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Summary Checkpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumor-cell-intrinsic and microenvironmental features underpinning CPI sensitization. Here, we collated whole-exome and transcriptomic data for >1,000 CPI-treated patients across seven tumor types, utilizing standardized bioinformatics workflows and clinical outcome criteria to validate multivariable predictors of CPI sensitization. Clonal tumor mutation burden (TMB) was the strongest predictor of CPI response, followed by total TMB and CXCL9 expression. Subclonal TMB, somatic copy alteration burden, and histocompatibility leukocyte antigen (HLA) evolutionary divergence failed to attain pan-cancer significance. Dinucleotide variants were identified as a source of immunogenic epitopes associated with radical amino acid substitutions and enhanced peptide hydrophobicity/immunogenicity. Copy-number analysis revealed two additional determinants of CPI outcome supported by prior functional evidence: 9q34 (TRAF2) loss associated with response and CCND1 amplification associated with resistance. Finally, single-cell RNA sequencing (RNA-seq) of clonal neoantigen-reactive CD8 tumor-infiltrating lymphocytes (TILs), combined with bulk RNA-seq analysis of CPI-responding tumors, identified CCR5 and CXCL13 as T-cell-intrinsic markers of CPI sensitivity., Graphical Abstract, Highlights • Large-scale meta-analysis of >1,000 CPI-treated cases with exome/transcriptome data • Clonal TMB and CXCL9/CXCL13 expression are the strongest predictors of CPI response • A multivariable predictor of CPI response significantly outperforms TMB • 9q34 loss and CCND1 amplification are additional determinants of CPI response, A whole-exome and transcriptome meta-analysis of over 1,000 patients treated with immune checkpoint blockade across seven tumor types highlights the potential of multivariable prediction models that consider both tumor- and T-cell-intrinsic mechanisms of response.

Impaired DNA damage repair (DDR) is increasingly recognised as a hallmark in pancreatic ductal adenocarcinoma (PDAC). It is estimated that around 14% of human PDACs harbour mutations in genes involved in DDR, including, amongst others, BRCA1/2, PALB2, ATM, MSH2, MSH6 and MLH1. Recently, DDR intervention by PARP inhibitor therapy has demonstrated effectiveness in germline BRCA1/2-mutated PDAC. Extending this outcome to the significant proportion of human PDACs with somatic or germline mutations in DDR genes beyond BRCA1/2 might be beneficial, but there is a lack of data, and consequently, no clear recommendations are provided in the field. Therefore, an expert panel was invited by the European Society of Digestive Oncology (ESDO) to assess the current knowledge and significance of DDR as a target in PDAC treatment. The aim of this virtual, international expert meeting was to elaborate a set of consensus recommendations on testing, diagnosis and treatment of PDAC patients with alterations in DDR pathways. Ahead of the meeting, experts completed a 27-question survey evaluating the key issues. The final recommendations herein should aid in facilitating clinical practice decisions on the management of DDR-deficient PDAC., SCOPUS: ar.j, info:eu-repo/semantics/published

Molecular characterization of tumors has shifted cancer treatment strategies away from nonspecific cytotoxic treatment of histology-specific tumors toward targeting of actionable mutations that can be found across multiple cancer types. The development of high-throughput technologies such as next-generation sequencing, combined with decision support applications and availability of patient databases, has provided tools that optimize disease management. Precision oncology has proven success in improving outcomes and quality of life, as well as identifying and overcoming mechanisms of drug resistance and relapse. Addressing challenges that impede its use will improve matching of therapies to patients. Here we review the current status of precision oncology medicine, emphasizing its impact on patients - what they understand about precision oncology medicine and their hopes for the future.Lay abstract Precision oncology offers individualized treatment of cancer on a per-patient basis, based on the unique DNA fingerprint of a patient’s cancer. New, advanced technologies for DNA sequencing have led to rapid advancement in developing novel therapies. Decision-making tools have followed pace, leading to improved matching of therapy to patient, ultimately improving outcomes (including quality of life), building trust between patient and physician, and increasing hope for the future.

There are currently five programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) inhibitors approved for the treatment of locally advanced or metastatic urothelial carcinoma (UC) of the bladder. For platinum-ineligible patients, testing of tumor specimens for PD-L1 expression is required. However, scoring of PD-L1 immunohistochemistry is complex due to different antibodies used, the requirement to score expression in different cellular compartments and intratumoral heterogeneity. It can also be difficult to obtain and test longitudinal tumor samples, which would be desirable to monitor treatment responses and tumor evolution under treatment-induced selective pressure. In the present proof-of concept study, we provide evidence that PD-L1 can be detected in the urine of patients with non-muscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Urine PD-L1 levels were significantly higher in NMIBC and MIBC patients when compared to patients with various non-malignant urological diseases. Further prospective and independent studies are required to assess the value of PD-L1 in the urine as a novel biomarker with potential for the early detection, prediction and therapeutic monitoring of patients with UC of the bladder.

