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Background & Aims Colorectal cancers (CRCs) are rare in adolescents and adults ages 25 years or younger. We analyzed clinical, pathology, and molecular features of colorectal tumors from adolescents and young adults in an effort to improve genetic counseling, surveillance, and, ultimately, treatment and outcomes. Methods We analyzed clinical data and molecular and genetic features of colorectal tumor tissues from 139 adolescents or young adults (age, ≤25 y; median age, 23 y; 58% male), collected from 2000 through 2017; tumor tissues and clinical data were obtained from the nationwide network and registry of histopathology and cytopathology and The Netherlands Cancer Registry, respectively. DNA samples from tumors were analyzed for microsatellite instability, mutations in 56 genes, and genome-wide somatic copy number aberrations. Results Mucinous and/or signet ring cell components were observed in 33% of tumor samples. A genetic tumor risk syndrome was confirmed for 39% of cases. Factors associated with shorter survival time included younger age at diagnosis, signet ring cell carcinoma, the absence of a genetic tumor risk syndrome, and diagnosis at an advanced stage of disease. Compared with colorectal tumors from patients ages 60 years or older in the Cancer Genome Atlas, higher proportions of tumors from adolescents or young adults were microsatellite stable with nearly diploid genomes, or contained somatic mutations in TP53 and POLE, whereas lower proportions contained mutations in APC. Conclusions We found clinical, molecular, and genetic features of CRCs in adolescents or young adults to differ from those of patients older than age 60 years. In 39% of patients a genetic tumor risk syndrome was identified. These findings provide insight into the pathogenesis of CRC in young patients and suggest new strategies for clinical management. Performing genetic and molecular analyses for every individual diagnosed with CRC at age 25 years or younger would aid in this optimization.

Contains fulltext : 218296.pdf (Publisher’s version ) (Closed access) Endometrial carcinoma (EC) is traditionally diagnosed by a histopathological assessment of an endometrial biopsy, leaving up to 30% of patients undiagnosed due to technical failure or an inadequate amount of tissue. The aim of the current study is to assess whether mutational analysis of cervical cytology or pipelle endometrial biopsies improves the diagnostic accuracy of traditional histopathological diagnosis of EC. This prospective multicentre cohort study included patients surgically treated for EC or a benign gynaecological condition (control group). A Pap brush sample, cervicovaginal self-sample, pipelle endometrial biopsy and surgical specimen of either the EC or normal endometrium were obtained. A targeted next-generation sequencing panel was used to analyse these samples for mutations in eight genes. Sensitivity, specificity and predictive values were calculated. Fifty-nine EC patients and 31 control patients were included. In these patients, traditional histopathological diagnosis by pipelle had a sensitivity of 79% and a specificity of 100%. For EC patients, 97% of surgical specimens contained at least one mutation. Mutational analysis of Pap brush samples, self-samples and pipelle endometrial biopsies yielded a sensitivity of 78, 67 and 96% with a specificity of 97, 97 and 94%, respectively. Combining one of these three methods with histopathological pipelle endometrial biopsy evaluations yielded a sensitivity of 96, 93 and 96%, respectively. Our study has shown that mutational analysis of either cervical cytology or pipelle endometrial biopsies improves diagnosis of EC. Prospective validation will support implementation in clinical practice. 8 p.

Clear cell odontogenic carcinoma (CCOC) is a rare, low-grade malignant epithelial neoplasm, occurring in the jawbones, mainly affecting the mandible of elderly patients. In addition to hyalinizing clear cell carcinoma of the salivary gland, it is one of the epithelial neoplasms known to harbor an EWSR1-ATF1 fusion. Therefore, a link between these tumors seems plausible. We describe six cases of CCOC showing EWSR1 rearrangements, with two cases being positive for the ATF1 partner gene using FISH analysis. In one case, an EWSR1-CREB1 fusion was identified using RT-PCR, which we report for the first time in this tumor type. The other three cases investigated by FISH were negative for ATF1, CREB1 and CREB3L2. In conclusion, our data show that EWSR1-CREB1 is an alternative fusion gene to EWSR1-ATF1 in CCOC.

