Detection of circulating tumour DNA after neoadjuvant chemoradiotherapy in patients with locally advanced oesophageal cancer.

Active surveillance instead of standard surgery after neoadjuvant chemoradiotherapy (nCRT) has been proposed for patients with oesophageal cancer. Circulating tumour DNA (ctDNA) may be used to facilitate selection of patients for surgery. We show that detection of ctDNA after nCRT seems highly suggestive of major residual disease. Tumour biopsies and blood samples were taken before, and 6 and 12 weeks after, nCRT. Biopsies were analysed with regular targeted next-generation sequencing (NGS). Circulating cell-free DNA (cfDNA) was analysed using targeted NGS with unique molecular identifiers and digital polymerase chain reaction. cfDNA mutations matching pre-treatment biopsy mutations confirmed the presence of ctDNA. In total, 31 patients were included, of whom 24 had a biopsy mutation that was potentially detectable in cfDNA (77%). Pre-treatment ctDNA was detected in nine of 24 patients (38%), four of whom had incurable disease progression before surgery. Pre-treatment ctDNA detection had a sensitivity of 47% (95% CI 24-71) (8/17), specificity of 85% (95% CI 42-99) (6/7), positive predictive value (PPV) of 89% (95% CI 51-99) (8/9), and negative predictive value (NPV) of 40% (95% CI 17-67) (6/15) for detecting major residual disease (>10% residue in the resection specimen or progression before surgery). After nCRT, ctDNA was detected in three patients, two of whom had disease progression. Post-nCRT ctDNA detection had a sensitivity of 21% (95% CI 6-51) (3/14), specificity of 100% (95% CI 56-100) (7/7), PPV of 100% (95% CI 31-100) (3/3), and NPV of 39% (95% CI 18-64) (7/18) for detecting major residual disease. The addition of ctDNA to the current set of diagnostics did not lead to more patients being clinically identified with residual disease. These results indicate that pre-treatment and post-nCRT ctDNA detection may be useful in identifying patients at high risk of disease progression. The addition of ctDNA analysis to the current set of diagnostic modalities may not improve detection of residual disease after nCRT. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Cholangiocarcinoma cell proliferation is enhanced in primary sclerosing cholangitis: A role for IL-17A.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the biliary tree and a risk factor for development of cholangiocarcinoma (CCA). The pathogenesis of PSC-related CCA is largely unclear, although it is assumed that chronic inflammatory environment plays a pivotal role. We aimed to investigate the effect of inflammation-related cytokines in PSC on the proliferation rate of cancer cells. For this, the proliferation index in PSC-CCA and sporadic CCA was determined by Ki-67 immunohistochemistry. The percentage of Ki-67 positivity in cancer cells was significantly higher in PSC-CCA than in sporadic CCA (41.3% ± 5.7% vs 25.8% ± 4.1%; P = .038). To assess which cytokines in the inflammatory environment have the potential to stimulate cancer cell proliferation, patient-derived CCA organoids (CCAOs) were exposed to five cytokines related to PSC (Interleukin (IL)-1ß, IL-6, IL-17A, interferon gamma and tumor necrosis factor alpha). Only IL-17A showed a significant stimulatory effect on cell proliferation in CCAOs, increasing organoid size by 45.9% ± 16.4% (P < .01) and proliferation rate by 38% ± 16% (P < .05). IL-17A immunohistochemistry demonstrated that PSC-CCA might express more IL-17A than sporadic CCA. Moreover, correlation analysis in sporadic CCA and PSC-CCA found a significant correlation between IL-17A expression and proliferation. In conclusion, tumor cell proliferation is increased in PSC-CCA cells compared with sporadic CCA cells. IL-17A increases CCA cell proliferation in vitro and may contribute to the high proliferation rate in PSC-CCA in situ. Therefore, IL-17A represents a new potential therapeutic target in (PSC-)CCA, to be tested in future trials.

Next-generation sequencing mutation analysis on biliary brush cytology for differentiation of benign and malignant strictures in primary sclerosing cholangitis.

