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Introduction: RET gene fusions are established oncogenic drivers in 1% of NSCLC. Accurate detection of advanced patients with RET fusions is essential to ensure optimal therapy choice. We investigated the performance of fluorescence in situ hybridization (FISH) as a diagnostic test for detecting functional RET fusions.Methods: Between January 2016 and November 2019, a total of 4873 patients with NSCLC were routinely screened for RET fusions using either FISH (n = 2858) or targeted RNA next-generation sequencing (NGS) (n = 2015). If sufficient material was available, positive cases were analyzed by both methods (n = 39) and multiple FISH assays (n = 17). In an independent cohort of 520 patients with NSCLC, whole-genome sequencing data were investigated for disruptive structural variations and functional fusions in the RET and compared with ALK and ROS1 loci.Results: FISH analysis revealed RET rearrangement in 48 of 2858 cases; of 30 rearranged cases double tested with NGS, only nine had a functional RET fusion. RNA NGS yielded RET fusions in 14 of 2015 cases; all nine cases double tested by FISH had RET locus rearrangement. Of these 18 verified RET fusion cases, 16 had a split signal and two a complex rearrangement by FISH. By whole-genome sequencing, the prevalence of functional fusions compared with all disruptive events was lower in the RET (4 of 9, 44%) than the ALK (27 of 34, 79%) and ROS1 (9 of 12, 75%) loci.Conclusions: FISH is a sensitive but unspecific technique for RET screening, always requiring a confirmation using an orthogonal technique, owing to frequently occurring RET rearrangements not resulting in functional fusions in NSCLC. (C) 2021 International Association for the Study of Lung Cancer. Published by Elsevier Inc.

Primary high-risk human papillomavirus (hrHPV) DNA testing has been introduced in several countries worldwide, including The Netherlands. The objective of this study was to compare three automated workflow procedures for hrHPV testing of which the hrHPV detection assays meet the international guidelines for HPV testing. To mimic a realistic screening situation, we aimed to process 15 000 residual PreservCyt cervical samples in a period of 3 months. During a 3 months period, four technicians were involved in processing 5000 specimens per month on three automated platforms, (1) Qiagen Digene HC2 HPV DNA test (HC2, signal amplification); (2) Roche Cobas HPV test (DNA amplification), and (3) Hologic Aptima HPV test (RNA amplification). We measured and scored general aspects (time-to-results, hands-on-time (HOT)), maintenance, pre-run, run and post-run aspects, inventory (orders, storage), and number of errors on a scale from 1 to 10. As determined for one complete workflow each, maximum processing capacity and HOT were 296 samples and 2 h:55 m, 282 samples and 3 h:20 m, and 264 samples and 4 h:15 m for Aptima, Cobas, and HC2, respectively. The mean throughput time per run was 5 h:51 m for Cobas in which 94 samples could be processed. For Aptima, the mean throughput time per run was 6 h:30 m for 60 samples. Mean throughput time for HC2 is longer since results were provided on day 2. In this study, the fully automated Aptima workflow scores best with a 7.2, followed by Cobas with a score of 7.1 and HC2 with a score of 5.8. Although all HPV tests used in this comparison meet the international test guidelines, the performance (workflow) characteristics of the assays vary widely. A specific choice of a laboratory for high-throughput testing can be different based on the laboratory’s demands, but also hands-on-time, time-to-results/ # samples, maintenance, pre-run, run and post-run parameters, consumables, technical support, and number of errors are important operational factors for the selection of a fully automated workflow for hrHPV testing.

Objectives The oncogenic MET exon 14 skipping mutation (METex14del) is described to drive 1.3 %–5.7 % of non-small-cell lung cancer (NSCLC) and multiple studies with cMET inhibitors show promising clinical responses. RNA-based analysis seems most optimal for METex14del detection, however, acquiring sufficient RNA material is often problematic. An alternative is DNA-based analysis, but commercially available DNA-based panels only detect up to 63 % of known METex14del alterations. The goal of this study is to describe an optimized DNA-based diagnostic test for METex14del in NSCLC, including clinical features and follow-up of patients treated with cMET-targeted therapy and consequent resistance mechanisms. Material and methods Routinely processed diagnostic pathology non-squamous NSCLC specimens were investigated by a custom-made DNA-based targeted amplicon-based next generation sequencing (NGS) panel, which includes 4 amplicons for METex14del detection. Retrospectively, histopathological characteristics and clinical follow up were investigated for advanced non-squamous NSCLC with METex14del. Results In silico analysis showed that our NGS panel is able to detect 96 % of reported METex14 alterations. METex14del was found in 2 % of patients with non-squamous NSCLC tested for therapeutic purposes. In total, from May 2015 - Sep 2018, METex14del was found in 46 patients. Thirty-six of these patients had advanced non-squamous NSCLC, they were predominantly elderly (76.5 years [53–90]), male (25/36) and (ex)-smokers (23/36). Five patients received treatment with crizotinib (Pfizer Oncology), in a named patient based program, disease control was achieved for 4/5 patients (3 partial responses, 1 stable disease) and one patient had a mixed response. Two patients developed a MET D1228N mutation during crizotinib treatment, inducing a resistance mechanism to crizotinib. Conclusions This study shows that METex14del can be reliably detected by routine DNA NGS analysis. Although a small cohort, patients responded well to targeted treatment, underlining the need for routine testing of METex14del in advanced non-squamous NSCLC to guarantee optimal personalized treatment.

