[Contribution of connected prescriptions (NetSIG) for the management of molecular pathology exams]. / Apport de la prescription connectée (NetSIG) dans la prise en charge et la gestion des examens de pathologie moléculaire.

INTRODUCTION: This study describes our experience implementing a connected prescription software (NetSIG, Terascop) for molecular pathology exams. MATERIAL AND METHODS: NetSIG was set up for liquid biopsies and tissue testing. After registration and activation of regional pathology laboratories, NetSIG was implemented for external then internal prescriptions. RESULTS: NetSIG allows users to follow up on all prescriptions on the website, to interact through messages and to consult reports after validation. External set up was quick (3-4 months) and comprehensive (>70%). Prescriptions were made by physicians or more often by secretaries or referring pathologists. Internal prescriptions were made by pathologists then registered in NetSIG by our secretaries. This deployment strategy has resulted in very good completeness of prescriptions (>90%). DISCUSSION AND CONCLUSION: Connected prescriptions made this complex circuit more fluid and facilitated the redistribution of different administrative and technical tasks. The number of phone calls decreased sharply. Half of the prescriptions were made by pathologists and half by oncologists (physicians or secretaries). The mean dearchiving duration for blocks was one day. Mean forwarding of blocks was 2.5 days. Mean turnaround time was 8 days for targeted techniques and 13 days for Next Generation Sequencing. Physicians appreciated the interactivity of the software and the fact that they could consult it on a smartphone.

Durable response of lung carcinoma patients to EGFR tyrosine kinase inhibitors is determined by germline polymorphisms in some immune-related genes.

BACKGROUND: Non-small cell lung cancer is a very poor prognosis disease. Molecular analyses have highlighted several genetic alterations which may be targeted by specific therapies. In clinical practice, progression-free survival on EGFR TKI treatment is between 12 and 14 months. However, some patients progress rapidly in less than 6 months, while others remain free of progression for 16 months or even longer during EGFR TKI treatment. METHODS: We sequenced tumor exomes from 135 lung cancer patients (79 with EGFR-wildtype (WT), 56 with EGFR-mutant tumors) enrolled in the ALCAPONE trial (genomic analysis of lung cancers by next generation sequencing for personalized treatment). RESULTS: Some germline polymorphisms were enriched in the EGFR-mutant subset compared to EGFR-WT tumors or to a reference population. However, the most interesting observation was the negative impact of some germline SNPs in immunity-related genes on survival on EGFR TKI treatment. Indeed, the presence of one of three particular SNPs in the HLA-DRB5 gene was associated with a decreased PFS on EGFR TKI. Moreover, some SNPs in the KIR3DL1 and KIR3DL2 genes were linked to a decrease in both progression-free and overall survival of patients with EGFR-mutant tumors. CONCLUSION: Our data suggest that SNPs in genes expressed by immune cells may influence the response to targeted treatments, such as EGFR TKIs. This indicates that the impact of these cells may not be limited to modulating the response to immunotherapies. Further studies are needed to determine the exact mechanisms underlying this influence and to identify the associated predictive and prognostic markers that would allow to refine treatments and so improve lung cancer patient outcomes. TRIAL REGISTRATION: NCT02281214: NGS Genome Analysis in Personalization of Lung Cancer Treatment (ALCAPONE).

Idylla EGFR assay on extracted DNA: advantages, limits and place in molecular screening according to the latest guidelines for non-small-cell lung cancer (NSCLC) patients.

AIMS: Idylla epidermal growth factor receptor (EGFR) is a fast and fully automated mutation assay that is easy to implement. However, under the Biocartis-recommended technical conditions, tissue sections are directly introduced into the cartridge, at the risk of exhausting the tumour sample. In this study, we evaluate the performance of Idylla EGFR on extracted DNA and discuss its place within the global non-small-cell lung cancer (NSCLC) screening strategy. METHODS: 577 comparative tests between Idylla EGFR on extracted DNA and next-generation sequencing (NGS) were performed across two centres. RESULTS: Preanalytical thresholds were established (20% tumour cell content, 50 ng DNA input) and challenged prospectively in routine practice. 16.8% of samples referred for screening were considered non eligible for Idylla EGFR testing. Due to discordant by design cases, Idylla EGFR sensitivity was 86.9% for currently actionable EGFR mutations. Idylla EGFR specificity was 100% in first-line screening. NGS was always feasible on the same DNA. CONCLUSION: Idylla EGFR on extracted DNA is feasible and enables tumour material to be saved compared with tissue section use. It is not necessary to replace the analytical thresholds of the Biocartis algorithm. Due to both the limits of the mutational repertoire and the high increase of targetable genes in NSCLC, the use of Idylla EGFR should be restricted to clinical emergency situations accompanied by NGS.