BACKGROUND AND AIMS: Differentiation of benign and malignant biliary tract strictures on brush material remains highly challenging but is essential for adequate clinical management of patients with primary sclerosing cholangitis (PSC). In this case-control study, biliary brush cytology samples from PSC patients with cholangiocarcinoma (PSC-CCA) were compared with samples from PSC patients without CCA (PSC-control subjects) using next-generation sequencing (NGS). METHODS: Cells on archived slides were dissected for DNA extraction. NGS was performed using a gene panel containing 242 hotspots in 14 genes. Repeated brush samples from the same patient were analyzed to study the consistency of NGS results. In PSC-CCA cases that underwent surgical resection, molecular aberrations in brush samples were compared with NGS data from subsequent resection specimens. RESULTS: Forty patients (20 PSC-CCA and 20 PSC-control subjects) were included. The gene panel detected 22 mutations in 15 of 20 PSC-CCA brush samples, including mutations in TP53 (8 brush samples), K-ras (5), G-nas (3), ERBB2 (1), APC (1), PIK3CA (1), and SMAD4 (1). One G-nas and 3 K-ras mutations were found in 3 of 20 PSC-control brush samples. The sensitivity of the NGS panel was 75% (95% confidence interval, 62%-80%) and specificity 85% (95% confidence interval, 64%-95%). Repeated brush samples showed identical mutations in 6 of 9 cases. Three repeated brush samples demonstrated additional mutations as compared with the first brush sample. In 6 of 7 patients, mutations in brush samples were identical to mutations in subsequent resection specimens. CONCLUSIONS: NGS mutation analysis of PSC brush cytology detects oncogenic mutations with high sensitivity and specificity and seems to constitute a valuable adjunct to cytologic assessment of brush samples.

Genetic alterations during the neoplastic cascade towards cholangiocarcinoma in primary sclerosing cholangitis.

Carcinogenesis of primary sclerosing cholangitis (PSC)-associated cholangiocarcinoma (CCA) is largely unexplored. Improved understanding of the molecular events involved may guide development of novel avenues for rational clinical management. We aimed to assess the genetic alterations during progression of the neoplastic cascade from biliary dysplasia towards CCA in PSC. Forty-four resection specimens or biopsies of PSC patients with biliary dysplasia (n = 2) and/or CCA (n = 42) were included. DNA was extracted from sections of formalin-fixed paraffin-embedded tissue blocks with dysplasia (n = 23), CCA (n = 69), and nonneoplastic tissue (n = 28). A custom-made next-generation sequencing (NGS) panel of 28 genes was used for mutation and copy number variation (CNV) detection. In addition, CNVs of CDKN2A, EGFR, MCL1, and MYC were examined by fluorescence in situ hybridization. Alterations in 16 low-grade dysplasia samples included loss of FGFR1 (19%), CDKN2A (13%), and SMAD4 (6%), amplification of FGFR3 (6%), EGFR (6%), and ERBB2 (6%), and mutations in SMAD4 (13%). High-grade dysplasia (n = 7) is characterized by MYC amplification (43%), and mutations in ERBB2 (71%) and TP53 (86%). TP53 mutations are the most common aberrations in PSC-CCA (30%), whereas mutations in KRAS (16%), GNAS (14%), and PIK3CA (9%) are also common. In conclusion, PSC-CCA exhibits a variety of genetic alterations during progression of the neoplastic cascade, with mainly CNVs being present early, whereas mutations in ERBB2, TP53, and KRAS appear later in the development of CCA. These findings are promising for the development of NGS-guided diagnostic strategies in PSC-CCA. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Hedgehog pathway mutations are involved in the pathogenesis of plaque-type "trichoblastoma": A report of two cases.

We present two cases of plaque-type trichoblastoma with atypical foci. A rare variant of trichoblastoma is the plaque variant, which is characterized by poor circumscription and locally infiltrative growth pattern. These lesions mostly require multiple stages of Mohs micrographic surgery. Debate still exists whether this variant should be considered as a benign entity or as "low-grade" malignant counterpart of trichoblastoma. In this report we describe two cases of plaque-type trichoblastoma with atypical foci, which harbored somatic mutations in the Hedgehog pathway, thus should be acknowledged as intermediate malignancies. In addition, extensive molecular workup of both the trichoblastic and atypical component in sequential lesions in the same patient was performed.

In-depth molecular analysis of combined and co-primary pulmonary large cell neuroendocrine carcinoma and adenocarcinoma.