Whole genome sequencing (WGS) using fresh frozen tissue and matched blood samples from cancer patients is becoming in reach as the most complete genetic tumor test. With a trend towards the availability of small biopsies and the need to screen an increasing number of (complex) biomarkers, the use of a single all-inclusive test is preferred over multiple consecutive assays. To meet high-quality diagnostics standards, we optimized and clinically validated WGS sample and data processing procedures resulting in a technical success rate of 95.6% for fresh-frozen samples with sufficient (≥20%) tumor content.Independent validation of identified biomarkers against commonly used diagnostic assays showed a high sensitivity (recall) (98.5%) and precision (positive predictive value) (97.8%) for detection of somatic SNV and indels (across 22 genes), and high concordance for detection of gene amplification (97.0%, EGRF and MET) as well as somatic complete loss (100%, CDKN2A/p16). Gene fusion analysis showed a concordance of 91.3% between DNA-based WGS and an orthogonal RNA-based gene fusion assay. Microsatellite (in)stability assessment showed a sensitivity of 100% with a precision of 94%, and virus detection (HPV) an accuracy of 100% compared to standard testing.In conclusion, whole genome sequencing has a >95% sensitivity and precision compared to routinely used DNA techniques in diagnostics and all relevant mutation types can be detected reliably in a single assay.

ObjectiveHodgkin'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.Design54 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).ResultsKRAS 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%, pMLH1 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.ConclusionsWe 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.

Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to also identify actionable copy number gains (gene amplifications) in addition to sequence variants. While guidelines for the reporting of sequence variants are available, guidance with respect to reporting copy number gains from gene-panel NGS data is limited. Here, we report on Dutch consensus recommendations obtained in the context of the national Predictive Analysis for THerapy (PATH) project, which aims to optimize and harmonize routine diagnostics in molecular pathology. We briefly discuss two common approaches to detect gene copy number gains from NGS data, i.e., the relative coverage and B-allele frequencies. In addition, we provide recommendations for reporting gene copy gains for clinical purposes. In addition to general QC metrics associated with NGS in routine diagnostics, it is recommended to include clinically relevant quantitative parameters of copy number gains in the clinical report, such as (i) relative coverage and estimated copy numbers in neoplastic cells, (ii) statistical scores to show significance (e.g., z-scores), and (iii) the sensitivity of the assay and restrictions of NGS-based detection of copy number gains. Collectively, this information can guide clinical and analytical decisions such as the reliable detection of high-level gene amplifications and the requirement for additional in situ assays in case of borderline results or limited sensitivity. Electronic supplementary material The online version of this article (10.1007/s00428-019-02555-3) contains supplementary material, which is available to authorized users.

textabstractBackground: Lynch syndrome (LS) is caused by germline mismatch repair (MMR) gene mutations. De novo MMR gene mutations are rare, and somatic mosaicism in LS is thought to be infrequent. We describe the first case of somatic mosaicism by a de novo MLH1 mutation for a patient diagnosed with a rectosigmoid adenocarcinoma at age 31. Methods: Twelve years after initial colorectal cancer diagnosis, tumor tissue of the patient was tested with sensitive next generation sequencing (NGS) analysis for the presence of somatic MMR mutations. Results: In tumor tissue, an inactivating MLH1 mutation (c.518_519del; p.(Tyr173Trpfs*18)) was detected, which was also present at low level in the blood of the patient. In both parents, as well as the patient's sisters, the mutation was not present. Conclusion: We show that low‐level mosaicism can be detected by using high‐coverage targeted NGS panels on constitutional and/or tumor DNA. This report illustrates that by using sensitive sequencing techniques, more cases of genetic diseases driven by mosaic mutations may be identified, with important clinical consequences for patients and family members.