Integrative Clinical and DNA Methylation Analyses in a Population-Based Cohort Identifies CDH17 and LRP2 as Risk Recurrence Factors in Stage II Colon Cancer.

Stage II colon cancer (CC), although diagnosed early, accounts for 16% of CC deaths. Predictors of recurrence risk could mitigate this but are currently lacking. By using a DNA methylation-based clinical screening in real-world (n = 383) and in TCGA-derived cohorts of stage II CC (n = 134), we have devised a novel 40 CpG site-based classifier that can segregate stage II CC into four previously undescribed disease sub-classes that are characterised by distinct molecular features, including activation of MYC/E2F-dependant proliferation signatures. By multivariate analyses, hypermethylation of 2 CpG sites at genes CDH17 and LRP2, respectively, was found to independently confer either significantly increased (CDH17; p-value, 0.0203) or reduced (LRP2; p-value, 0.0047) risk of CC recurrence. Functional enrichment and immune cell infiltration analyses, on RNAseq data from the TCGA cohort, revealed cases with hypermethylation at CDH17 to be enriched for KRAS, epithelial-mesenchymal transition and inflammatory functions (via IL2/STAT5), associated with infiltration by 'exhausted' T cells. By contrast, LRP2 hypermethylated cases showed enrichment for mTORC1, DNA repair pathways and activated B cell signatures. These findings will be of value for improving personalised care paths and treatment in stage II CC patients.

Do frozen embryo transfers modify the epigenetic control of imprinted genes and transposable elements in newborns compared with fresh embryo transfers and natural conceptions?

OBJECTIVE: To determine whether the epigenetic control of imprinted genes (IGs) and transposable elements (TEs) differs at birth between fresh or frozen embryo transfers and natural conceptions. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): A total of 202 singleton births were divided into three groups: 84 natural pregnancies (controls), 66 in vitro fertilization/intracytoplasmic sperm injection with fresh embryo transfers, and 52 vitro fertilization/intracytoplasmic sperm injection with frozen embryo transfers. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Pyrosequencing was used to assess the DNA methylation profiles of three IGs (H19/IGF2:IG-DMR [two sequences], KCNQ1OT1:TSS-DMR, and SNURF:TSS-DMR) and two TEs (LINE-1 and HERV-FRD) in cord blood and placenta. The quantitative reverse transcriptase polymerase chain reaction was used to study the transcription of three IGs (H19, KCNQ1, and SNRPN) and two TEs (LINE-1 and ORF2). RESULT(S): After adjustment, the placental DNA methylation levels of H19/IGF2 were lower in the fresh embryo transfer group than in the control (H19/IGF2-seq1) and frozen embryo transfer (H19/IGF2-seq2) groups. The DNA methylation rate for LINE-1 was lower in placentas from the fresh embryo transfer group than in placentas from the control and frozen embryo transfer groups and for HERV-FRD compared with controls. In cord blood, DNA methylation levels were not significantly associated with the mode of conception. The relative expression of LINE-1 and ORF2 was decreased in both cord blood and placental tissues from fresh embryo transfer conceptions compared with natural conceptions and frozen embryo transfer conceptions. CONCLUSION(S): Compared with natural conceptions and frozen embryo transfers, fresh embryo transfers were associated with methylation and/or transcription changes in some TEs and IGs, mostly in placental samples, which could indicate altered placental epigenetic regulation resulting from ovarian stimulation protocols.