Background and aims Due to the high load of immunogenic frameshift neoantigens, tumors arising in individuals with Lynch syndrome (LS), the most common inherited colorectal cancer (CRC) syndrome, are characterized by a pronounced immune infiltration. However, the immune status of normal colorectal mucosa in LS is not well characterized. We assessed the immune infiltrate in tumor-distant normal colorectal mucosa from LS CRC patients, sporadic microsatellite-unstable (MSI) and microsatellite-stable (MSS) CRC patients, and cancer-free LS carriers. Methods CD3-positive, FOXP3-positive and CD8-positive T cells were quantified in 219, 233 and 201 formalin-fixed paraffin-embedded (FFPE) normal colonic mucosa tissue sections from CRC patients and LS cancer-free carriers and 26, 22 and 19 LS CRCs, respectively. CD3-positive T cells were also quantified in an independent cohort of 97 FFPE normal rectal mucosa tissue sections from LS carriers enrolled on the CAPP2 clinical trial. The expression of 770 immune-relevant genes was analyzed in a subset of samples using the NanoString nCounter platform. Results LS normal mucosa specimens showed significantly elevated CD3-, FOXP3- and CD8-positive T cell densities compared to non-LS controls. Gene expression profiling and cluster analysis revealed distinct immune profiles in LS carrier mucosa with and without cancer manifestation. Long term follow-up of LS carriers within the CAPP2 trial found a correlation between mucosal T cell infiltrate and time to subsequent tumor occurrence. Conclusion LS carriers show elevated mucosal T cell infiltration even in the absence of cancer. The normal mucosa immune profile may be a temporary or permanent tumor risk modifier in LS carriers.

Background Targeted therapies have improved survival and quality of life for patients with non-small-cell lung cancer with actionable driver mutations. However, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 gene (HER2, also known as ERBB2) exon 20 insertions (Ex20mut) are characterized by a poor response to currently approved tyrosine kinase inhibitors and immunotherapies. The underlying immune biology is not well understood. Materials and methods We carried out messenger RNA expression profiling of lung adenocarcinomas (ADCs) with ERBB2 (n = 19) and EGFR exon 20-insertion mutations (n = 13) and compared these to tumors with classical EGFR mutations (n = 40, affecting EGFR exons 18, 19 or 21) and EGFR/ERBB2 mutation-negative lung ADC (EGFR/ERBB2wt, n = 26) focusing on immunologically relevant transcripts. Tumor-infiltrating immune cells were estimated from gene expression profiles. Results Cytotoxic cells were significantly lower in EGFR-mutated tumors regardless of the affected exon, while Th1 cells were significantly lower in EGFR-Ex20mut compared to EGFR/ERBB2wt tumors. We assessed the differentially expressed genes of ERBB2-Ex20mut and EGFR-Ex20mut tumors compared to EGFR-Ex18/19/21mut and EGFR/ERBB2wt tumors. Of these, the genes GUSB, HDAC11, IFNGR2, PUM1, RASGRF1 and RBL2 were up-regulated, while a lower expression of CBLC, GBP1, GBP2, GBP4 and MYC was observed in all three comparison groups. The omnibus test revealed 185 significantly (FDR = 5%) differentially expressed genes and we found these four most significant gene expression changes in the study cohort: VHL and JAK1 were overexpressed in ERBB2-Ex20mut and EGFR-Ex20mut tumors compared to both EGFR-Ex18/19/21mut and EGFR/ERBB2wt tumors. RIPK1 and STK11IP showed the highest expression in ERBB2-Ex20mut tumors. Conclusions Targeted gene expression profiling is a promising tool to read out the characteristics of the tumor microenvironment from routine diagnostic lung cancer biopsies. Significant immune reactivity and specific immunosuppressive characteristics in ERBB2-Ex20mut and EGFR-Ex20mut lung ADC with at least some degree of immune infiltration support further clinical evaluation of immune-modulators as partners of immune checkpoint inhibitors in such tumors., Highlights • Gene expression profiling to characterize the tumor microenvironment is feasible using diagnostic lung cancer biopsies. • EGFR exon 20-mutated tumors show a higher expression of VHL and an immunologic ‘colder’ phenotype than EGFR/ERBB2wt tumors. • ERBB2 exon 20-mutated tumors show an overexpression of RIPK1 and STK11IP and a reduction of cytotoxic natural killer cells. • Drugs targeting these alterations are potential partners of checkpoint blockade in exon 20-mutated non-small-cell lung cancer.

Background Single-site carcinoma of unknown primary (CUP) is recognised as a distinct favourable subtype in the European Society of Medical Oncology (ESMO) classification. There is broad consensus that these patients are candidates for local ablative treatment strategies with surgery and/or radiotherapy, but data on their outcomes are scarce. Patients and methods In this study, we have addressed the prospects of cure and prognostic factors in a retrospective cohort of 63 patients who were eligible for local treatment at our centre. Results Median event-free (EFS) and overall survival (OS) were 15.6 months and 52.5 months, respectively. Of 61 patients who received local treatment, 20 (32.8%) remained event-free over a median follow-up of 28 months. Baseline clinical parameters including affected organ, number, volume and histology of metastases had no significant impact on prognosis, whereas deleterious TP53 mutations and DNA copy number loss emerged as independent adverse risk factors with respect to EFS. Surgical treatment was associated with improved OS as compared to radiation-based therapy. Conclusion Our study advocates to pursue localised treatment with surgery and/or radiotherapy whenever feasible and implies that genetic parameters might additionally determine the clinical course of single-site CUP patients.