Patients with multiple tumors, either synchronous or metachronous, can have metastatic disease or suffer from multiple independent primary tumors. While proper diagnosis of these patients is important for prognosis and treatment, this can be challenging using only clinical and histological criteria. The aim of the present study was to evaluate the value of mitochondrial D310 mutation analysis in diagnostic questions regarding tumor clonality for a wide range of tumor types. Sanger sequencing of D310 was performed on a diagnostic cohort of 382 patients with 857 tumors that were previously analyzed using routine molecular analysis on genomic DNA. The D310 mononucleotide repeat was frequently somatically mutated (56/321, 17 %) in several tumor types, including breast, head and neck, gynecological, lung, colorectal, and skin tumors. For 84/327 (26 %) patients, a D310 mutation was detected in at least one of their tumors; for these patients, D310 can be used to determine the clonal relationship between their multiple tumors. Clonality assessments based on mitochondrial DNA (mtDNA) and routine genomic DNA analysis were concordant in 52/73 (71 %) patients. We conclude that D310 mutation status might aid in determining clonality of clinically challenging synchronous or metachronous tumors. To this end, next generation sequencing targeted genomic DNA assays should be complemented with mtDNA markers, such as the D310 repeat. Electronic supplementary material The online version of this article (doi:10.1007/s00428-015-1817-5) contains supplementary material, which is available to authorized users.

Many hyperplasias and lymphomas of marginal zone B-cells are associated with infection. We identified six children and one adolescent with cervical lymphadenopathy showing prominent polyclonal nodal marginal zone hyperplasia (pNMZH) and four adolescents with monoclonal paediatric nodal marginal zone lymphoma (pNMZL). The clonality status was assessed using BIOMED-2-IG PCR analysis. Haemophilus influenzae was identified in all six cases of pNMZH that could be tested by direct culture (N = 3) or a very sensitive PCR for the H. influenzae gyrase gene in frozen materials (N = 5). H. influenzae was not detected in three pNMZLs and 28 non-specific reactive cervical lymph nodes of age-matched controls, except for a single control node that was obtained during oropharyngeal surgery for a cleft palate showing very low copy numbers of H. influenzae. pNMZH patients were younger than pNMZL patients (median age 12 versus 21 years). pNMZH showed a prominent nodular appearance with variable fibrosis without acute inflammation. Within the nodules, the expanded germinal centres and variably sized marginal zones were colonized by activated B-cells with weak expression of IgD and lack of CD10 and/or BCL6 expression. Some areas showed skewed light chain expression in plasma cells (4/5 cases lambda). In four cases tested, this was confirmed by flow cytometry for surface Ig (3/4 cases lambda). In contrast, pNMZL showed more extensive expansion of marginal zones by centrocytoid cells and often expression of BCL2 protein. Several H. influenzae strains are known to interact with the constant part of IgD on human B-cells, leading to their polyclonal proliferation and activation. We speculate that in vivo stimulation of IgD+ marginal zone B-cells by this bacterium may be implicated in this particular lymphadenopathy that should be distinguished from monoclonal pNMZL.

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.

Female BRCA1/2 mutation carriers affected with breast and/or ovarian cancer may develop new tumor deposits over time. It is of utmost importance to know the clonal relationships between multiple tumor localizations, enabling differentiation between multiple primaries or metastatic disease with consequences for therapy and prognosis. We evaluated the value of targeted next generation sequencing in the diagnostic workup of BRCA1/2 mutation carriers with ≥ 2 tumor localizations and uncertain tumor origins. Forty-two female BRCA1/2 mutation carriers with ≥2 tumor localizations were selected. Patients with inconclusive tumor origin after histopathological revision were 'cases'; patients with certain tumor origin of ≥3 tumors served as 'controls'. Tumors of cases and controls were analyzed by targeted next generation sequencing using a panel including CDKN2A, PTEN and TP53, hotspot mutation sites for 27 different genes and 143 single nucleotide polymorphisms for detection of loss of heterozygosity. Based on prevalence of identical or different mutations and/or loss of heterozygosity patterns, tumors were classified as 'multiple primaries' or 'one entity'. Conventional histopathology yielded a conclusive result in 38/42 (90%) of patients. Four cases and 10 controls were analyzed by next generation sequencing. In 44 tumor samples, 48 mutations were found; 39 (81%) concerned TP53 mutations. In all 4 cases, the intra-patient clonal relationships between the tumor localizations could be unequivocally identified by molecular analysis. In all controls, molecular outcomes matched the conventional histopathological results. In most BRCA1/2 mutation carriers with multiple tumors routine pathology work-up is sufficient to determine tumor origins and relatedness. In case of inconclusive conventional pathology results, molecular analyses using next generation sequencing can reliably determine clonal relationships between tumors, enabling optimal treatment of individual patients.