c-MYC and p53 expression highlight starry-sky pattern as a favourable prognostic feature in R-CHOP-treated diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogeneous entity, in which the first-line treatment currently consists of an immuno-chemotherapy regimen (R-CHOP). However, around 30% of patients will not respond or will relapse. Overexpression of c-MYC or p53 is frequently found in DLBCL, but an association with prognosis remains controversial, as for other biomarkers previously linked with DLBCL aggressivity (CD5, CD23, or BCL2). The aim of this study was to explore the expression of these biomarkers and their correlation with outcome, clinical, or pathological features in a DLBCL cohort. Immunohistochemical (c-MYC, p53, BCL2, CD5, and CD23), morphological ('starry-sky' pattern [SSP]), targeted gene panel sequencing by next-generation sequencing (NGS), and fluorescence in situ hybridisation analyses were performed on tissue microarray blocks for a retrospective cohort of 94 R-CHOP-treated de novo DLBCL. In univariate analyses, p53 overexpression (p53high ) was associated with unfavourable outcome (p = 0.04) and with c-MYC overexpression (p = 0.01), whereas c-MYC overexpression was linked with an SSP (p = 0.004), but only tended towards an inferior prognosis (p = 0.06). Presence of a starry-sky morphology was found to be correlated with better survival in p53high DLBCL (p = 0.03) and/or c-MYC-positive DLBCL (p = 0.002). Furthermore, NGS data revealed that these three variables were associated with somatic mutations (PIM1, TNFRSF14, FOXO1, and B2M) involved in B-cell proliferation, survival, metabolism, and immune signalling. Taken together, these results show that the SSP pattern seems to be a protective factor in high-risk DLBCL subgroups and highlight cell death as a built-in failsafe mechanism to control tumour growth.

Identification of novel, clonally stable, somatic mutations targeting transcription factors PAX5 and NKX2-3, the epigenetic regulator LRIF1, and BRAF in a case of atypical B-cell chronic lymphocytic leukemia harboring a t(14;18)(q32;q21).

Diagnosis of B-cell chronic lymphocytic leukemia (B-CLL) is usually straightforward, involving clinical, immunophenotypic (Matutes score), and (immuno)genetic analyses (to refine patient prognosis for treatment). CLL cases with atypical presentation (e.g., Matutes ≤ 3) are also encountered, and for these diseases, biology and prognostic impact are less clear. Here we report the genomic characterization of a case of atypical B-CLL in a 70-yr-old male patient; B-CLL cells showed a Matutes score of 3, chromosomal translocation t(14;18)(q32;q21) (BCL2/IGH), mutated IGHV, deletion 17p, and mutations in BCL2, NOTCH1 (subclonal), and TP53 (subclonal). Quite strikingly, a novel PAX5 mutation that was predicted to be loss of function was also seen. Exome sequencing identified, in addition, a potentially actionable BRAF mutation, together with novel somatic mutations affecting the homeobox transcription factor NKX2-3, known to control B-lymphocyte development and homing, and the epigenetic regulator LRIF1, which is implicated in chromatin compaction and gene silencing. Neither NKX2-3 nor LRIF1 mutations, predicted to be loss of function, have previously been reported in B-CLL. Sequencing confirmed the presence of these mutations together with BCL2, NOTCH1, and BRAF mutations, with the t(14;18)(q32;q21) translocation, in the initial diagnostic sample obtained 12 yr prior. This is suggestive of a role for these novel mutations in B-CLL initiation and stable clonal evolution, including upon treatment withdrawal. This case extends the spectrum of atypical B-CLL with t(14;18)(q32;q21) and highlights the value of more global precision genomics for patient follow-up and treatment in these patients.

[Contribution and limits of lean management in the organization and working of a pathology department]. / Apports et limites du lean management dans l'organisation et le fonctionnement d'un service de Pathologie.

INTRODUCTION: In order to validate our strategy of continuous improvement and to identify new ways to increase performance, an evaluation of all the procedures was conducted in our department using the principles of lean management. MATERIAL AND METHODS: Lean-6-sigma methodology (Gemba Walk, Value StreamMapping, spaghetti diagram, Kaizen workshop and priorization matrix) was used to analyze the procedures of the conventional and molecular sectors, and to identify bottlenecks, actions without added value and solutions. RESULTS: The audit identified bottlenecks in pre-analytical (registration), analytical (cytology, immunohistochemistry, sequencing, pathologists) and post-analytical processes (absence of secretaries, delivery of reports by mail). It underlined a suboptimal flow of people and materials, the heavy impact of an increasing work load (8%/year) in reception and microscopy even though we had outsourced, and an often critical work place schedule for technicians which prevent them from achieving tasks without added value (quality control, validation of methods and protocols) or even daily tasks (cutting, immunohistochemistry). After completing the 72 actions aimed at managing overproduction, improving working conditions and developing new activities, turn-around time was partially under control and the automation process was well advanced. DISCUSSION AND CONCLUSION: The audit validated our strategy of continuous improvement and advanced the standardization of our working conditions. Even if the turn-around time for reports was shortened, the audit initiated a positive medical and technical dynamic that should help us to implement the next steps of our reorganization (automation and extension of the department).