Background Sensitive and reliable molecular diagnostics is needed to guide therapeutic decisions for cancer patients. Although less material becomes available for testing, genetic markers are rapidly expanding. Simultaneous detection of predictive markers, including mutations, gene amplifications and MSI, will save valuable material, time and costs. Methods Using a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach, we developed an NGS panel allowing detection of predictive mutations in 33 genes, gene amplifications of 13 genes and microsatellite instability (MSI) by the evaluation of 55 microsatellite markers. The panel was designed to target all clinically relevant single and multiple nucleotide mutations in routinely available lung cancer, colorectal cancer, melanoma, and gastro-intestinal stromal tumor samples, but is useful for a broader set of tumor types. Results The smMIP-based NGS panel was successfully validated and cut-off values were established for reliable gene amplification analysis (i.e. relative coverage ≥3) and MSI detection (≥30% unstable loci). After validation, 728 routine diagnostic tumor samples including a broad range of tumor types were sequenced with sufficient sensitivity (2.4% drop-out), including samples with low DNA input (EGFR, BRAF, MET, ERBB2, KIT, PDGFRA). Amplifications were observed in 5.5% of the samples, comprising clinically relevant amplifications (e.g. MET, ERBB2, FGFR1). 1.5% of the tumor samples were classified as MSI-high, including both MSI-prone and non-MSI-prone tumors. Conclusions We developed a comprehensive workflow for predictive analysis of diagnostic tumor samples. The smMIP-based NGS analysis was shown suitable for limited amounts of histological and cytological material. As smMIP technology allows easy adaptation of panels, this approach can comply with the rapidly expanding molecular markers.

Item does not contain fulltext Benign/low-grade fibroblastic tumors encompass a broad spectrum of tumors with different morphologies and molecular genetic abnormalities. However, despite significant progress in recent genomic characterization, there are still tumors in this histologic spectrum that are difficult to classify, lacking known molecular characteristics. Triggered by a challenging congenital spindle cell neoplasm arising in the heel of a 1-year-old boy, we applied RNA sequencing for genetic discovery and identified a novel EWSR1-SMAD3 gene fusion. On the basis of the index case superficial acral location and fibroblastic appearance with a nonspecific immunophenotype, we searched our files for similar cases and screened them by fluorescence in situ hybridization for these abnormalities. Thus an identical EWSR1-SMAD3 fusion was identified in 2 additional spindle cell tumors with similar clinicopathologic features. Both cases occurred in the feet of adult women (58 and 61 y old) and were characterized by distinctive nodular growth with zonation pattern of peripheral hypercellular areas arranged in short fascicles, transitioning to hypocellular central areas of hyalinization and infarction. Focal stippled calcification in the collagenous area was present in 1 case. All 3 tumors had similar immunoprofiles, being negative for SMA, CD34, CD31, and S100, but showing consistent ERG positivity of uncertain significance. Follow-up information was available in 2 patients who developed local recurrences after incomplete initial excisions, at 5 and 14 months, respectively. None developed metastatic disease. In summary, we report a group of locally recurrent superficial acral tumors, characterized by bland spindle cell fascicular growth, occasional zonation pattern, ERG positivity, and recurrent EWSR1-SMAD3 gene fusions.

Synchronous primary endometrial and ovarian cancers (SEOs) represent 10% of all endometrial and ovarian cancers and are assumed to develop as independent entities. We investigated the clonal relationship between endometrial and ovarian carcinomas in a large cohort classified as SEOs or metastatic disease (MD). The molecular profiles were compared to The Cancer Genome Atlas (TCGA) data to explore primary origin. Subsequently, the molecular profiles were correlated with clinical outcome. To this extent, a retrospective multicenter study was performed comparing patients with SEOs (n = 50), endometrial cancer with synchronous ovarian metastasis (n = 19) and ovarian cancer with synchronous endometrial metastasis (n = 20). Targeted next‐generation sequencing was used, and a clonality index was calculated. Subsequently, cases were classified as POLE mutated, mismatch repair deficient (MMR‐D), TP53‐wild‐type or TP53‐mutated. In 92% of SEOs (46/50), the endometrial and concurrent ovarian carcinoma shared at least one somatic mutation, with a clonality index above 0.95, supporting a clonal origin. The SEO molecular profiles showed striking similarities with the TCGA endometrial carcinoma set. SEOs behaved distinctly different from metastatic disease, with a superior outcome compared to endometrial MD cases (p, What's new? When primary endometrial and ovarian tumors are found simultaneously in the same patient, it has been assumed that they are separate cancers that developed independently. However, in this study, the authors found that these tumors share a clonal origin 92% of the time. They also found that these “synchronous” cancers tend to have a favorable prognosis, with far better outcomes than metastatic disease. Some subgroups, including TP53 mutations and extra‐utero‐ovarian disease, were independent predictors for poor clinical outcome, which may impact adjuvant treatment planning.