Lynch syndrome (LS) is caused by germline mutations in mismatch repair (MMR) genes, resulting in microsatellite-unstable tumours. Approximately 35% of suspected LS (sLS) patients test negative for germline MMR gene mutations, hampering conclusive LS diagnosis. The aim of this study was to investigate somatic MMR gene aberrations in microsatellite-unstable colorectal and endometrial cancers of sLS patients negative for germline MMR gene mutations. Suspected LS cases were selected from a retrospective Clinical Genetics Department diagnostic cohort and from a prospective multicentre population-based study on LS in The Netherlands. In total, microsatellite-unstable tumours of 40 sLS patients (male/female 20/20, median age 57 years) were screened for somatic MMR gene mutations by next-generation sequencing. In addition, loss of heterozygosity (LOH) of the affected MMR genes in these tumours as well as in 68 LS-associated tumours and 27 microsatellite-unstable tumours with MLH1 promoter hypermethylation was studied. Of the sLS cases, 5/40 (13%) tumours had two pathogenic somatic mutations and 16/40 (40%) tumours had a (likely) pathogenic mutation and LOH. Overall, LOH of the affected MMR gene locus was observed in 24/39 (62%) tumours with informative LOH markers. Of the LS cases and the tumours with MLH1 promoter hypermethylation, 39/61 (64%) and 2/21 (10%) tumours, respectively, demonstrated LOH. Half of microsatellite-unstable tumours of sLS patients without germline MMR gene mutations had two (likely) deleterious somatic MMR gene aberrations, indicating their sporadic origin. Therefore, we advocate adding somatic mutation and LOH analysis of the MMR genes to the molecular diagnostic workflow of LS.

Introduction Lynch syndrome (LS) is an autosomal dominantly inherited form of colorectal cancer (CRC) and is implicated in 2%–4% of CRC cases. LS develops from a mutation in one allele of one of the DNA mismatch repair (MMR) genes, most commonly MLH1 and MSH2, or less frequently MSH6 and PMS2. Loss of functional MMR proteins leads to defects in DNA repair and, subsequently, high DNA microsatellite instability (MSI-High). LS diagnostics currently consists of an analysis of MMR protein expression by means of Immunohistochemistry (IHC) and a molecular analysis to detect MSI. During our 10 year experience in performing LS diagnostics we have encountered few cases where IHC and MSI analyses do not match. This study is to clarify these inconsistent molecular alterations which will contribute to our understanding of LS diagnostics. Material and methods Of 2335 LS cases 27 (1.1%) showed discrepant IHC and MSI results. To clarify this, IHC and MSI analyses were repeated if possible with different antibodies and different MSI markers (mononucleotide instead of dinucleotide markers). Using MS-MLPA MLH1 hypermethylation, BRAF V600E mutation status and LOH of the MMR genes was analysed. Finally, germline and somatic mutations in MMR genes were analysed by using NGS. Protein modelling was also performed to visualise structural protein changes. Results and discussions Among these 27 cases, due to the decline of patients for further testing, 4 cases were excluded from further analyses. Of the remaining cases in 2 cases using a different antibody explained the unexpected results and in 2 cases switching from dinucleotide to mononucleotide MSI markers. In respectively 8 and 4 cases somatic and germline mutations explained the IHC and MSI results. Finally, in 2 cases protein modelling of identified mutations explained the presence of protein staining in MSI-high cases. The rest cases cannot be further studied due to the lack of tissues. Conclusion Based on these results, we have concluded: 1. Mononucleotide markers are more sensitive to detect microsatellite instability. 2. Protein modelling can explain presence of protein staining for mutations that do not cause a major conformational change. 3. Somatic sequencing is a valuable addition to Lynch syndrome diagnostics.