BACKGROUND The potential of deep learning to support radiologist prostate magnetic resonance imaging (MRI) interpretation has been demonstrated. PURPOSE The aim of this study was to evaluate the effects of increased and diversified training data (TD) on deep learning performance for detection and segmentation of clinically significant prostate cancer-suspicious lesions. MATERIALS AND METHODS In this retrospective study, biparametric (T2-weighted and diffusion-weighted) prostate MRI acquired with multiple 1.5-T and 3.0-T MRI scanners in consecutive men was used for training and testing of prostate segmentation and lesion detection networks. Ground truth was the combination of targeted and extended systematic MRI-transrectal ultrasound fusion biopsies, with significant prostate cancer defined as International Society of Urological Pathology grade group greater than or equal to 2. U-Nets were internally validated on full, reduced, and PROSTATEx-enhanced training sets and subsequently externally validated on the institutional test set and the PROSTATEx test set. U-Net segmentation was calibrated to clinically desired levels in cross-validation, and test performance was subsequently compared using sensitivities, specificities, predictive values, and Dice coefficient. RESULTS One thousand four hundred eighty-eight institutional examinations (median age, 64 years; interquartile range, 58-70 years) were temporally split into training (2014-2017, 806 examinations, supplemented by 204 PROSTATEx examinations) and test (2018-2020, 682 examinations) sets. In the test set, Prostate Imaging-Reporting and Data System (PI-RADS) cutoffs greater than or equal to 3 and greater than or equal to 4 on a per-patient basis had sensitivity of 97% (241/249) and 90% (223/249) at specificity of 19% (82/433) and 56% (242/433), respectively. The full U-Net had corresponding sensitivity of 97% (241/249) and 88% (219/249) with specificity of 20% (86/433) and 59% (254/433), not statistically different from PI-RADS (P > 0.3 for all comparisons). U-Net trained using a reduced set of 171 consecutive examinations achieved inferior performance (P < 0.001). PROSTATEx training enhancement did not improve performance. Dice coefficients were 0.90 for prostate and 0.42/0.53 for MRI lesion segmentation at PI-RADS category 3/4 equivalents. CONCLUSIONS In a large institutional test set, U-Net confirms similar performance to clinical PI-RADS assessment and benefits from more TD, with neither institutional nor PROSTATEx performance improved by adding multiscanner or bi-institutional TD.

Mutations in the tumor suppressor gene TP53 are among the most common genetic aberrations in cancer. In prostate cancer, the role of mutant TP53 remains incompletely understood. Initially, mutations in TP53 were considered late events during malignant progression and associated with metastatic dissemination and castration resistance. However, recent studies report an inactivation of TP53 at an unexpectedly high frequency in primary as well as metastatic castration-naive prostate cancer. In this chapter, we discuss the biology of p53, the relevance of TP53 mutations for prostate cancer progression and therapy resistance, and its potential role as a marker to identify patients who require more intensified treatment.

Modern concepts in precision cancer medicine are based on increasingly complex genomic analyses and require standardized criteria for the functional evaluation and reporting of detected genomic alterations in order to assess their clinical relevance. In this article, we propose and address the necessary steps in systematic variant evaluation consisting of bioinformatic analysis, functional annotation and clinical interpretation, focusing on the latter two aspects. We discuss the role and clinical application of current variant classification systems and point out their scope and limitations. Finally, we highlight the significance of the molecular tumor board as a platform for clinical decision-making based on genomic analyses.

BACKGROUND: Liquid rebiopsies can detect resistance mutations to guide therapy of anaplastic lymphoma kinase-rearranged (ALK(+)) non-small-cell lung cancer (NSCLC) failing tyrosine kinase inhibitors (TKI). Here, we analyze how their results relate to the anatomical pattern of disease progression and patient outcome. METHODS: Clinical, molecular, and radiologic characteristics of consecutive TKI-treated ALK(+) NSCLC patients were analyzed using prospectively collected plasma samples and the 17-gene targeted AVENIO kit, which covers oncogenic drivers and all TP53 exons. RESULTS: In 56 patients, 139 instances of radiologic changes were analyzed, of which 133 corresponded to disease progression. Circulating tumor DNA (ctDNA) alterations were identified in most instances of extracranial progression (58/94 or 62%), especially if concomitant intracranial progression was also present (89%, P

Background Soft-tissue sarcomas (STS) are a heterogeneous group of mesenchymal tumors for which response to immunotherapies is not well established. Therefore, it is important to risk-stratify and identify STS patients who will most likely benefit from these treatments. Results To reveal shared and distinct methylation signatures present in STS, we performed unsupervised deconvolution of DNA methylation data from the TCGA sarcoma and an independent validation cohort. We showed that leiomyosarcoma can be subclassified into three distinct methylation groups. More importantly, we identified a component associated with tumor-infiltrating leukocytes, which suggests varying degrees of immune cell infiltration in STS subtypes and an association with prognosis. We further investigated the genomic alterations that may influence tumor infiltration by leukocytes including RB1 loss in undifferentiated pleomorphic sarcomas and ELK3 amplification in dedifferentiated liposarcomas. Conclusions In summary, we have leveraged unsupervised methylation-based deconvolution to characterize the immune compartment and molecularly stratify subtypes in STS, which may benefit precision medicine in the future.

BACKGROUND: Targeted genetic profiling of tissue samples is paramount to detect druggable genetic aberrations in patients with non-squamous non-small cell lung cancer (NSCLC). Accurate upfront estimation of tumor cell content (TCC) is a crucial pre-analytical step for reliable testing and to avoid false-negative results. As of now, TCC is usually estimated on hematoxylin-eosin (H&E) stained tissue sections by a pathologist, a methodology that may be prone to substantial intra- and interobserver variability. Here we the investigate suitability of digital pathology for TCC estimation in a clinical setting by evaluating the concordance between semi-automatic and conventional TCC quantification. METHODS: TCC was analyzed in 120 H&E and thyroid transcription factor 1 (TTF-1) stained high-resolution images by 19 participants with different levels of pathological expertise as well as by applying two semi-automatic digital pathology image analysis tools (HALO and QuPath). RESULTS: Agreement of TCC estimations [intra-class correlation coefficients (ICC)] between the two software tools (H&E: 0.87; TTF-1: 0.93) was higher compared to that between conventional observers (0.48; 0.47). Digital TCC estimations were in good agreement with the average of human TCC estimations (0.78; 0.96). Conventional TCC estimators tended to overestimate TCC, especially in H&E stainings, in tumors with solid patterns and in tumors with an actual TCC close to 50%. CONCLUSIONS: Our results determine factors that influence TCC estimation. Computer-assisted analysis can improve the accuracy of TCC estimates prior to molecular diagnostic workflows. In addition, we provide a free web application to support self-training and quality improvement initiatives at other institutions.