Background/Aim: Differentiated vulvar intraepithelial neoplasia (dVIN) and lichen sclerosus (LS) can give rise to vulvar squamous cell carcinoma (VSCC), but genetic evidence is currently still limited. We aimed to determine genetic abnormalities in VSCC and backtrack these abnormalities in the dVIN and LS lesions. Materials and Methods: DNA from VSCC and patient-matched dVIN and LS samples of twelve patients was collected. High-resolution genome-wide copy number analysis was performed and subsequently, we sequenced TP53. Results: Copy number alterations were identified in all VSCC samples. One dVIN lesion presented with three copy number alterations that were preserved in the paired VSCC sample. Targeted sequencing of TP53 identified mutations in five VSCCs. All five mutations were traced back in the dVIN (n=5) or the LS (n=1) with frequencies ranging from 3-19%. Conclusion: Our data provide genetic evidence for a clonal relationship between VSCC and dVIN or LS.

Kaposiform hemangioendothelioma (KHE) is a locally aggressive vascular condition of childhood and is clinicopathologically related to tufted angioma (TA), a benign skin lesion. Due to their rarity molecular data are scarce. We investigated 7 KHE and 3 TA by comprehensive mutational analysis and genome-wide methylation profiling and compared the clustering, also with vascular malformations. Lesions were from 7 females and 3 males. The age range was 2 months to 9 years with a median of 10 months. KHEs arose in the soft tissue of the thigh (n = 2), retroperitoneum (n = 1), thoracal/abdominal (n = 1), supraclavicular (n = 1) and neck (n = 1). One patient presented with multiple lesions without further information. Two patients developed a Kasabach-Merritt phenomenon. TAs originated in the skin of the shoulder (n = 2) and nose/forehead (n = 1). Of the 5 KHEs and 2 TAs investigated by DNA sequencing, one TA showed a hot spot mutation in NRAS, and one KHE a mutation in RAD50. Unsupervised hierarchical clustering analysis indicated a common methylation pattern of KHEs and TAs, which separated from the homogeneous methylation pattern of vascular malformations. In conclusion, methylation profiling provides further evidence for KHEs and TAs potentially forming a spectrum of one entity. Using next generation sequencing, heterogeneous mutations were found in a subset of cases (2/7) without the presence of GNA14 mutations, previously reported in KHE and TA.

Introduction/Background Endometrial carcinoma (EC) is traditionally diagnosed by histopathological assessment of an endometrial biopsy, leaving up to 30% of patients undiagnosed due to technical failure of the biopsy procedure or an inadequate amount of tissue. The aim of the current study is to assess whether mutational analyses of cervical cytology including self-samples or pipelle endometrial biopsies can improve the diagnostic accuracy of traditional histopathological diagnosis of EC. Methodology A prospective multicentre cohort study was performed in three hospitals in the Netherlands. Patients surgically treated for EC or a benign gynecological condition (control group) were included. A Pap brush sample, a cervicovaginal self-sample, a pipelle endometrial biopsy and the surgical specimen of the endometrial carcinoma (endometrial carcinoma group) or normal endometrium (control group) were obtained. A targeted next-generation sequencing panel was used to analyze these samples for mutations in eight genes. Diagnostic accuracy was determined by calculating sensitivity, specificity and predictive values. Results Fifty-nine EC patients and 31 control patients were included. In these patients traditional histopathological diagnosis by pipelle had a sensitivity of 78.9% and a specificity of 100%. For EC patients, 96.6% of surgical specimens contained at least 1 mutation. Mutational analysis of Pap brush samples, self-samples, and pipelle endometrial biopsies yielded a sensitivity of 78.0%, 67.3% and 96.5% with a specificity of 96.8%, 96.8% and 93.5%, respectively. Combining these three methods individually with histopathological pipelle endometrial biopsy evaluations yielded a sensitivity of 95.8%, 93.0% and 96.5, respectively. Conclusion This study has shown that mutational analysis of either cervical cytology, self-samples or pipelle endometrial biopsies improves diagnosis of EC, and is feasible for detection of endometrial pathology. Prospective validation will support implementation in routine clinical practice. Disclosure M.L.: advisory board AstraZeneca, Bayer, Bristol-Myers Squibb, Janssen pharmaceuticals, Roche; sponsored research by Astra Zeneca, Bristol-Myers Squibb, llumina; royalties by Nimagen. Furthermore, no completing interests were declared.