Up to 14% of large cell neuroendocrine carcinomas (LCNECs) are diagnosed in continuity with nonsmall cell lung carcinoma. In addition to these combined lesions, 1% to 7% of lung tumors present as co-primary tumors with multiple synchronous lesions. We evaluated molecular and clinicopathological characteristics of combined and co-primary LCNEC-adenocarcinoma (ADC) tumors. Ten patients with LCNEC-ADC (combined) and five patients with multiple synchronous ipsilateral LCNEC and ADC tumors (co-primary) were included. DNA was isolated from distinct tumor parts, and 65 cancer genes were analyzed by next generation sequencing. Immunohistochemistry was performed including neuroendocrine markers, pRb, Ascl1 and Rest. Pure ADC (N = 37) and LCNEC (N = 17) cases were used for reference. At least 1 shared mutation, indicating tumor clonality, was found in LCNEC- and ADC-parts of 10/10 combined tumors but only in 1/5 co-primary tumors. A range of identical mutations was observed in both parts of combined tumors: 8/10 contained ADC-related (EGFR/KRAS/STK11 and/or KEAP1), 4/10 RB1 and 9/10 TP53 mutations. Loss of pRb IHC was observed in 6/10 LCNEC- and 4/10 ADC-parts. The number and intensity of expression of Ascl1 and neuroendocrine markers increased from pure ADC (low) to combined ADC (intermediate) and combined and pure LCNEC (high). The opposite was true for Rest expression. In conclusion, all combined LCNEC-ADC tumors were clonally related indicating a common origin. A relatively high frequency of pRb inactivation was observed in both LCNEC- and ADC-parts, suggesting an underlying role in LCNEC-ADC development. Furthermore, neuroendocrine differentiation might be modulated by Ascl1(+) and Rest(-) expression.

Personalised selection of experimental treatment in patients with advanced solid cancer is feasible using whole-genome sequencing.

BACKGROUND: Biomarker-guided therapy in an experimental setting has been suggested to improve patient outcomes. However, trial-specific pre-screening tests are time and tissue consuming and complicate the personalised treatment of patients eligible for early-phase clinical trials. In this study the feasibility of whole-genome sequencing (WGS) as a one-test-for-all for guided inclusion in early-phase trials was investigated. METHODS: Phase I Molecular Tumor Board (MTB) at the Erasmus MC Cancer Institute reviewed patients with advanced cancer without standard-of-care treatment (SOC) options for a 'fresh-frozen' (FF) tumour biopsy for WGS based on clinical-pathological features. Clinical grade WGS was performed by Hartwig Medical Foundation. MTB matched the patient with a trial, if available. RESULTS: From September 2019-March 2021, 31 patients with highly diverse tumour types underwent a tumour biopsy for WGS. The median turnaround time (TAT) was 15 days [10-42 days]. At least one actionable event was found in 84% of the patients (26/31). One-third of the patients (11/31) received matched experimental treatment. CONCLUSIONS: WGS on fresh FF biopsies is a feasible tool for the selection of personalised experimental therapy in patients with advanced cancer without SOC options. WGS is now possible in an acceptable TAT and thus could fulfil the role of a universal genomic pre-screening test.

Universal Immunohistochemistry for Lynch Syndrome: A Systematic Review and Meta-analysis of 58,580 Colorectal Carcinomas.

BACKGROUND & AIMS: Lynch syndrome is a form of hereditary colorectal cancer (CRC) caused by pathogenic germline variants (PV) in DNA mismatch repair (MMR) genes. Currently, many Western countries perform universal immunohistochemistry testing on CRC to increase the identification of Lynch syndrome patients and their relatives. For a clear understanding of health benefits and costs, data on its outcomes are required: proportions of Lynch syndrome, sporadic MMR-deficient (MMRd) cases, and unexplained MMRd cases. METHODS: Ovid Medline, Embase, and Cochrane CENTRAL were searched for studies reporting on universal MMR immunohistochemistry, followed by MMR germline analysis, until March 20, 2020. Proportions were calculated, subgroup analyses were performed based on age and diagnostics used, and random effects meta-analyses were conducted. Quality was assessed using the Joanna Briggs Critical Appraisal Tool for Prevalence Studies. RESULTS: Of 2723 identified articles, 56 studies covering 58,580 CRCs were included. In 6.22% (95% CI, 5.08%-7.61%; I2 = 96%) MMRd was identified. MMR germline PV was present in 2.00% (95% CI, 1.59%-2.50%; I2 = 92%), ranging from 1.80% to 7.27% based on completeness of diagnostics and age restriction. Immunohistochemistry outcomes were missing in 11.81%, and germline testing was performed in 76.30% of eligible patients. In 7 studies, including 6848 CRCs completing all diagnostic stages, germline PV and biallelic somatic MMR inactivation were found in 3.01% and 1.75%, respectively; 0.61% remained unexplained MMRd. CONCLUSIONS: Age, completeness, and type of diagnostics affect the percentage of MMR PV and unexplained MMRd percentages. Complete diagnostics explain almost all MMRd CRCs, reducing the amount of subsequent multigene panel testing. This contributes to optimizing testing and surveillance in MMRd CRC patients and relatives.

