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A patient with gastrointestinal stroma tumor (GIST) and KIT p.V559D and BRAF p.G469A alterations was referred to our institutional molecular tumor board (MTB) to discuss therapeutic implications. The patient had been diagnosed with B-cell chronic lymphocytic leukemia (CLL) years prior to the MTB presentation. GIST had been diagnosed 1 month earlier. After structured clinical annotation of the molecular alterations and interdisciplinary discussion, we considered BRAF/KIT co-mutation unlikely in a treatment-naïve GIST. Discordant variant allele frequencies furthermore suggested a second malignancy. NGS of a CLL sample revealed the identical class 2 BRAF alteration, thus supporting admixture of CLL cells in the paragastric mass, leading to the detection of 2 alterations. Following the MTB recommendation, the patient received imatinib and had a radiographic response. Structured annotation and interdisciplinary discussion in specialized tumor boards facilitate the clinical management of complex molecular findings. Coexisting malignancies and clonal hematopoiesis warrant consideration in case of complex and uncommon molecular findings., (© The Author(s) 2024. Published by Oxford University Press.)

Background: β-catenin activation plays a crucial role for tumourigenesis in the large intestine but except for Lynch syndrome (LS) associated cancers stabilizing mutations of β-catenin gene (CTNNB1) are rare in colorectal cancer (CRC). Previous animal studies provide an explanation for this observation. They showed that CTNNB1 mutations induced transformation in the colon only when CTNNB1 was homozygously mutated or when membranous β-catenin binding was hampered by E-cadherin haploinsufficiency. We were interested, if these mechanisms are also found in human CTNNB1 mutated CRCs., Results: Among 869 CRCs stabilizing CTNNB1 mutations were found in 27 cases. Homo- or hemizygous CTNNB1 mutations were detected in 74% of CTNNB1 mutated CRCs (13 microsatellite instabile (MSI-H), 7 microsatellite stabile (MSS)) but only in 3% (1/33) of extracolonic CTNNB1 mutated cancers. In contrast to MSS CRC, CTNNB1 mutations at codon 41 or 45 were highly selected in MSI-H CRC. Of the examined three CRC cell lines, β-catenin and E-cadherin expression was similar in cell lines without or with hetereozygous CTNNB1 mutations (DLD1 and HCT116), while a reduced E-cadherin expression combined with cytoplasmic accumulation of β-catenin was found in a cell line with homozygous CTNNB1 mutation (LS180). Reduced expression of E-cadherin in human MSI-H CRC tissue was identified in 60% of investigated cancers, but no association with the CTNNB1 mutational status was found., Conclusions: In conclusion, this study shows that in contrast to extracolonic cancers stabilizing CTNNB1 mutations in CRC are commonly homo- or hemizygous indicating a higher threshold of β-catenin stabilization to be required for transformation in the colon as compared to extracolonic sites. Moreover, we found different mutational hotspots in CTNNB1 for MSI-H and MSS CRCs suggesting a selection of different effects on β-catenin stabilization according to the molecular pathway of tumourigenesis. Reduced E-cadherin expression in CRC may further contribute to higher levels of transcriptionally active β-catenin, but it is not directly linked to the CTNNB1 mutational status.

Purpose: Next-generation sequencing (NGS) can be used for comprehensive investigation of molecular events in breast cancer. We evaluated the relevance of genomic alterations for response to neoadjuvant chemotherapy (NACT) in the GeparSepto trial., Experimental Design: Eight hundred fifty-one pretherapeutic formalin-fixed paraffin-embedded (FFPE) core biopsies from GeparSepto study were sequenced. The panel included 16 genes for mutational ( AKT1, BRAF, CDH1, EGFR, ERBB2, ESR1, FBXW7, FGFR2, HRAS, KRAS, NRAS, SF3B1, TP53, HNF1A, PIK3CA , and PTEN ) and 8 genes for copy-number alteration analysis ( CCND1, ERBB2, FGFR1, PAK1, PIK3CA, TOP2A, TP53 , and ZNF703 )., Results: The most common genomic alterations were mutations of TP53 (38.4%) and PIK3CA (21.5%), and 8 different amplifications ( TOP2A 34.9%; ERBB2 30.6%; ZNF703 30.1%; TP53 21.9%; PIK3CA 24.1%; CCND1 17.7%; PAK1 14.9%; FGFR 12.6%). All other alterations had a prevalence of less than 5%. The genetic heterogeneity in different breast cancer subtypes [lum/HER2neg vs. HER2pos vs. triple-negative breast cancer (TNBC)] was significantly linked to differences in NACT response. A significantly reduced pathologic complete response rate was observed in PIK3CA -mutated breast cancer [ PIK3CA mut: 23.0% vs. wild-type (wt) 38.8%, P < 0.0001] in particular in the HER2pos subcohort [multivariate OR = 0.43 (95% CI, 0.24-0.79), P = 0.006]. An increased response to nab-paclitaxel was observed only in PIK3CA wt breast cancer, with univariate significance for the complete cohort ( P = 0.009) and the TNBC ( P = 0.013) and multivariate significance in the HER2pos subcohort (test for interaction P = 0.0074)., Conclusions: High genetic heterogeneity was observed in different breast cancer subtypes. Our study shows that FFPE-based NGS can be used to identify markers of therapy resistance in clinical study cohorts. PIK3CA mutations could be a major mediator of therapy resistance in breast cancer., (©2019 American Association for Cancer Research.)