Objectives Endometrial carcinoma (EC) is traditionally diagnosed by histopathological assessment of endometrial biopsies, leaving up to 22% of patients undiagnosed. This study explores the feasibility of the clinical implementation of detecting EC using mutational analysis of cervical cytology. Methods This prospective multicentre study included patients that underwent a hysterectomy for histopathologically proven EC or a benign gynecological condition (control group). A Pap brush sample, cervicovaginal self sample, pipelle and hysterectomy specimen were obtained from each patient. A targeted next-generation sequencing panel was used to screen these samples for mutations in eight genes. Diagnostic accuracy was calculated, including sensitivity, specificity and predictive values. Results Fifty-nine EC patients and 31 control patients were included. In these patients traditional histopathological diagnosis had a sensitivity of 78.9% and a specificity of 100%. For EC patients, 96.6% of surgical specimens contained at least 1 mutation. Blinded mutational analysis of Pap brush samples, self-samples, and pipelle endometrial biopsies yielded a sensitivity of 78.0%, 67.3% and 96.5% with a specificity of 96.8%, 96.8% and 93.5%, respectively. Combining these three methods with histopathological pipelle endometrial biopsies evaluations yielded a sensitivity of 95.8%, 93.0% and 96.5 respectively. Conclusions This study has shown the potential of mutational analysis of cervical cytology and pipelle endometrial biopsies to improve diagnosis of EC, whether or not in additional to traditional histopathological assessment. Prospective evaluation is required for validation and clinical implementation.

Tetraspanin CD37 is predominantly expressed on the cell surface of mature B lymphocytes and is currently being studied as novel therapeutic target for B-cell lymphoma. Recently, we demonstrated that loss of CD37 induces spontaneous B-cell lymphoma in Cd37-knockout mice and correlates with inferior survival in patients with diffuse large B-cell lymphoma (DLBCL). Here, CD37 mutation analysis was performed in a cohort of 137 primary DLBCL samples, including 44 primary immune-privileged site-associated DLBCL (IP-DLBCL) samples originating in the testis or central nervous system. CD37 mutations were exclusively identified in IP-DLBCL cases (10/44, 23%) but absent in non-IP-DLBCL cases. The aberrations included 10 missense mutations, 1 deletion, and 3 splice-site CD37 mutations. Modeling and functional analysis of CD37 missense mutations revealed loss of function by impaired CD37 protein expression at the plasma membrane of human lymphoma B cells. This study provides novel insight into the molecular pathogenesis of IP-DLBCL and indicates that anti-CD37 therapies will be more beneficial for DLBCL patients without CD37 mutations.

Desmoid-type fibromatosis, also called desmoid tumor, is a locally aggressive myofibroblastic neoplasm that usually arises in deep soft tissue with significant potential for local recurrence. It displays an unpredictable clinical course. β-Catenin, the genetic key player of desmoid tumors shows nuclear accumulation due to mutations that prevent its degradation leading to activation of Wnt signaling and myofibroblastic cell proliferation. The corresponding hot spot mutations are located in exon 3 of the CTNNB1 gene or alternatively, in the APC tumor suppressor gene, most often as a germline mutation. Multifocal desmoid tumors are very rare and clinical characteristics are poorly understood. Here we present six sporadic and one familial case of multifocal desmoid tumors. Four female and three male patients, aged between 7 and 30 years (mean 18.4 years) were identified in a cohort of 1392 cases. Tumors were located in (distal) extremities, thorax, breast, abdominal wall, shoulder, and neck. Four cases showed a CTNNB1 mutation and one an APC germline mutation. In two sporadic cases no CTNNB1 mutation was identified. Four patients showed (multiple) recurrences and one patient was lost to follow-up. In conclusion, multifocal desmoid tumors are a very rare disease and may occur in sporadic cases that are characterized by recurrent CTNNB1 mutations. However, the underlying pathogenesis of multifocal desmoid tumors remains poorly understood with often aggressive clinical behavior and challenging therapeutical management.