The clonal relation of primary upper urinary tract urothelial carcinoma and paired urothelial carcinoma of the bladder.

The risk of developing urothelial carcinoma of the bladder (UCB) in patients treated by radical nephroureterectomy (RNU) for an upper urinary tract urothelial carcinoma (UTUC) is 22% to 47% in the 2 years after surgery. Subject of debate remains whether UTUC and the subsequent UCB are clonally related or represent separate origins. To investigate the clonal relationship between both entities, we performed targeted DNA sequencing of a panel of 41 genes on matched normal and tumor tissue of 15 primary UTUC patients treated by RNU who later developed 19 UCBs. Based on the detected tumor-specific DNA aberrations, the paired UTUC and UCB(s) of 11 patients (73.3%) showed a clonal relation, whereas in four patients the molecular results did not indicate a clear clonal relationship. Our results support the hypothesis that UCBs following a primary surgically resected UTUC are predominantly clonally derived recurrences and not separate entities.

Multicenter Comparison of Molecular Tumor Boards in The Netherlands: Definition, Composition, Methods, and Targeted Therapy Recommendations.

BACKGROUND: Molecular tumor boards (MTBs) provide rational, genomics-driven, patient-tailored treatment recommendations. Worldwide, MTBs differ in terms of scope, composition, methods, and recommendations. This study aimed to assess differences in methods and agreement in treatment recommendations among MTBs from tertiary cancer referral centers in The Netherlands. MATERIALS AND METHODS: MTBs from all tertiary cancer referral centers in The Netherlands were invited to participate. A survey assessing scope, value, logistics, composition, decision-making method, reporting, and registration of the MTBs was completed through on-site interviews with members from each MTB. Targeted therapy recommendations were compared using 10 anonymized cases. Participating MTBs were asked to provide a treatment recommendation in accordance with their own methods. Agreement was based on which molecular alteration(s) was considered actionable with the next line of targeted therapy. RESULTS: Interviews with 24 members of eight MTBs revealed that all participating MTBs focused on rare or complex mutational cancer profiles, operated independently of cancer type-specific multidisciplinary teams, and consisted of at least (thoracic and/or medical) oncologists, pathologists, and clinical scientists in molecular pathology. Differences were the types of cancer discussed and the methods used to achieve a recommendation. Nevertheless, agreement among MTB recommendations, based on identified actionable molecular alteration(s), was high for the 10 evaluated cases (86%). CONCLUSION: MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational cancer profiles. We propose a "Dutch MTB model" for an optimal, collaborative, and nationally aligned MTB workflow. IMPLICATIONS FOR PRACTICE: Interpretation of genomic analyses for optimal choice of target therapy for patients with cancer is becoming increasingly complex. A molecular tumor board (MTB) supports oncologists in rationalizing therapy options. However, there is no consensus on the most optimal setup for an MTB, which can affect the quality of recommendations. This study reveals that the eight MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational profiles. The Dutch MTB model is based on a collaborative and nationally aligned workflow with interinstitutional collaboration and data sharing.

Master transcription factors form interconnected circuitry and orchestrate transcriptional networks in oesophageal adenocarcinoma.

