including 35 cases of chronic pancreatitis).
Inconclusive or doubtful (low-grade dysplasia or atypia) cytopathology was found in 68 cases. Of these, 29 patients who had adenocarcinoma were subsequently diagnosed, including 19 cases with a former KRAS mutation. Sensitivity, specificity, positive and negative predictive values, and overall accuracy of cytopathology alone to diagnose adenocarcinoma were 73%, 100%, 100%, 75%, and 85%, respectively. When KRAS mutation analysis was combined with pathology, these values reached 88%, 99%, 99%, 89%, and 93%, respectively. The performance of EUS-FNA to diagnose malignancy was similarly improved after the KRAS-mutation assay (negative predictive value increased from 67% to 88%; accuracy increased from 85% to 94%).
EUS-FNA plus KRAS-mutation analysis, using allelic discrimination, is accurate and improves the diagnosis of pancreatic adenocarcinoma when pathology is inconclusive or doubtful.
No preview · Article · May 2014 · Journal of clinical gastroenterology
[Show abstract][Hide abstract] ABSTRACT: It remains presently unclear whether disease progression in colorectal carcinoma (CRC), from early, to invasive and metastatic forms, is associated to a gradual increase in genetic instability and to a scheme of sequentially occurring Copy Number Alterations (CNAs).
In this work we set to determine the existence of such links between CRC progression and genetic instability and searched for associations with patient outcome. To this aim we analyzed a set of 162 Chromosomal Instable (CIN) CRCs comprising 131 primary carcinomas evenly distributed through stage 1 to 4, 31 metastases and 14 adenomas by array-CGH. CNA profiles were established according to disease stage and compared. We, also, asked whether the level of genomic instability was correlated to disease outcome in stage 2 and 3 CRCs. Two metrics of chromosomal instability were used; (i) Global Genomic Index (GGI), corresponding to the fraction of the genome involved in CNA, (ii) number of breakpoints (nbBP).
Stage 1, 2, 3 and 4 tumors did not differ significantly at the level of their CNA profiles precluding the conventional definition of a progression scheme based on increasing levels of genetic instability. Combining GGI and nbBP,we classified genomic profiles into 5 groups presenting distinct patterns of chromosomal instability and defined two risk classes of tumors, showing strong differences in outcome and hazard risk (RFS: p = 0.012, HR = 3; OS: p < 0.001, HR = 9.7). While tumors of the high risk group were characterized by frequent fractional CNAs, low risk tumors presented predominantly whole chromosomal arm CNAs. Searching for CNAs correlating with negative outcome we found that losses at 16p13.3 and 19q13.3 observed in 10% (7/72) of stage 2-3 tumors showed strong association with early relapse (p < 0.001) and death (p < 0.007, p < 0.016). Both events showed frequent co-occurrence (p < 1x10-8) and could, therefore, mark for stage 2-3 CRC susceptible to negative outcome.
Our data show that CRC disease progression from stage 1 to stage 4 is not paralleled by increased levels of genetic instability. However, they suggest that stage 2-3 CRC with elevated genetic instability and particularly profiles with fractional CNA represent a subset of aggressive tumors.
[Show abstract][Hide abstract] ABSTRACT: Inflammatory hepatocellular adenomas (IHCAs) are benign liver lesions that can be characterized histologically by the presence of an inflammatory infiltrate and at the molecular level by the overexpression of acute phase inflammatory response genes. Recurrent somatic mutations of the interleukin-6 (IL-6) signal transducer (IL6ST) locus, encoding the critical component of the IL-6 signal transduction machinery gp130, are present in 60% of IHCAs and in a subset (2%) of hepatocellular carcinoma (HCCs). By screening of 256 human hepatic adenoma specimens (the largest genetic analysis of IL6ST performed to date in this setting), we identified 24 distinct somatic IL6ST mutations among 66 mutant adenomas. The functional analysis of nine different gp130 mutants expressed in hepatic cancer cell lines consistently revealed the constitutive and IL-6-independent activation of the JAK/STAT signaling pathway. We further demonstrated that the signaling activity of mutant gp130 in IHCA remains responsive to suppressor of cytokine signaling 3 (SOCS3), a physiological gp130 inhibitor. Specifically, cells expressing a double mutant variant of gp130 with a disrupted SOCS3-binding site at residue 759 (Y186/Y759F) displayed a hyperactivation of signal transducer and activator of transcription 3 (STAT3) as compared with cells expressing the endogenous IHCA-associated Y186 gp130 mutant. Notably, we identified that constitutive signaling via gp130 in IHCA requires the Janus kinase family member JAK1, but not JAK2 or tyrosine kinase 2. In support of this notion, AG490, a tyrosine kinase inhibitor that selectively blocks JAK2, had no effect on gp130 activity. In stark contrast, we showed that ruxolitinib, a JAK1/JAK2-selective tyrosine kinase inhibitor used to treat patients with myelofibrosis, dramatically impaired JAK1-STAT signaling downstream of all IHCA-associated gp130 mutants. In conclusion, our findings provide a rationale for the use of JAK1 inhibitors for the treatment of HCAs expressing mutant gp130 as well as a subset of HCCs that bear similar mutations.