Fusion proteins involving the BRAF serine/threonine kinase occur in many cancers. The oncogenic potential of BRAF fusions has been attributed to the loss of critical N-terminal domains that mediate BRAF autoinhibition. We used whole-exome and RNA sequencing in a patient with glioblastoma multiforme to identify a rearrangement between TTYH3, encoding a membrane-resident, calcium-activated chloride channel, and BRAF intron 1, resulting in a TTYH3–BRAF fusion protein that retained all features essential for BRAF autoinhibition. Accordingly, the BRAF moiety of the fusion protein alone, which represents full-length BRAF without the amino acids encoded by exon 1 (BRAFΔE1), did not induce MEK/ERK phosphorylation or transformation. Likewise, neither the TTYH3 moiety of the fusion protein nor full-length TTYH3 provoked ERK pathway activity or transformation. In contrast, TTYH3–BRAF displayed increased MEK phosphorylation potential and transforming activity, which were caused by TTYH3-mediated tethering of near-full-length BRAF to the (endo)membrane system. Consistent with this mechanism, a synthetic approach, in which BRAFΔE1 was tethered to the membrane by fusing it to the cytoplasmic tail of CD8 also induced transformation. Furthermore, we demonstrate that TTYH3–BRAF signals largely independent of a functional RAS binding domain, but requires an intact BRAF dimer interface and activation loop phosphorylation sites. Cells expressing TTYH3–BRAF exhibited increased MEK/ERK signaling, which was blocked by clinically achievable concentrations of sorafenib, trametinib, and the paradox breaker PLX8394. These data provide the first example of a fully autoinhibited BRAF protein whose oncogenic potential is dictated by a distinct fusion partner and not by a structural change in BRAF itself.

Das Prinzip der Prazisionsonkologie beruht heute im Wesentlichen auf einer sich standig verfeinernden molekularen Charakterisierung von Tumoren. Im Idealfall bedingt diese Charakterisierung eine personalisierte, zielgerichtete, molekular gesteuerte medikamentose Behandlung. In diesem Kontext verschiebt sich das diagnostische Spektrum zunehmend in Richtung einer breiten genomischen Charakterisierung mittels neuer massiv paralleler Sequenziertechnologien („next generation sequencing“). Diese Weiterentwicklung birgt jedoch nicht nur Chancen fur die onkologische Patientenversorgung, sondern bringt auch eine Reihe von inhaltlichen und strukturellen Herausforderungen mit sich, die nur durch eine enge interdisziplinare Zusammenarbeit gemeistert werden konnen. In diesem Ubersichtsartikel sollen verschiedene Aspekte der breiteren Genomanalyse maligner Tumoren zusammenfassend beleuchtet und diskutiert werden. Zudem wird – auf dieser Basis – ein Blick in die nahe Zukunft der Prazisionsonkologie geworfen.

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 [PIK3CAmut: 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 PIK3CAwt 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.

Summary About 60–70% of hidradenoma papilliferum (HP), a benign tumour of the anogenital region, were recently described to harbour mutations in major driver genes of the PI3K/AKT/MAPK-signalling pathways. However, the underlying genetic defects of the non-mutant cases are still unknown. Using a 409 gene panel, we employed targeted next generation sequencing to investigate the mutational landscape in a cohort of seven PI3K/AKT-negative cases and five cases with known hotspot mutations in either PIK3CA or AKT1. In total, we identified 29 mutations in 22 of 409 genes. The four cases with PIK3CA hotspot mutations carried no or only few additional mutations. The AKT1 hotspot mutated case harboured additional mutations in four genes (SYNE1, ADAMTS20, EP400 and CASC5). At least two of these genes are involved in or contribute to the PI3K/AKT-pathway. In the seven non-hotspot mutated cases we observed 18 mutations. Each case carried at least one mutation in a gene contributing to or involved in PI3K/AKT-signalling. Affected genes were PIK3CA (n=1, non-hotspot mutation), PIK3R1 (n=3), SYNE1, AR, IL6ST, PDGFRB, KMT2C, AR, BTK, DST, KAT6A, BRD3, RNF213, USP9X, ADGRB3, MAGI1, and IL7R (each gene mutated once). The identified PIK3CA and PIK3R1 mutations lead to constitutive activated PI3K/AKT-signalling. In conclusion, we demonstrate the genetic basis of HP in all cases. Our data suggest that tumourigenic alterations in the PI3K/AKT-pathway are indispensable in HP and establish a homogenous morphomolecular entity with a functionally converging and selecting tumourigenic mechanism.

Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancers (NSCLC) have the best prognosis among metastatic pulmonary malignancies, with a median patient survival currently exceeding 5 years. While this is definitely a major therapeutic success for thoracic oncology, it may not be entirely attributable to rapid drug development and the strenuous clinical efforts. At the genetic level, ALK+ disease is also unique, distinguished by the lowest tumor mutational burden (mean below 3 mutations/Mbp), the lowest frequency of TP53 mutations (20-25%) and very few other co-mutations compared to other NSCLC. The relative simplicity and stability of the genetic landscape not only contribute to the relatively favourable clinical course, but also make study of the effects from individual molecular features easier. EML4-ALK fusion variant 3 (E6;A20) and TP53 mutations were recently identified as main molecular determinants of adverse outcome: they occur in about 30-40% and 20-25% of newly-diagnosed cases, respectively, have possibly synergistic effects and are independently associated with more aggressive disease, shorter progression-free survival under treatment with ALK inhibitors and worse overall survival. Secondary detection of TP53 mutations at disease progression in previously negative patients defines another subset (about 20%) with similarly poor outcome, while detection of ALK resistance mutations guides next-line therapy. As our biological understanding deepens, additional molecular risk factors will be identified and refine our concepts further. The translation of clinical risk at the molecular level and the ability to predict early events are of key importance for individualized patient management and preclinical modeling in order to advance therapeutic options.