OBJECTIVE: While oesophageal squamous cell carcinoma remains infrequent in Western populations, the incidence of oesophageal adenocarcinoma (EAC) has increased sixfold to eightfold over the past four decades. We aimed to characterise oesophageal cancer-specific and subtypes-specific gene regulation patterns and their upstream transcription factors (TFs). DESIGN: To identify regulatory elements, we profiled fresh-frozen oesophageal normal samples, tumours and cell lines with chromatin immunoprecipitation sequencing (ChIP-Seq). Mathematical modelling was performed to establish (super)-enhancers landscapes and interconnected transcriptional circuitry formed by master TFs. Coregulation and cooperation between master TFs were investigated by ChIP-Seq, circularised chromosome conformation capture sequencing and luciferase assay. Biological functions of candidate factors were evaluated both in vitro and in vivo. RESULTS: We found widespread and pervasive alterations of the (super)-enhancer reservoir in both subtypes of oesophageal cancer, leading to transcriptional activation of a myriad of novel oncogenes and signalling pathways, some of which may be exploited pharmacologically (eg, leukemia inhibitory factor (LIF) pathway). Focusing on EAC, we bioinformatically reconstructed and functionally validated an interconnected circuitry formed by four master TFs-ELF3, KLF5, GATA6 and EHF-which promoted each other's expression by interacting with each super-enhancer. Downstream, these master TFs occupied almost all EAC super-enhancers and cooperatively orchestrated EAC transcriptome. Each TF within the transcriptional circuitry was highly and specifically expressed in EAC and functionally promoted EAC cell proliferation and survival. CONCLUSIONS: By establishing cancer-specific and subtype-specific features of the EAC epigenome, our findings promise to transform understanding of the transcriptional dysregulation and addiction of EAC, while providing molecular clues to develop novel therapeutic modalities against this malignancy.

Molecular and pathological basis of HPV-negative cervical adenocarcinoma seen in a global study.

International surveys find HPV-negativity in up to 30% of cervical adenocarcinomas. We investigated the pathological diagnosis by expert consensus with immunohistochemistry and the presence of somatic mutations in recognised tumour genes in HPV-positive and negative cervical adenocarcinomas (CADC). A sample was selected of 45 paraffin-embedded cervical blocks diagnosed locally as usual cervical adenocarcinoma from a global study. These represented different diagnoses made at previous diagnostic review and HPV status. All were suitable for analysis for somatic tumour associated gene mutations. Three pathologists examined H/E slides and immunohistochemistry for p16, progesterone receptor and p53 and classified the cases. L1 genes from high-risk HPVs and low-risk HPVs were analysed by SPF10 PCR-DEIA-LiPA25 version 1 in whole tissue sections and microdissected tumour and retested by PCR for E6/E7 genes of hrHPVs if negative. Cases were analysed for microsatellite instability and next-generation sequencing mutation analysis. From the 45 cases, 20 cases of usual CADC were confirmed of which 17 (85%) were HPV-positive in tumour cells. The other 25 cases were reclassified as endometrial, serous, clear-cell and gastric-type adenocarcinomas and all were HPV-negative in tumour cells. Careful retesting for HPV DNA and IHC leads to more accurate identification of HPV-positive usual cervical adenocarcinomas. Endometrioid endometrial adenocarcinomas, other uterine adenocarcinoma with multiple somatic mutations were important in misclassification of HPV-negative cases locally managed as cervical adenocarcinoma, as was gastric-type adenocarcinoma with germline STK11 mutation in East Asia. Few consensuses confirmed HPV-negative usual cervical adenocarcinomas showed somatic tumorigenic mutations also seen in some HPV-positive usual CADC.

Impact of panel design and cut-off on tumour mutational burden assessment in metastatic solid tumour samples.

Tumour mutational burden (TMB) has emerged as a promising biomarker to predict immune checkpoint inhibitors (ICIs) response in advanced solid cancers. However, harmonisation of TMB reporting by targeted gene panels is lacking, especially in metastatic tumour samples. To address this issue, we used data of 2841 whole-genome sequenced metastatic cancer biopsies to perform an in silico analysis of TMB determined by seven gene panels (FD1CDx, MSK-IMPACT™, Caris Molecular Intelligence, Tempus xT, Oncomine Tumour Mutation Load, NeoTYPE Discovery Profile and CANCERPLEX) compared to exome-based TMB as a golden standard. Misclassification rates declined from up to 30% to <1% when the cut-point for high TMB was increased. Receiver operating characteristic analysis demonstrated that, for correct classification, the cut-point for each gene panel may vary more than 20%. In conclusion, we here demonstrate that a major limitation for the use of gene panels is inter-assay variation and the need for dynamic thresholds to compare TMB outcomes.