Multi-center real-world comparison of the fully automated Idylla™ microsatellite instability assay with routine molecular methods and immunohistochemistry on formalin-fixed paraffin-embedded tissue of colorectal cancer.

Microsatellite instability (MSI) is present in 15-20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla™ MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla™ testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla™ results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla™ MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had ≥ 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla™ MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla™ MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla™ MSI Assay and routine tests.

A case report of Muir-Torre syndrome in a woman with breast cancer and MSI-Low skin squamous cell carcinoma.

BACKGROUND: The tumor spectrum in the Lynch syndrome is well defined, comprising an increased risk of developing colonic and extracolonic malignancies. Muir-Torre syndrome is a variant with a higher risk of skin disease. Patients have been described carrying mutations in the mismatch repair genes and presenting tumors with unusual histology or affected organ not part of the Lynch syndrome spectrum. Hence, the real link between Lynch syndrome, or Muir-Torre syndrome, and these tumors remains difficult to assess. CASE PRESENTATION: We present the case of a 45-year-old-woman, diagnosed with breast cancer at 39 years of age and skin squamous cell carcinoma (SCC) at 41 years of age, without personal history of colorectal cancer. The microsatellite instability analysis performed on the skin SCC showed a low-level of microsatellite instability (MSI-Low). The immunohistochemical expression analysis of the four DNA mismatch repair proteins MLH1, MSH2, MSH6 and PMS2 showed a partial loss of the expression of MSH2 and MSH6 proteins. Germline deletion was found in MSH2 gene (c.1277-? _1661 + ?del), exon 8 to 10. Then, at 45 years of age, she presented hyperplastic polyps of the colon and a sebaceous adenoma. CONCLUSION: Squamous cell carcinomas have been described in Lynch syndrome and Muir-Torre syndrome in two studies and two case reports. This new case further supports a possible relationship between Lynch syndrome and squamous cell carcinoma.

Copy number variations in DCC/18q and ERBB2/17q are associated with disease-free survival in microsatellite stable colon cancer.

We conducted a prospective study to assess the prognostic impact of selected copy number variations (CNVs) in Stage II-III microsatellite stable (MSS) colon cancer. A total of 401 patients were included from 01/2004 to 01/2009. The CNVs in 8 selected target genes, DCC/18q, EGFR/7p, TP53/17p, BLK/8p, MYC/8q, APC/5q, ERBB2/17q and STK6/20q, were detected using a quantitative multiplex polymerase chain reaction of short fluorescent fragment (QMPSF) method. The primary end-point was the impact of the CNVs on the 4-year disease-free survival (DFS). The recurrence rate at 4 years was 20.9%, corresponding to 14% Stage II patients versus 31% Stage III patients (p < 0.0001). The 4-year DFS was significantly decreased in patients with a loss at DCC/18q (p = 0.012) and a gain at ERBB2/17q (p = 0.041). The multivariate analysis demonstrated that Stage III, a loss at DCC/18q and a gain at ERBB2/17q were independent factors associated with DFS. A combination of DCC/18q and ERBB2/17q was also associated with relapse, with the hazard ratio increasing from 1 to 2.4 (95% confidence interval (CI), 1.5-4.1) and 3.1 (95% CI, 1.2-8.4) in the presence of 0, 1 or 2 alterations, respectively (p = 0.0013). CNVs in DCC/18q and ERBB2/17q are significantly associated with DFS in Stage II-III MSS colon cancer.

Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome.

BACKGROUND: Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. METHODS: In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up. RESULTS: Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested-MSP, pyrosequencing, and MS-HRM varied, the prognostic effect seemed similar (HR 1.74, 95 % CI 0.97-3.15; HR 1.85, 95 % CI 0.93-3.86; HR 1.83, 95 % CI 0.92-3.65, respectively). CONCLUSIONS: Our results show that upon optimizing and aligning four RET methylation assays with regard to primer location and sensitivity, differences in methylation frequencies and clinical sensitivities are observed; however, the effect on the marker's prognostic outcome is minimal.