Cancer of unknown primary (CUP) denotes cancer cases where metastatic spread is histologically confirmed, but no respective primary tumor can be identified. The challenging diagnosis of CUP is further complicated in cases with previously identified malignancies or with dubious clonal relationship between metastatic sites due to ambiguous histology. Our study aims at elucidating clonal relationships by comparing the respective mutational spectra. Targeted next-generation sequencing (NGS) employing formalin-fixed and paraffin-embedded (FFPE) tumor tissue was performed on 174 consecutive CUP patients. Among these, 43/174 (24.7%) patients had a documented prior malignancy. Data on pairwise targeted NGS testing to address clonal relationships between the previous malignancy and the presumed CUP (n = 11) or between different CUP metastatic sites (n = 7) was available in 18 patients. NGS could clarify clonal relationships in 16/18 cases. Among the 11 CUP patients with antecedent malignancies, four cases were clonally independent of the previous malignancy but harbored deleterious germline mutations in BRCA/BAP1/ATM genes. Seven CUP cases were clonally related to the antecedent malignancy, changing the CUP diagnosis to relapse of the prior malignancy. In the seven CUP cases, with doubtfully related metastatic sites, NGS confirmed clonal relationship in five cases and was inconclusive in two. In conclusion, NGS proved an efficient tool to elucidate clonal relationships in clinically challenging CUP cases. Our study cautions against a premature diagnosis of CUP. Relapses of antecedent malignancies should be carefully considered. CUPs clonally independent from the antecedent malignancy should raise a red flag of a potential cancer-predisposing germline mutation.

INTRODUCTIONCirculating tumor cells (CTCs) may be used to improve cancer diagnosis, prognosis, and treatment. However, because knowledge regarding CTC biology is limited and the numbers of CTCs and CTC-positive cancer patients are low, progress in this field is slow. We addressed this limitation by combining diagnostic leukapheresis (DLA) and microfluidic enrichment to obtain large numbers of viable CTCs from metastasized breast cancer patients.METHODSDLA was applied to 9 patients, and 7.5 mL of peripheral blood was drawn. CTCs were enriched with the Parsortix™ system. The quality of CTCs from fresh and cryopreserved DLA products was tested, and CTCs were cultured in vitro. Single uncultured and cultured CTCs were isolated by micromanipulation to determine different parameters, such as genomic aberrations and mutation profiles of selected tumor-associated genes. Expression levels of estrogen receptor and HER2/neu were monitored during in vitro culture.RESULTSViable CTCs from peripheral blood and fresh or frozen DLA products could be enriched. DLA increased the likelihood of successful CTC culture. Cryopreserved DLA products could be stored with minimal CTC loss and no overt reduction in the tumor cell quality and viability during an observation period of up to 3 years. The analyzed parameters did not change during in vitro culture. DLA samples with high CTC numbers and lower ratios of apoptotic CTCs were more likely to grow in culture.CONCLUSIONSThe increased CTC numbers from fresh or cryopreserved DLA products facilitate multiple functional and molecular analyses and, thus, could improve our knowledge of their biology.

Characterization of tumors utilizing next‐generation sequencing methods, including assessment of the number of somatic mutations (tumor mutational burden [TMB]), is currently at the forefront of the field of personalized medicine. Recent clinical studies have associated high TMB with improved patient response rates and survival benefit from immune checkpoint inhibitors; hence, TMB is emerging as a biomarker of response for these immunotherapy agents. However, variability in current methods for TMB estimation and reporting is evident, demonstrating a need for standardization and harmonization of TMB assessment methodology across assays and centers. Two uniquely placed organizations, Friends of Cancer Research (Friends) and the Quality Assurance Initiative Pathology (QuIP), have collaborated to coordinate efforts for international multistakeholder initiatives to address this need. Friends and QuIP, who have partnered with several academic centers, pharmaceutical organizations, and diagnostic companies, have adopted complementary, multidisciplinary approaches toward the goal of proposing evidence‐based recommendations for achieving consistent TMB estimation and reporting in clinical samples across assays and centers. Many factors influence TMB assessment, including preanalytical factors, choice of assay, and methods of reporting. Preliminary analyses highlight the importance of targeted gene panel size and composition, and bioinformatic parameters for reliable TMB estimation. Herein, Friends and QuIP propose recommendations toward consistent TMB estimation and reporting methods in clinical samples across assays and centers. These recommendations should be followed to minimize variability in TMB estimation and reporting, which will ensure reliable and reproducible identification of patients who are likely to benefit from immune checkpoint inhibitors.