Yield of Lynch Syndrome Surveillance for Patients With Pathogenic Variants in DNA Mismatch Repair Genes.

BACKGROUND & AIMS: Patients with Lynch syndrome are offered the same colorectal cancer (CRC) surveillance programs (colonoscopy every 2 years), regardless of the pathogenic DNA mismatch repair gene variant the patient carries. We aimed to assess the yield of surveillance for patients with these variants in MLH1, MSH2, MSH6, and PMS2. METHODS: We analyzed data on colonoscopy surveillance, including histopathology analysis, from all patients diagnosed with Lynch syndrome (n = 264) at a single center. We compared the development of (advanced) adenomas and CRC among patients with pathogenic variants in the DNA mismatch repair genes MLH1 (n = 55), MSH2 (n = 44), MSH6 (n = 143), or PMS2 (n = 22) over 1836 years of follow-up (median follow-up of 6 years per patient). RESULTS: At first colonoscopy, CRC was found in 8 patients. During 916 follow-up colonoscopies, CRC was found in 9 patients. No CRC was found in patients with variants in MSH6 or PMS2 over the entire follow-up period. There were no significant differences in the number of colonoscopies with adenomas or advanced adenomas among the groups. The median time of adenoma development was 3 years (IQR, 2-6 years). There were no significant differences in time to development of adenoma. However, patients with variants in MSH6 had a significant longer time to development of advanced neoplasia (advanced adenoma or CRC) than patients in the other groups. Six carriers died during follow up (5 from cancer, of which 3 from pancreatic cancer). CONCLUSIONS: No CRC was found during follow-up of patients with Lynch syndrome carrying pathogenic variants in MSH6; advanced neoplasia developed over shorter follow-up time periods in patients with pathogenic variants in MLH1 or MSH2. The colonoscopy interval for patients with pathogenic variants in MSH6 might be increased to 3 years from the regular 2-year interval.

Routine Molecular Analysis for Lynch Syndrome Among Adenomas or Colorectal Cancer Within a National Screening Program.

BACKGROUND & AIMS: It is important to identify individuals with Lynch syndrome because surveillance programs can reduce their morbidity and mortality from colorectal cancer (CRC). We assessed the diagnostic yield of immunohistochemistry to detect Lynch syndrome in patients with advanced and multiple adenomas within our national CRC screening program. METHODS: We performed a prospective study of all participants (n = 1101; 55% male; median age, 66 years; interquartile range, 61-70 years) referred to the Erasmus MC in The Netherlands after a positive result from a fecal immunohistochemical test, from December 2013 to December 2016. Colon tissues were collected from patients with advanced adenomas, ≥4 nonadvanced adenomas, or CRC, and analyzed by immunohistochemistry to identify patients with loss of mismatch repair (MMR) proteins (MLH1, MSH2, MSH6, or PMS2): a marker of Lynch syndrome. Specimens from patients with loss of MLH1 were analyzed for MLH1 promoter hypermethylation. Patients with an MMR-deficient tumor or adenoma without MLH1 promoter hypermethylation were referred for genetic analysis. RESULTS: At colonoscopy, 456 patients (41%) (65% male; mean age, 67 years; interquartile range, 63-71 years) were found to have CRC and/or an adenoma eligible for analysis by immunohistochemistry. Of 56 CRCs, 7 (13%) had lost an MMR protein and 5 had hypermethylation of the MLH1 promoter. Analyses of tumor DNA revealed that 2 patients without MLH1 promoter hypermethylation had developed sporadic tumors. In total, 400 patients with adenomas were analyzed. Of the examined adenomas, 208 (52%) had a villous component and/or high-grade dysplasia: 186 (47%) had a villous component and 41 (10%) had high-grade dysplasia. Only 1 adenoma had lost an MMR protein. This adenoma was found to have 2 somatic mutations in MSH6. CONCLUSIONS: In a CRC screening program in The Netherlands for individuals aged 55 to 75 years, routine screening for Lynch syndrome by immunohistochemistry analysis of colon tissues from patients with advanced and multiple adenomas identified no individuals with this genetic disorder.