Background: TP53 mutations are frequent in breast cancer, however their clinical relevance in terms of response to chemotherapy is controversial., Methods: 450 pre-therapeutic, formalin-fixed, paraffin-embedded core biopsies from the phase II neoadjuvant GeparSixto trial that included HER2-positive and triple negative breast cancer (TNBC) were subjected to Sanger sequencing of exons 5-8 of the TP53 gene. TP53 status was correlated to response to neoadjuvant anthracycline/taxane-based chemotherapy with or without carboplatin and trastuzumab/lapatinib in HER2-positive and bevacizumab in TNBC. p53 protein expression was evaluated by immunohistochemistry in the TNBC subgroup., Results: Of 450 breast cancer samples 297 (66.0%) were TP53 mutant. Mutations were significantly more frequent in TNBC (74.8%) compared to HER2-positive cancers (55.4%, P < 0.0001). Neither mutations nor different mutation types and effects were associated with pCR neither in the whole study group nor in molecular subtypes (P > 0.05 each). Missense mutations tended to be associated with a better survival compared to all other types of mutations in TNBC (P = 0.093) and in HER2-positive cancers (P = 0.071). In TNBC, missense mutations were also linked to higher numbers of tumor-infiltrating lymphocytes (TILs, P = 0.028). p53 protein overexpression was also linked with imporved survival (P = 0.019)., Conclusions: Our study confirms high TP53 mutation rates in TNBC and HER2-positive breast cancer. Mutations did not predict the response to an intense neoadjuvant chemotherapy in these two molecular breast cancer subtypes.

With the approval of olaparib as monotherapy treatment in platinum-sensitive, relapsed high-grade serous ovarian cancer by the European Medical Agency (EMA), comprehensive genotyping of BRCA1 and BRCA2 in tumor tissue has become a mandatory pre-therapeutic test. This requires significant advances in routine tumor test methodologies due to the large size of both genes and the lack of mutational hot spots. Classical focused screening approaches, like Sanger sequencing, do not allow for a sensitive, rapid, and economic analysis of tumor tissue. Next-generation sequencing (NGS) approaches employing targeted panels for BRCA1/2 to interrogate formalin-fixed and paraffin-embedded tumor samples from either surgical resection or biopsy specimens can overcome these limitations. Although focused NGS methods have been implemented by few centers in routine molecular diagnostics for the analysis of some druggable oncogenic mutations, the reliable diagnostic testing of the entire coding regions of BRCA1 and BRCA2 was a new challenge requiring extensive technological improvement and quality management. Here, we describe the implementation and results of the first round robin trial for BRCA1/2 mutation testing in tumor tissue that was conducted in central Europe on May 2015, shortly after the approval and prior to the official release of olaparib. The high success rate of 81 % (21/26 test centers) demonstrates that BRCA1/2 multicenter mutation testing is well feasible in FFPE tumor tissue, extending to other tumor entities beyond ovarian cancer. The high number of test centers passing the trial demonstrates the success of the concerted efforts by German, Swiss, and Austrian pathology centers to ensure quality-controlled NGS-based testing and proves the potential of this technology in routine molecular pathology. On the basis of our results, we provide recommendations for predictive testing of tumor tissue for BRCA1/2 to clinical decision making in ovarian cancer patients.

The mutated BRAF oncogene represents a therapeutic target in malignant melanoma. Because BRAF mutations are also involved in the pathogenesis of other human malignancies, the use of specific BRAF inhibitors might also be extended to other diseases in the future. A prerequisite for the clinical application of BRAF inhibitors is the reliable detection of activating BRAF mutations in routine histopathological samples. In a multicenter approach, we evaluated a novel and fully automated PCR-based system (Idylla) capable of detecting BRAF V600 mutations in formalin-fixed, paraffin-embedded tissue within 90 minutes with high sensitivity. We analyzed a total of 436 samples with the Idylla system. Valid results were obtained in 421 cases (96.56%). Its performance was compared with conventional methods (pyrosequencing or Sanger sequencing). Concordant results were obtained in 406 cases (96.90%). Reanalysis of eight discordant samples by next-generation sequencing and/or pyrosequencing with newly extracted DNA and the BRAF RGQ Kit confirmed the Idylla result in seven cases, resulting in an overall agreement of 98.57%. In conclusion, the Idylla system is a highly reliable and sensitive platform for detection of BRAF V600 mutations in formalin-fixed, paraffin-embedded material, providing an efficient alternative to conventional diagnostic methods, particularly for routine diagnostics laboratories with limited experience in molecular pathology., (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group with Sézary syndrome (SS) as one of the most aggressive variants. Recently, we identified a loss of E2A as a recurrent event in SS, which enhanced proliferation via upregulation of the proto-oncogene MYC. MYC-induced transformation usually requires deleterious alterations of key apoptotic genes including p53; however, p53 functionality and mutation status in SS are unclear., Objectives: We investigated functionality of p53 signalling by pharmacological treatment with the MDM2 antagonist nutlin-3, which might result in p53 activation. Furthermore, we analysed the TP53 mutation status in CTCL cell lines and highly purified tumour cells from patients with SS by mRNA and DNA sequencing., Methods: We analysed the apoptosis induction due to nutlin-3 treatment in various SS cell lines and primary patient samples by annexin V/propidium iodide staining. Induction of p53 target genes was analysed by immunoblotting, and TP53 was sequenced at the mRNA and DNA level., Results: We identified various TP53 mutations and an impaired p53 signalling in the vast majority of the investigated cell lines and primary SS cells., Conclusions: In accordance with the importance of MYC deregulation in SS, p53 signalling is frequently nonfunctional in SS. However, although most likely ineffective as exclusive treatment in SS, it remains possible that pharmacological p53 activation could be beneficial in combination with other approaches including classical chemotherapeutics., (© 2012 The Authors. BJD © 2012 British Association of Dermatologists.)