Tyrosine kinase inhibitors currently confer the greatest survival gain for nonsmall cell lung cancer (NSCLC) patients with actionable genetic alterations. Simultaneously, the increasing number of targets and compounds poses the challenge of reliable, broad and timely molecular assays for the identification of patients likely to benefit from novel treatments. Here, we demonstrate the feasibility and clinical utility of comprehensive, NGS-based genetic profiling for routine workup of advanced NSCLC based on the first 3,000 patients analyzed in our department. Following automated extraction of DNA and RNA from formalin-fixed, paraffin-embedded tissue samples, parallel sequencing of DNA and RNA for detection of mutations and gene fusions, respectively, was performed using PCR-based enrichment with an ion semiconductor sequencing platform. Overall, 807 patients (27%) were eligible for currently approved, EGFR-/BRAF-/ALK- and ROS1-directed therapies, while 218 additional cases (7%) with MET, ERBB2 (HER2) and RET alterations could potentially benefit from experimental targeted compounds. In addition, routine capturing of comutations, e.g. TP53 (55%), KEAP1 (11%) and STK11 (11%), as well as the precise typing of fusion partners and involved exons in case of actionable translocations including ALK and ROS1, are prognostic and predictive tools currently gaining importance for further refinement of therapeutic and surveillance strategies. The reliability, low dropout rates (

ObjectiveWe aimed at the identification of genetic alterations that may functionally substitute for CTNNB1 mutation in ß-catenin-activated hepatocellular adenomas (HCAs) and hepatocellular carcinoma (HCC).DesignLarge cohorts of HCA (n=185) and HCC (n=468) were classified using immunohistochemistry. The mutational status of the CTNNB1 gene was determined in ß-catenin-activated HCA (b-HCA) and HCC with at least moderate nuclear CTNNB1 accumulation. Ultra-deep sequencing was used to characterise CTNNB1wild-type and ß-catenin-activated HCA and HCC. Expression profiling of HCA subtypes was performed.ResultsA roof plate-specific spondin 2 (RSPO2) gene rearrangement resulting from a 46.4 kb microdeletion on chromosome 8q23.1 was detected as a new morphomolecular driver of β-catenin-activated HCA. RSPO2 fusion positive HCA displayed upregulation of RSPO2 protein, nuclear accumulation of β-catenin and transcriptional activation of β-catenin-target genes indicating activation of Wingless-Type MMTV Integration Site Family (WNT) signalling. Architectural and cytological atypia as well as interstitial invasion indicated malignant transformation in one of the RSPO2 rearranged b-HCAs. The RSPO2 gene rearrangement was also observed in three β-catenin-activated HCCs developing in context of chronic liver disease. Mutations of the human telomerase reverse transcriptase promoter—known to drive malignant transformation of CTNNB1-mutated HCA—seem to be dispensable for RSPO2 rearranged HCA and HCC.ConclusionThe RSPO2 gene rearrangement leads to oncogenic activation of the WNT signalling pathway in HCA and HCC, represents an alternative mechanism for the development of b-HCA and may drive malignant transformation without additional TERT promoter mutation.

ZusammenfassungUndifferenzierte Karzinome der Gallenwege sind eine Rarität und manifestieren sich häufiger in der Gallenblase als in den extrahepatischen Gallenwegen. Wir berichten hier von einem äußerst seltenen undifferenzierten Karzinom des distalen Gallengangs vom Spindel- und Riesenzelltyp. Der Tumor ging in Form atypischer Drüsen und Einzelzellkomplexe aus dem biliären Epithel hervor und graduell in eine vorwiegend spindelzellige Architektur über, die mehr als 98 % des Tumorvolumens ausmachte. Fokal gelangten osteoklastäre, multinukleäre Riesenzellen zur Darstellung. Die immunhistochemischen Untersuchungen zeigten in den besser differenzierten, drüsigen Tumoranteilen eine kräftige Expression epithelialer Marker, die in den spindelzelligen Arealen bei Hinzutreten einer Positivität für Vimentin nur noch einzelzellig erhalten blieb. Der Tumor wies eine hohe Ki-67-Proliferationsaktivität auf und zeigte Mutationen in den Genen Cyclin D3 (CCND3), Fibroblast Growth Factor Receptor 4 (FGFR4), Neurofibromin 1 (NF1) und NOTCH3. Der hier vorgestellte Fall unterstreicht die Relevanz dieser Tumorentität jenseits von Pankreas und Gallenblase und verdeutlicht die zur Diagnosefindung unverzichtbare Kombination aus Morphologie und Immunhistochemie.

Homologous recombination deficiency (HRD) leads to DNA double strand breaks and can be exploited by the use of PARP inhibitors to induce synthetic lethality of cancer cells. Over the last years, the clinical utility of this therapeutic concept was successfully demonstrated in ovarian, breast, prostate and pancreatic cancer. Currently, the role of HRD is investigated in trials testing immune checkpoint blockers alone or in combination with PARP inhibitors. But the relationship between HRD and immune cell contexture in cancer is incompletely understood. Here, we analyzed the association of HRD with tumor mutational burden (TMB), immune cell composition and gene expression in 10,000 tumors of 32 solid cancer types from the TCGA project. For each of the tumors, the HRD sum score (HRDsum) was calculated from allele-specific copy numbers (derived from genotyping data) and the TMB was calculated from the missense mutation calls (derived from WES data). HRDsum and tumor mutational burden (TMB) correlated positively pan-cancer (R=0.42) and within most cancer types. By contrast, HRD was absent in ultra-hypermutated (TMB >= 100 mut/Mb) tumors. Ultra-hypermutation was typically associated with microsatellite instability (MSI) or POLE/POLD1 mutation. Significant positive correlation of the HRDsum and immune cell infiltration was observed pan-cancer, but only within a few cancer types. Significant positive correlation of HRDsum and the T-cell inflamed gene expression profile was observed only in 7 of 32 cancer types: in breast cancer, ovarian cancer, low grade glioma, testicular germ cell tumors and three kinds of kidney cancer. A functional genomics analysis was carried out by correlating genome-wide gene expression data (derived from RNA-Seq) with HRDsum. The resulting lists of significantly correlating genes were analyzed for enrichment of 50 hallmark gene sets (catalog H, MSigDB). We detected simultaneous enrichment of two proliferation-related categories, E2F_TARGETS and G2M_checkpoint, for 13 of the 32 cancer types (ACC, BLCA, BRCA, KIRC, KIRP, LGG, LIHC, LUAD, LUSC, MESO, PRAD, SARC, THYM). The study shows that HRD is associated with immunological activation of the tumor microenvironment only in a minority of cancer types. Our data support further studies of immune activating therapies in combination with immune checkpoint blockade in the not intrinsically activated cancer types and advocate to combine different genomic and transcriptomic biomarkers (including HRD and TMB) for comprehensive molecular diagnostics and therapy guidance. Citation Format: Jan Budczies, Klaus Kluck, Susanne Beck, Iordanis Ouralidis, Michael Allgäuer, Michael Menzel, Eugen Rempel, Daniel Kazdal, Lukas Perkhofer, Alexander Kleger, Peter Schirmacher, Thomas Seufferlein, Albrecht Stenzinger. HRD is inversely correlated with MSI and identifies immunologically cold tumors in most cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 810.