Double somatic mutations in mismatch repair genes are frequent in colorectal cancer after Hodgkin's lymphoma treatment.

OBJECTIVE: Hodgkin's lymphoma survivors who were treated with infradiaphragmatic radiotherapy or procarbazine-containing chemotherapy have a fivefold increased risk of developing colorectal cancer (CRC). This study aims to provide insight into the development of therapy-related CRC (t-CRC) by evaluating histopathological and molecular characteristics. DESIGN: 54 t-CRCs diagnosed in a Hodgkin's lymphoma survivor cohort were analysed for mismatch repair (MMR) proteins by immunohistochemistry, microsatellite instability (MSI) and KRAS/BRAF mutations. MSI t-CRCs were evaluated for promoter methylation and mutations in MMR genes. Pathogenicity of MMR gene mutations was evaluated by in silico predictions and functional analyses. Frequencies were compared with a general population cohort of CRC (n=1111). RESULTS: KRAS and BRAF mutations were present in 41% and 15% t-CRCs, respectively. Compared with CRCs in the general population, t-CRCs had a higher MSI frequency (24% vs 11%, p=0.003) and more frequent loss of MSH2/MSH6 staining (13% vs 1%, p<0.001). Loss of MLH1/PMS2 staining and MLH1 promoter methylation were equally common in t-CRCs and the general population. In MSI CRCs without MLH1 promoter methylation, double somatic MMR gene mutations (or loss of heterozygosity as second hit) were detected in 7/10 (70%) t-CRCs and 8/36 (22%) CRCs in the general population (p=0.008). These MMR gene mutations in t-CRCs were classified as pathogenic. MSI t-CRC cases could not be ascribed to Lynch syndrome. CONCLUSIONS: We have demonstrated a higher frequency of MSI among t-CRCs, which results from somatic MMR gene mutations. This suggests a novel association of somatic MMR gene mutations with prior anticancer treatment.

Interim results from the CATNON trial (EORTC study 26053-22054) of treatment with concurrent and adjuvant temozolomide for 1p/19q non-co-deleted anaplastic glioma: a phase 3, randomised, open-label intergroup study.

BACKGROUND: The role of temozolomide chemotherapy in newly diagnosed 1p/19q non-co-deleted anaplastic gliomas, which are associated with lower sensitivity to chemotherapy and worse prognosis than 1p/19q co-deleted tumours, is unclear. We assessed the use of radiotherapy with concurrent and adjuvant temozolomide in adults with non-co-deleted anaplastic gliomas. METHODS: This was a phase 3, randomised, open-label study with a 2 × 2 factorial design. Eligible patients were aged 18 years or older and had newly diagnosed non-co-deleted anaplastic glioma with WHO performance status scores of 0-2. The randomisation schedule was generated with the electronic EORTC web-based ORTA system. Patients were assigned in equal numbers (1:1:1:1), using the minimisation technique, to receive radiotherapy (59·4 Gy in 33 fractions of 1·8 Gy) alone or with adjuvant temozolomide (12 4-week cycles of 150-200 mg/m2 temozolomide given on days 1-5); or to receive radiotherapy with concurrent temozolomide 75 mg/m2 per day, with or without adjuvant temozolomide. The primary endpoint was overall survival adjusted for performance status score, age, 1p loss of heterozygosity, presence of oligodendroglial elements, and MGMT promoter methylation status, analysed by intention to treat. We did a planned interim analysis after 219 (41%) deaths had occurred to test the null hypothesis of no efficacy (threshold for rejection p<0·0084). This trial is registered with ClinicalTrials.gov, number NCT00626990. FINDINGS: At the time of the interim analysis, 745 (99%) of the planned 748 patients had been enrolled. The hazard ratio for overall survival with use of adjuvant temozolomide was 0·65 (99·145% CI 0·45-0·93). Overall survival at 5 years was 55·9% (95% CI 47·2-63·8) with and 44·1% (36·3-51·6) without adjuvant temozolomide. Grade 3-4 adverse events were seen in 8-12% of 549 patients assigned temozolomide, and were mainly haematological and reversible. INTERPRETATION: Adjuvant temozolomide chemotherapy was associated with a significant survival benefit in patients with newly diagnosed non-co-deleted anaplastic glioma. Further analysis of the role of concurrent temozolomide treatment and molecular factors is needed. FUNDING: Schering Plough and MSD.