HSP110 T17 simplifies and improves the microsatellite instability testing in patients with colorectal cancer.

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.

CHFR promoter methylation indicates poor prognosis in stage II microsatellite stable colorectal cancer.

PURPOSE: Data on the prognostic significance of promoter CpG island methylation in colorectal cancer (CRC) are conflicting, possibly due to associations between methylation and other factors affecting survival such as genetic alterations and use of adjuvant therapy. Here, we examine the prognostic impact of promoter methylation in patients with CRC treated with surgery alone in the context of microsatellite instability (MSI), BRAF and KRAS mutations. EXPERIMENTAL METHODS: One hundred and seventy-three CRCs were analyzed for promoter methylation of 19 tumor suppressor and DNA repair genes, the CpG island methylator phenotype (CIMP), MSI, the exon 15 V600E BRAF mutation and KRAS codon 12 and 13 mutations. RESULTS: Unsupervised hierarchical clustering based on methylation status of 19 genes revealed three subgroups: cluster 1 [CL1, 57% (98/173) of CRCs], cluster 2 [CL2, 25% (43/173) of CRCs], and cluster 3 [CL3, 18% (32/173) of CRCs]. CL3 had the highest methylation index (0.25, 0.49, and 0.69, respectively, P = <0.01) and was strongly associated with CIMP (P < 0.01). Subgroup analysis for tumor stage, MSI, and BRAF status showed no statistically significant differences in survival between CL1, CL2, and CL3 nor between CIMP and non-CIMP CRCs. Analyzing genes separately revealed that CHFR promoter methylation was associated with a poor prognosis in stage II, microsatellite stability (MSS), BRAF wild-type (WT) CRCs: multivariate Cox proportional HR = 3.89 [95% confidence interval (CI), 1.58-9.60, P < 0.01; n = 66] and HR = 2.11 (95% CI, 0.95-4.69, P = 0.068, n = 136) in a second independent population-based study. CONCLUSIONS: CHFR promoter CpG island methylation, which is associated with MSI, also occurs frequently in MSS CRCs and is a promising prognostic marker in stage II, MSS, BRAF WT CRCs.

Promoter CpG island methylation of RET predicts poor prognosis in stage II colorectal cancer patients.

Improved prognostic stratification of patients with TNM stage II colorectal cancer (CRC) is desired, since 20-30% of high-risk stage II patients may die within five years of diagnosis. This study was conducted to investigate REarranged during Transfection (RET) gene promoter CpG island methylation as a possible prognostic marker for TNM stage II CRC patients. The utility of RET promoter CpG island methylation in tumors of stage II CRC patients as a prognostic biomarker for CRC related death was studied in three independent series (including 233, 231, and 294 TNM stage II patients, respectively) by using MSP and pyrosequencing. The prognostic value of RET promoter CpG island methylation was analyzed by using Cox regression analysis. In the first series, analyzed by MSP, CRC stage II patients (n = 233) with RET methylated tumors had a significantly worse overall survival as compared to those with unmethylated tumors (HRmultivariable = 2.51, 95%-CI: 1.42-4.43). Despite a significant prognostic effect of RET methylation in stage III patients of a second series, analyzed by MSP, the prognostic effect in stage II patients (n = 231) was not statistically significant (HRmultivariable = 1.16, 95%-CI 0.71-1.92). The third series (n = 294), analyzed by pyrosequencing, confirmed a statistically significant association between RET methylation and poor overall survival in stage II patients (HRmultivariable = 1.91, 95%-CI: 1.04-3.53). Our results show that RET promoter CpG island methylation, analyzed by two different techniques, is associated with a poor prognosis in stage II CRC in two independent series and a poor prognosis in stage III CRC in one series. RET methylation may serve as a useful and robust tool for clinical practice to identify high-risk stage II CRC patients with a poor prognosis. This merits further investigation.

Patients with colorectal tumors with microsatellite instability and large deletions in HSP110 T17 have improved response to 5-fluorouracil­based chemotherapy.