6053 Background: Several studies have demonstrated a negative prognostic impact of tumor cell budding (TCB) in HPV- head and neck squamous cell carcinoma (HNSCC), but analyses of its prognostic impact in HPV+ HNSCC and of the underlying molecular alterations are lacking. Methods: The study cohort included 331 HPV- and HPV+ HNSCC from TCGA with digitalized H&E-stained slides available. Corresponding mutation, methylation, and gene expression data were obtained from the pan-cancer atlas web page. Tumor buds were defined as clusters of up to four tumor cells separated from the tumor mass. The numbers of tumor buds were evaluated in ten digital high-power fields by a senior pathologist. A two-tier cellular dissociation grading system was introduced by separating tumors with six or more tumor buds (TCB-high) from tumors with fewer tumor buds (TCB-low). The impact of TCB on overall survival (OS) was analyzed using Cox proportional hazard models. Results with p < 0.05 were considered significant. Association of TCB with mutations was analyzed using the Wilcoxon test. Association of TCB with gene expression and methylation was analyzed using Spearman correlations. Lists of altered genes were compiled correcting the p-values with the Benjamin-Hochberg method and controlling the FDR at 5%. Results: In a univariate analysis, OS was significantly shorter in TCB-high tumors compared to TCB-low tumors in HPV- HNSCC (HR = 1.4, 95% CI 1.0-2.3) and HPV+ HNSCC (HR = 5.0, 95% CI 1.9-13.7). Shorter OS in TCB-high HNSCC was confirmed in a multivariate analysis including age, sex, HPV status, tumor site, tumors stage, and tumor margin status (HR = 1.7, 95% CI 1.2-2.4). Significant association of TCB with mutations was detected for two genes: NSD1 mutations correlated negatively with TCB in HPV- HNSCC, while TP53 mutations correlated positively with TCB in HPV+ HNSCC. Methylation of 126 (1%) genes was associated with TCB in HPV- HNSCC, while methylation of 511 (3%) genes was associated with TCB in HPV+ HNSCC. Expression of 422 (2%) genes was associated with TCB in HPV- HNSCC, while expression of 786 (4%) genes was associated with TCB in HPV+ HNSCC. Among these genes, those annotated to the epithelial mesenchymal transition were highly significantly enriched in both HPV- HNSCC (5.5-fold enrichment) and HPV+ HNSCC (2.7-fold enrichment). Conclusions: Evaluation of TCB based on digital HE slides was conducted in a large clinically and molecularly characterized HNSCC cohort. The prognostic impact of TCB could be validated in HPV- HNSCC, while a prognostic impact of TCB could be demonstrated for the first time in HPV+ HNSCC. TCB correlated with mutations of only two genes and theses correlations were imperfect. A plethora of genes correlated with TCB on the level of methylation and gene expression level. These genes should be further analyzed and prioritized for the evaluation of therapeutic targeting.

e21141 Background: First-line pembrolizumab monotherapy is a standard treatment for metastatic non-small-cell lung cancer (NSCLC) with high PD-L1 expression (TPS ≥50%). However, many patients do not respond, and reliable biomarkers are lacking. Methods: Blood samples of 33 patients with metastatic NSCLC were analyzed at baseline (BL) and after four cycles of pembrolizumab monotherapy (FU). The expression of 12 genes involved in tumor-specific immune responses ( FAS, RORgt, FOXP3, IFNγ, FASLv1, PRF1, GATA3, PD1, CD247, GZMB, MKI67, TBX21) in whole blood RNA was quantified by RT-PCR in absolute terms using plasmids as standards. Results: Gene expression was significantly higher after immunotherapy for 11/12 genes analyzed ( FAS, RORgt, FOXP3, IFNγ, FASLv1, PRF1, GATA3, PD1, CD247, GZMB, TBX21 with fold changes [FC] 1.99 – 5.45; all p < 0.001). Blood neutrophil and total leukocyte counts were significantly decreased (ANC BL:7.65/nl vs. FU:5.36/nl, p = 0.024; Leu BL:10.65/nl vs. FU:7.78/nl, p = 0.032), while lymphocyte counts did not change (ALC BL:1.41/nl vs. FU:1.47/nl, p = 0.267). Increases in the expression of RORgt, FASLv1, PRF1, PD1, CD247, GZMB and TBX21 remained significant (FC 1.9 – 3.7; all p < 0.001) even after correction for the blood lymphocyte/leukocyte ratio (Ly/Lc BL:0.151 vs. FU:0.197, p = 0.003). Patients with long-term response (LTR, i.e. lasting > 6 months) had significantly higher increases in the expression of 8 genes ( RORgt, FOXP3, IFNγ, FASLv1, GATA3, PD1, CD247, TBX21) compared with patients with rapid disease progression within 3 months (RP) (FC 2.34 – 4.62, all p < 0.05). There were no significant differences (FU-BL) between LTR and RP regarding ANC (RP:-1.00/nl vs. LTR:-2.63/nl, p = 0.50), ALC (RP:-0,14/nl vs. LTR:+0,12/nl, p = 0,33) or Ly/Lc (RP:0.015 vs. LR:0.0542, p = 0.259). Conclusions: Pembrolizumab monotherapy of metastatic NSCLC causes significant increases in the blood expression for several genes including FAS, RORgt, FOXP3, IFNγ, FASLv1, PRF1, GATA3, PD1, CD247, GZMB, TBX21, as well as a decrease in circulating neutrophiles and total leucocytes. Long-term responders have more pronounced blood gene expression changes, which appear to correlate with long-term benefit, in contrast to blood count changes, which are not indicative. These results are currently being validated in a larger prospective study.