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Contains fulltext : 193424.pdf (Publisher’s version ) (Open Access) Myositis ossificans is defined as a self-limiting pseudotumor composed of reactive hypercellular fibrous tissue and bone. USP6 rearrangements have been identified as a consistent genetic driving event in aneurysmal bone cyst and nodular fasciitis. It is therefore an integral part of the diagnostic workup when dealing with (myo)fibroblastic lesions of soft tissue and bone. Two cases of myositis ossificans with USP6 rearrangement were published so far. We determine herein the incidence of USP6 rearrangement in myositis ossificans using USP6 fluorescence in situ hybridization analysis (FISH). Of the 11 cases included, seven patients were female and four were male. Age ranged from 6 to 56years (mean 27years). Lesions were located in the thigh (n=5), knee (n=1), lower leg (n=1), lower arm (n=1), perineum (n=1), gluteal (n=1) and thoracic wall (n=1). All assessable cases except one (8/9) showed rearrangement of USP6 providing evidence that myositis ossificans is genetically related to nodular fasciitis and aneurysmal bone cyst.

With the recent introduction of Poly(ADP‐ribose) polymerase inhibitors, a promising novel therapy has become available for ovarian carcinoma (OC) patients with inactivating BRCA1 or BRCA2 mutations in their tumor. To select patients who may benefit from these treatments, assessment of the mutation status of BRCA1 and BRCA2 in the tumor is required. For reliable evaluation of germline and somatic mutations in these genes in DNA derived from formalin‐fixed, paraffin‐embedded (FFPE) tissue, we have developed a single‐molecule molecular inversion probe (smMIP)‐based targeted next‐generation sequencing (NGS) approach. Our smMIP‐based NGS approach provides analysis of both strands of the open reading frame of BRCA1 and BRCA2, enabling the discrimination between real variants and formalin‐induced artefacts. The single molecule tag enables compilation of unique reads leading to a high analytical sensitivity and enabling assessment of the reliability of mutation‐negative results. Multiplex ligation‐dependent probe amplification (MLPA) and Methylation‐specific multiplex ligation‐dependent probe amplification (MS‐MLPA) were used to detect exon deletions of BRCA1 and methylation of the BRCA1 promoter, respectively. Here, we show that this combined approach allows the rapid and reliable detection of both germline and somatic aberrations affecting BRCA1 and BRCA2 in DNA derived from FFPE OCs, enabling improved hereditary cancer risk assessment and clinical treatment of ovarian cancer patients.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Standard treatment of advanced EOC is surgery in combination with chemotherapy, consisting of a platinum-based drug (carboplatin or cisplatin) and paclitaxel. Although most women show a good response to first line treatment, tumors do not respond in 15-20% of patients, whereas in 80% of cases of advanced EOC, the disease recurs within three years. Patients who have responded to first line treatment are referred to as ‘platinum sensitive’, and are treated with a PARP inhibitor in second line. Several other new targeted inhibitors are investigated in clinical trials, including PI3-kinase and bromodomain inhibitors. There is great need of new, more effective therapies with improved long-term treatment outcome, and diagnostic assays and biomarkers to predict chemotherapy response in the clinic. We have determined whether tumor cells isolated from ascites of patients with EOC can be used to develop in vitro cell proliferation assays. We also characterized the immune status of ascites by cytokine analysis and used flow cytometry to analyze the immune cell types in ascites. In a pilot study, ascites was gathered from 18 patients with advance stage EOC. Cells were isolated by centrifugation and tumor cells were separated from immune cells by adhering them to tissue culture plates. Sensitivity of tumor cells to platinum compounds, paclitaxel, PARP inhibitors, and other second line and investigational drugs, as well as drug combinations, was determined in cell proliferation assays, using intracellular ATP content as an indirect read-out of cell number [1]. The mutant status of a number of known oncogenes and tumor suppressor genes in the patient-derived cell samples was determined by DNA sequence analysis. Mutation status was related to histopathological data and in vitro drug response. We demonstrate that ascites from EOC patients contains many tumor cells and that these tumor cells can be used to perform drug sensitivity assays in vitro. In an ongoing study, in which ascites from hundred EOC patients will be included, the in vitro sensitivity to standard-of-care chemotherapy will be related to the immune status of the ascites and to the clinical outcome of the patient, which is defined by platinum sensitivity. An update of this study will be presented at the conference. [1] Uitdehaag et al. (2014) PLoS ONE 9(3): e92146 Citation Format: Guido Zaman, Judith E. den Ouden, Jelle Dylus, Antoon M. van Doornmalen, Rogier C. Buijsman, Astrid Eijkelenboom, Leon F. Massuger, Anne M. van Altena. Chemotherapy sensitivity of tumor cells from ascites of ovarian cancer patients: Relationship with immune status and clinical response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2221.

5036Background: Predictive biomarkers are needed to improve the proportion of pts with mCRPC that may benefit from anti-PD-1/PD-L1 ICB. A comprehensive characterization of both genomic alterations ...