BACKGROUND & AIMS: Patients with colorectal tumors with microsatellite instability (MSI) have better prognoses than patients with tumors without MSI, but have a poor response to 5-fluorouracil­based chemotherapy. A dominant-negative form of heat shock protein (HSP)110 (HSP110DE9) expressed by cancer cells with MSI, via exon skipping caused by somatic deletions in the T(17) intron repeat, sensitizes the cells to 5-fluorouracil and oxaliplatin.We investigated whether HSP110 T(17) could be used to identify patients with colorectal cancer who would benefit from adjuvant chemotherapy with 5-fluorouracil and oxaliplatin. METHODS: We characterized the interaction between HSP110 and HSP110DE9 using surface plasmon resonance. By using polymerase chain reaction and fragment analysis, we examined how the size of somatic allelic deletions in HSP110 T(17) affected the HSP110 protein expressed by tumor cells. We screened 329 consecutive patients with stage II­III colorectal tumors with MSI who underwent surgical resection at tertiary medical centers for HSP110 T(17). RESULTS: HSP110 and HSP110DE9 interacted in a1:1 ratio. Tumor cells with large deletions in T(17) had increased ratios of HSP110DE9:HSP110, owing to the loss of expression of full-length HSP110. Deletions in HSP110 T(17) were mostly biallelic in primary tumor samples with MSI. Patients with stage II­III cancer who received chemotherapy and had large HSP110 T(17) deletions (≥5 bp; 18 of 77 patients, 23.4%) had longer times of relapse-free survival than patients with small or no deletions (≤4 bp; 59 of 77 patients, 76.6%) in multivariate analysis (hazard ratio, 0.16; 95% confidence interval, 0.012­0.8; P = .03). We found a significant interaction between chemotherapy and T17 deletion (P =.009). CONCLUSIONS: About 25% of patients with stages II­III colorectal tumors with MSI have an excellent response to chemotherapy, due to large, biallelic deletions in the T(17) intron repeat of HSP110 in tumor DNA.

Diversity of the clinical presentation of the MMR gene biallelic mutations.

Constitutional mismatch repair-deficiency, due to biallelic mutations of MMR genes, results in a tumour spectrum characterized by leukaemias, lymphomas, brain tumours and adenocarcinomas of the gastro-intestinal tract, occurring mostly in childhood. We report here two families illustrating the phenotypic diversity associated with biallelic MMR mutations. In the first family, two siblings developed six malignancies including glioblastoma, lymphoblastic T cell lymphoma, rectal and small bowel adenocarcinoma with onset as early as 6 years of age. We showed that this dramatic clinical presentation was due to the presence of two complex genomic PMS2 deletions in each patient predicted to result into complete PMS2 inactivation. In the second family, the index case presented with an early form of Lynch syndrome with colorectal adenocarcinomas at ages 17 and 20 years, and urinary tract tumours at the age of 25 years. We identified in this patient two MSH6 mutations corresponding to a frameshift deletion and an in frame deletion. The latter was not predicted to result into complete inactivation of MSH6. These reports show that the clinical expression of biallelic MMR mutations depends on the biological impact of the second MMR mutation and that, in clinical practice, the presence of a second MMR mutation located in trans should also be considered in patients suspected to present a Lynch syndrome with an unusual early-onset of tumours.

Development of primary early-onset colorectal cancers due to biallelic mutations of the FANCD1/BRCA2 gene.

Fanconi anaemia (FA) is characterized by progressive bone marrow failure, congenital anomalies, and predisposition to malignancy. In a minority of cases, FA results from biallelic FANCD1/BRCA2 mutations that are associated with early-onset leukaemia and solid tumours. Here, we describe the clinical and molecular features of a remarkable family presenting with multiple primary colorectal cancers (CRCs) without detectable mutations in genes involved in the Mendelian predisposition to CRCs. We unexpectedly identified, despite the absence of clinical cardinal features of FA, a biallelic mutation of the FANCD1/BRCA2 corresponding to a frameshift alteration (c.1845_1846delCT, p.Asn615Lysfs*6) and a missense mutation (c.7802A>G, p.Tyr2601Cys). The diagnosis of FA was confirmed by the chromosomal analysis of lymphocytes. Reverse transcriptase (RT)-PCR analysis revealed that the c.7802A>G BRCA2 variation was in fact a splicing mutation that creates an aberrant splicing donor site and results partly into an aberrant transcript encoding a truncated protein (p.Tyr2601Trpfs*46). The atypical FA phenotype observed within this family was probably explained by the residual amount of BRCA2 with the point mutation c.7802A>G in the patients harbouring the biallelic FANCD1/BRCA2 mutations. Although this report is based in a single family, it suggests that CRCs may be part of the tumour spectrum associated with FANCD1/BRCA2 biallelic mutations and that the presence of such mutations should be considered in families with CRCs, even in the absence of cardinal features of FA.