Many hyperplasias and lymphomas of marginal zone B-cells are associated with infection. We identified six children and one adolescent with cervical lymphadenopathy showing prominent polyclonal nodal marginal zone hyperplasia (pNMZH) and four adolescents with monoclonal paediatric nodal marginal zone lymphoma (pNMZL). The clonality status was assessed using BIOMED-2-IG PCR analysis. Haemophilus influenzae was identified in all six cases of pNMZH that could be tested by direct culture (N = 3) or a very sensitive PCR for the H. influenzae gyrase gene in frozen materials (N = 5). H. influenzae was not detected in three pNMZLs and 28 non-specific reactive cervical lymph nodes of age-matched controls, except for a single control node that was obtained during oropharyngeal surgery for a cleft palate showing very low copy numbers of H. influenzae. pNMZH patients were younger than pNMZL patients (median age 12 versus 21 years). pNMZH showed a prominent nodular appearance with variable fibrosis without acute inflammation. Within the nodules, the expanded germinal centres and variably sized marginal zones were colonized by activated B-cells with weak expression of IgD and lack of CD10 and/or BCL6 expression. Some areas showed skewed light chain expression in plasma cells (4/5 cases lambda). In four cases tested, this was confirmed by flow cytometry for surface Ig (3/4 cases lambda). In contrast, pNMZL showed more extensive expansion of marginal zones by centrocytoid cells and often expression of BCL2 protein. Several H. influenzae strains are known to interact with the constant part of IgD on human B-cells, leading to their polyclonal proliferation and activation. We speculate that in vivo stimulation of IgD+ marginal zone B-cells by this bacterium may be implicated in this particular lymphadenopathy that should be distinguished from monoclonal pNMZL.

e20055 Background: Lung adenocarcinoma (LAC) is the most common histological type of non-small-cell lung cancer and is one of the malignancies with the most evolved personalized treatments based on molecular characteristics of the tumor. Mutations in EGFR, HER2 and BRAF, specific translocations of ALK, ROS1, RET and amplification of MET all have standard diagnostic importance and lead to specific treatment options for the individual LAC patients. Recently, in 2-4% of LAC MET gene mutations leading to skipping of exon 14 were found. These mutations were described to occur more frequently in tumors with sarcomatoid histology. LAC with MET exon 14 skipping mutations showed impressive, although temporary, responses to MET tyrosine kinase inhibitors (TKI) crizotinib, cabozantinib and capmatinib. We will present our experience with routine molecular diagnostic detection of the most common MET exon 14 skipping mutations. Methods: In January 2016 we included in our standard, DNA based, molecular diagnostics custom-made NGS analyses 4 amplicons for detection of MET skipping mutations. The analyses were performed on microdissected FFPE tissue sections or routine histology or cytology stained preparations. Nine different mutations were validated for their effect on splicing by RT-PCR on RNA isolated from the same tissue samples. Results: Between January 2016 and January 2017 676 routine molecular diagnostic analyses on LAC were performed. In 18 (2.7%) cases MET mutations were detected possibly resulting in exon 14 skipping. Nine out of 16 different mutations were tested by RT-PCR and all 9 were demonstrated to result in MET exon 14 skipping. Conclusions: MET exon 14 skipping mutations can reliably be detected in routine pathology tissue samples. These analyses can easily be included in routine molecular diagnostic NGS. When necessary, confirmation of the mutational effect on RNA splicing can be implemented as well. Routine identification of MET skipping mutations (2.7% of cases) adds substantially to the personalized targeted treatment strategies for LAC patients.

Objectives: To address whether successive B-cell lymphomas, diagnosed within a 5- to 15-year interval, are recurrences or unrelated primary lymphomas. Methods: Immunoglobulin heavy and κ light chain gene rearrangements were studied using multiplex polymerase chain reaction fragment assays and sequence analysis in 61 patients. Results: Clonal patterns of the multiple lymphomas from 36 patients were determined and classified accordingly: 30 recurrences, 2 possible recurrences, 2 different clones with a common origin, and 2 unrelated primary lymphomas. Conclusions: Regardless of subtype, 89% to 94% of late B-cell lymphoma relapses were recurrences of the primary tumor. Therefore, routinely investigating the possible clonal relationship between successive lymphomas may not be warranted except for specific lymphoma subtypes such as diffuse large B-cell lymphomas.