[Show abstract][Hide abstract] ABSTRACT: Point mutations of the Kras oncogene induce in cancerous cells an uncontrolled increase of cell proliferation and invasiveness. Mutation of Kras appears early during the process of the pancreatic carcinogenesis and is the most frequent genetic alteration in pancreatic adenocarcinoma (75 to 95 % of cases) as well as in precancerous lesions such as PanIN and IMPN. These latter lesions and tumour microenvironment are reproduced in transgenic models developed in mice. These models are induced on the basis of Kras mutation (Pdx1-Cre ; Kras(G12D) mice) associated or not to the inactivation of tumour suppressor genes (TP53, DPC4, INK4A). Kras mutation assay is easily performed in human biological samples, especially in the cellular material sampled in pancreatic masses under endoscopic ultrasound by fine needle aspiration biopsy. In the near future, searching for Kras mutation could be useful in clinical practice either for positive diagnosis of pancreatic adenocarcinoma in case of unconclusive/doubtful cytopathological analysis or for the differential diagnosis with chronic pancreatitis especially in its pseudotumoural form.
Preview · Article · Nov 2013 · Medecine sciences: M/S
[Show abstract][Hide abstract] ABSTRACT: Patients with colorectal tumors with microsatellite instability MSI) have better prognoses than patients with tumors without MSI, but have a poor response to 5-fluorouracil-based chemotherapy. A dominant-negative form of HSP110 HSP110DE9) expressed by cancer cells with MSI, via exon skipping caused by somatic deletions in the T17 intron repeat, sensitizes the cells to 5-fluorouracil and oxaliplatin. We investigated whether HSP110 T17 could be used to identify patients with colorectal cancer who would benefit from adjuvant chemotherapy with 5-fluorouracil and oxaliplatin.
We characterized the interaction between HSP110 and HSP110DE9 using surface plasmon resonance. Using PCR and fragment analysis, we examined how the size of somatic allelic deletions in HSP110 T17 affected the HSP110 protein expressed by tumor cells. We screened a 329 consecutive patients with stage II-III colorectal tumors with MSI who underwent surgical resection, at tertiary medical centers, for HSP110 T17.
HSP110 and HSP110DE9 interacted in a 1:1 ratio. Tumor cells with large deletions in T17 had increased ratios of HSP110DE9:HSP110, due to the loss of expression of full-length HSP110. Deletions in HSP110 T17 were mostly bi-allelic in primary tumor samples with MSI. Patients with stage II-III cancer who received chemotherapy and had large HSP110 T17 deletions ≥5 bp; 18/77 patients, 23.4%) had longer times of relapse-free survival than patients with small or no deletions ≤4 bp; 59/77 patients, 76.6%) in multivariate analysis hazard ratio, 0.16; 95% confidence interval, 0.012-0.8; P=.03). We found a significant interaction between chemotherapy and T17 deletion P=.009).
About 25% of patients with stage II-III colorectal tumors with MSI have an excellent response to chemotherapy, due to large, bi-allelic deletions in the T17 intron repeat of HSP110 in tumor DNA.
[Show abstract][Hide abstract] ABSTRACT: Colon cancer (CC) pathological staging fails to accurately predict recurrence, and to date, no gene expression signature has proven reliable for prognosis stratification in clinical practice, perhaps because CC is a heterogeneous disease. The aim of this study was to establish a comprehensive molecular classification of CC based on mRNA expression profile analyses.
Fresh-frozen primary tumor samples from a large multicenter cohort of 750 patients with stage I to IV CC who underwent surgery between 1987 and 2007 in seven centers were characterized for common DNA alterations, including BRAF, KRAS, and TP53 mutations, CpG island methylator phenotype, mismatch repair status, and chromosomal instability status, and were screened with whole genome and transcriptome arrays. 566 samples fulfilled RNA quality requirements. Unsupervised consensus hierarchical clustering applied to gene expression data from a discovery subset of 443 CC samples identified six molecular subtypes. These subtypes were associated with distinct clinicopathological characteristics, molecular alterations, specific enrichments of supervised gene expression signatures (stem cell phenotype-like, normal-like, serrated CC phenotype-like), and deregulated signaling pathways. Based on their main biological characteristics, we distinguished a deficient mismatch repair subtype, a KRAS mutant subtype, a cancer stem cell subtype, and three chromosomal instability subtypes, including one associated with down-regulated immune pathways, one with up-regulation of the Wnt pathway, and one displaying a normal-like gene expression profile. The classification was validated in the remaining 123 samples plus an independent set of 1,058 CC samples, including eight public datasets. Furthermore, prognosis was analyzed in the subset of stage II-III CC samples. The subtypes C4 and C6, but not the subtypes C1, C2, C3, and C5, were independently associated with shorter relapse-free survival, even after adjusting for age, sex, stage, and the emerging prognostic classifier Oncotype DX Colon Cancer Assay recurrence score (hazard ratio 1.5, 95% CI 1.1-2.1, p = 0.0097). However, a limitation of this study is that information on tumor grade and number of nodes examined was not available.
We describe the first, to our knowledge, robust transcriptome-based classification of CC that improves the current disease stratification based on clinicopathological variables and common DNA markers. The biological relevance of these subtypes is illustrated by significant differences in prognosis. This analysis provides possibilities for improving prognostic models and therapeutic strategies. In conclusion, we report a new classification of CC into six molecular subtypes that arise through distinct biological pathways. Please see later in the article for the Editors' Summary.