Why do results conflict regarding the prognostic value of the methylation status in colon cancers? The role of the preservation method.

BACKGROUND: In colorectal carcinoma, extensive gene promoter hypermethylation is called the CpG island methylator phenotype (CIMP). Explaining why studies on CIMP and survival yield conflicting results is essential. Most experiments to measure DNA methylation rely on the sodium bisulfite conversion of unmethylated cytosines into uracils. No study has evaluated the performance of bisulfite conversion and methylation levels from matched cryo-preserved and Formalin-Fixed Paraffin Embedded (FFPE) samples using pyrosequencing. METHODS: Couples of matched cryo-preserved and FFPE samples from 40 colon adenocarcinomas were analyzed. Rates of bisulfite conversion and levels of methylation of LINE-1, MLH1 and MGMT markers were measured. RESULTS: For the reproducibility of bisulfite conversion, the mean of bisulfite-to-bisulfite standard deviation (SD) was 1.3%. The mean of run-to-run SD of PCR/pyrosequencing was 0.9%. Of the 40 DNA couples, only 67.5%, 55.0%, and 57.5% of FFPE DNA were interpretable for LINE-1, MLH1, and MGMT markers, respectively, after the first analysis. On frozen samples the proportion of well converted samples was 95.0%, 97.4% and 87.2% respectively. For DNA showing a total bisulfite conversion, 8 couples (27.6%) for LINE-1, 4 couples (15.4%) for MLH1 and 8 couples (25.8%) for MGMT displayed significant differences in methylation levels. CONCLUSIONS: Frozen samples gave reproducible results for bisulfite conversion and reliable methylation levels. FFPE samples gave unsatisfactory and non reproducible bisulfite conversions leading to random results for methylation levels. The use of FFPE collections to assess DNA methylation by bisulfite methods must not be recommended. This can partly explain the conflicting results on the prognosis of CIMP colon cancers.

Hypermethylator phenotype in sporadic colon cancer: study on a population-based series of 582 cases.

The CpG island methylator phenotype (CIMP) is a distinct phenotype in colorectal cancer, associated with specific clinical, pathologic, and molecular features. However, most of the studies stratified methylation according to two subgroups (CIMP-High versus No-CIMP/CIMP-Low). In our study, we defined three different subgroups of methylation (No-CIMP, CIMP-Low, and CIMP-High) and evaluated the prognostic significance of methylation status on a population-based series of sporadic colon cancers. A total of 582 colon adenocarcinomas were evaluated using methylation-specific PCR for 5 markers (hMLH1, P16, MINT1, MINT2, and MINT31). No-CIMP status was defined as no methylated locus, CIMP-Low status as one to three methylated loci, and CIMP-High status as four or five methylated loci. Clinicopathologic and molecular characteristics were correlated to the methylation status. Crude and relative survival was compared according to methylation status. In the microsatellite-stable (MSS) group, CIMP-High was significantly associated with proximal location (P = 0.011) and BRAF mutation (P < 0.001). KRAS mutations were more associated with CIMP-High and CIMP-Low status (P = 0.008). A shorter 5-year survival was observed in MSS cancer patients with CIMP-Low or CIMP-High status. These results remained significant in multivariate analysis adjusted for age, stage, and BRAF and KRAS mutational status [CIMP-Low: hazard ratio (HR), 1.85; 95% confidence interval (95% CI), 1.37-2.51; CIMP-High, HR, 2.90; 95% CI, 1.53-5.49 compared with No-CIMP]. Within the high-level microsatellite instability group, no difference in survival was observed between the different CIMP groups. Our results show the interest of defining three subgroups of patients according to their methylation status (No-CIMP/CIMP-Low/CIMP-High). Methylation is an independent prognostic factor in MSS colon cancer.