Distinct Patterns of Somatic Mosaicism in the APC Gene in Neoplasms From Patients With Unexplained Adenomatous Polyposis.

We investigated the presence and patterns of mosaicism in the APC gene in patients with colon neoplasms not associated with any other genetic variants; we performed deep sequence analysis of APC in at least 2 adenomas or carcinomas per patient. We identified mosaic variants in APC in adenomas from 9 of the 18 patients with 21 to approximately 100 adenomas. Mosaic variants of APC were variably detected in leukocyte DNA and/or non-neoplastic intestinal mucosa of these patients. In a comprehensive sequence analysis of 1 patient, we found no evidence for mosaicism in APC in non-neoplastic intestinal mucosa. One patient was found to carry a mosaic c.4666dupA APC variant in only 10 of 16 adenomas, indicating the importance of screening 2 or more adenomas for genetic variants.

Prognostic relevance of mutations and copy number alterations assessed with targeted next generation sequencing in IDH mutant grade II glioma.

BACKGROUND: At current prognostication of low grade glioma remains suboptimal and might be improved with additional markers. These may guide treatment decisions, in particular on early adjuvant therapy versus wait and see after surgery. METHODS: We used a targeted Next-Generation Sequencing panel to assess mutational and copy number status of selected genes and chromosomes in a consecutive series of adult grade II supratentorial glioma, and assessed the impact of molecular markers of interest on overall survival. RESULTS: 207 IDH mutated grade II glioma samples were analyzed with a median follow-up of 6.9 years. Loss of region 9p21.3 did not show a correlation with outcome in IDH mutated 1p/19q-codeleted oligodendroglioma or IDH mutated astrocytoma. We found a significant shorter overall survival with univariable analysis in IDH mutated astrocytoma patients with trisomy of chromosome 7 (Log rank P = 0.044) and in IDH mutated 1p/19q-codeleted oligodendroglioma patients with a PTEN mutation (Log rank P = 0.033). We could not validate these findings in multivariate analysis or in the TCGA dataset. CONCLUSIONS: Loss of 9p21.3 is not associated with outcome in a molecularly defined cohort of grade II glioma and therefore it remains unclear if loss of 9p21.3 can be used as additional marker of anaplasia or to guide treatment decisions. Trisomy of chromosome 7 in IDH mutated astrocytoma and PTEN mutations in IDH mutated oligodendroglioma are potential markers of poor prognosis, but require confirmation in larger series.

A novel tissue-based ß-catenin gene and immunohistochemical analysis to exclude familial adenomatous polyposis among children with hepatoblastoma tumors.

BACKGROUND: The Wnt/ß-catenin pathway plays a central role in the pathogenesis of most hepatoblastomas (HBs), that is, up to 60-80% carry activating CTNNB1 mutations. HBs can however also be the first manifestation of familial adenomatous polyposis (FAP). As this is a severe disease, it is important for the patient and related family members to firmly exclude FAP at an early stage. Current diagnosis largely depends on APC germline mutation detection on genomic DNA, which is associated with 10-20% false-negative results. Here, we establish and validate a tissue-based ß-catenin gene and immunohistochemical analysis, which complements germline mutation screening to exclude the diagnosis of FAP among HB patients. METHODS: Tumor tissues of 18 HB patients, including three FAP cases were subjected to CTNNB1 exon 3 mutational analysis and immunohistochemistry comparing staining patterns for total and exon 3 specific ß-catenin antibodies. RESULTS: Our novel tissue-based method reliably identified all three FAP patients. Their tumors were characterized by a wild-type exon 3 sequence and a comparable nuclear staining for both antibodies. In contrast, the non-FAP tumors carried missense CTNNB1 mutations combined with a clearly reduced staining for the exon 3 antibody, or complete loss of staining in case of lesions with exon 3 deletions. CONCLUSION: We have successfully established and validated a novel ß-catenin gene and immunohistochemical diagnostic method, which, when combined with routine germline DNA testing, allows the exclusion of the diagnosis of FAP among HB patients.