PURPOSE Female BRCA1/2 mutation carriers commonly develop breast cancer and ovarian cancer. It is of utmost importance to know the clonal relationship between multiple tumor localizations, (multiple primaries or metastasized disease), since prognosis and treatment differ between these tumors and their metastases. We evaluated the value of targeted Next Generation Sequencing (NGS) in the diagnostic workup of BRCA1/2 mutation carriers with ≥2 tumor localizations and uncertain tumor origins. METHODS Forty-two female BRCA1/2 mutation carriers with ≥2 tumor locations with tumor material available for pathologic revision were selected. Median age at first cancer diagnosis was 48.0 years (range 30-68). Four patients with inconclusive tumor origin after histological and immunohistochemical analyses were ‘cases’; 10 patients with certain tumor origin of ≥3 tumors served as ‘controls’. Tumors of cases and controls were analyzed by targeted NGS using a panel including CDKN2A, PTEN and TP53, hotspot mutation sites for 27 different genes and 143 single nucleotide polymorphisms for detection of loss of heterozygosity (LOH). Based on prevalence of identical or different mutations and/or LOH patterns, tumors were classified as ‘multiple primaries’ or ‘one entity’. RESULTS In 44 tumors, 48 mutations were found; 39 (81%) concerned TP53 mutations. In all 10 controls and all 4 cases, the intrapatient clonal relationships between the tumors could be unequivocally identified by molecular analysis. In all controls, tumor origins based on molecular outcomes matched the conventional histopathological diagnosis. CONCLUSION In 90% of BRCA1/2 mutation carriers with multiple tumors routine pathology work-up is sufficient to determine tumor origins and relatedness. In case of inconclusive conventional pathology results, molecular analyses using NGS can reliably determine clonal relationships between tumors, enabling optimal treatment of individual patients. Citation Format: Willemina R.R. Geurts-Giele, Victorien M.T. van Verschuer, Carolien H.M. van Deurzen, Paul J. van Diest, Rute M. Pedrosa, J. Margriet Collee, Linetta B. Koppert, Caroline Seynaeve, Winand N.M. Dinjens. Molecular determination of the clonal relationship between multiple tumors in BRCA1/2-associated cancer patients has clinical relevance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3164.

Histological and molecular subtyping of non-small cell lung cancer (NSCLC) is important for predicting survival and drug response in these patients. Up to 8 % of NSCLC are multifocal and these tumor foci are often clonally related. Multiple foci can however also represent different primary tumors, with prognostic and therapeutic consequences. We describe a patient with multifocal NSCLC from which we obtained tissue from two separate lesions. With routine conventional molecular determinations, the clonal relationship between the two lesions was determined. In addition, targeted next generation sequencing with the Ion Torrent Personal Genome Machine (PGM) was performed to explore the accuracy and additional value of this relatively new technique. The two tumors of this patient showed different activating epidermal growth factor receptor (EGFR) mutations, EGFR amplification status, TP53 mutation status, and loss of heterozygosity patterns. With the PGM, all conventional detected mutations were confirmed, and an additional variant of unknown significance in ATM was detected in one of the tumors. The multifocal NSCLC of this patient represents two unrelated primary tumors. Our results suggest that multifocal NSCLC should be considered as potentially multiple primary tumors. As the presence of activating EGFR mutations has important therapeutic consequences, EGFR testing should be performed on all tumor foci present. In the present case, targeted next generation sequencing using the PGM appeared to be accurate and comparable with conventional molecular determinations. However, the application of the PGM in routine pathology molecular diagnostics needs validation in larger series of cases.

Heterozygous germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause Lynch syndrome. Biallelic mutations in the MMR genes are associated with a childhood cancer syndrome [constitutional mismatch repair-deficiency (CMMR-D)]. This is predominantly characterized by hematological malignancies and tumors of the bowel and brain, often associated with signs of neurofibromatosis type 1 (NF1). Diagnostic strategies for selection of patients for MMR gene analysis include analysis of microsatellite instability (MSI) and immunohistochemical (IHC) analysis of MMR proteins in tumor tissue. We report the clinical characterization and molecular analyses of tumor specimens from a family with biallelic PMS2 germline mutations. This illustrates the pitfalls of present molecular screening strategies. Tumor tissues of five family members were analyzed for MSI and IHC. MSI was observed in only one of the analyzed tissues. However, IHC analysis of brain tumor tissue of the index patient and his sister showed absence of PMS2 expression, and germline mutation analyses showed biallelic mutations in PMS2: p.Ser46IIe and p.Pro246fs. The same heterozygous mutations were confirmed in the father and mother, respectively. These data support the conclusion that in case of a clinical phenotype of CMMR-D, it is advisable to routinely combine MSI analysis with IHC analysis for the expression of MMR proteins. With inconclusive or conflicting results, germline mutation analysis of the MMR genes should be considered after thorough counselling of the patients and/or their relatives.