Tumor mutational burden (TMB) represents a new determinant of clinical benefit from immune checkpoint blockade that identifies responders independent of PD-L1 expression levels and is currently being explored in clinical trials. Although TMB can be measured directly by comprehensive genomic approaches such as whole-genome and exome sequencing, broad availability, short turnaround times, costs and amenability to formalin-fixed and paraffin-embedded tissue support the use of gene panel sequencing for approximating TMB in routine diagnostics. However, data on the parameters influencing panel-based TMB estimation are limited. Here, we report an extensive in silico analysis of the TCGA data set that simulates various panel sizes and compositions. We demonstrate that panel size is a critical parameter that influences confidence intervals (CIs) and cutoff values as well as important test parameters including sensitivity, specificity, and positive predictive value. Moreover, we evaluate the Illumina TSO500 panel, which will be made available for TMB estimation, and propose dynamic, entity-specific cutoff values based on current clinical trial data. Optimizing the cost-benefit ratio, our data suggest that panels between 1.5 and 3 Mbp are ideally suited to estimate TMB with small CIs, whereas smaller panels tend to deliver imprecise TMB estimates for low to moderate TMB (0-30 muts/Mbp), connected with insufficient separation of hypermutated tumors from non-hypermutated tumors.

Lung adenocarcinoma (ADC) is the most prevalent subtype of lung cancer and characterized by considerable morphological and mutational heterogeneity. However, little is known about the epigenomic intratumor variability between spatially separated histological growth patterns of ADC. In order to reconstruct the clonal evolution of histomorphological patterns, we performed global DNA methylation profiling of 27 primary tumor regions, seven matched normal tissues and six lymph node metastases from seven ADC cases. Additionally, we investigated the methylation data from 369 samples of the TCGA ADC cohort. All regions showed varying degrees of methylation changes between segments of different, but also of the same growth patterns. Similarly, copy number variations were seen between spatially distinct segments of each patient. Hierarchical clustering of promoter methylation revealed extensive heterogeneity within and between the cases. Intratumor DNA methylation heterogeneity demonstrated a branched clonal evolution of ADC regions driven by genomic instability with subclonal copy number changes. Notably, methylation profiles within tumors were not more similar to each other than to those from other individuals. In two cases, different tumor regions of the same individuals were represented in distant clusters of the TCGA cohort, illustrating the extensive epigenomic intratumor heterogeneity of ADCs. We found no evidence for the lymph node metastases to be derived from a common growth pattern. Instead, they had evolved early and separately from a particular pattern in each primary tumor. Our results suggest that extensive variation of epigenomic features contributes to the molecular and phenotypic heterogeneity of primary ADCs and lymph node metastases.

Summary A deeper understanding of melanoma biology has opened up new avenues for mechanistically informed therapies. However, data on the prevalence of druggable genetic lesions in melanoma are still conflicting and real-world performance data on high-throughput genetic profiling of melanoma cases using formalin fixed, paraffin embedded (FFPE) tissue with variable tumour cellularity and quality are lacking. We retrospectively analysed targeted next-generation sequencing data of 274 consecutive melanoma samples obtained for routine diagnostics between December 2013 and July 2017. Actionable mutations were detected in 197 cases (71.9%), of which activating BRAF (mostly p.V600E/K) and RAS (mostly p.Q61R/K) mutations occurred in 40.5% (n = 111) and 30.3% (n = 83) of cases, respectively. We identified driver mutations of the Triple-WT subgroup in 10.6% of cases (n = 29; 10 with activating KIT mutations). Median turnaround time was 5 working days with no dropouts. Tumour cellularity ranged from 5% to 95% and successful sequencing was possible at DNA concentrations as low as 0.03 ng/μL (median 10.58 ng/μL; range 0.03–209.05 ng/μL). Fast, quality-controlled high-throughput genetic profiling of FFPE melanoma samples is feasible and provides a landscape of genetic aberrations in melanoma that is currently relevant in clinical practice and approximates TCGA subtypes.

Increasingly extensive genomic diagnostics in cancer precision medicine require uniform evaluation criteria for the classification of variants with regard to their functional and therapeutic implications. In this review we present the most important guidelines and classification systems currently used in daily clinical practice, explain their advantages and disadvantages as well as differences and similarities, and present the step-by-step, systematic process that enables successful variant interpretation.Die immer umfangreichere genomische Diagnostik im Rahmen der Präzisionsonkologie erfordert einheitliche Bewertungskriterien für die Klassifizierung von Varianten im Hinblick auf ihre funktionelle Relevanz und therapeutischen Implikationen. In dieser Übersichtsarbeit stellen wir die wichtigsten derzeit gebräuchlichen Leitlinien und Klassifikationssysteme vor, erläutern Vor- und Nachteile sowie Unterschiede und Gemeinsamkeiten und beschreiben den schrittweisen, systematischen Prozess, der eine erfolgreiche Variantenbewertung ermöglicht.

Background Chemotherapy plus immune-checkpoint inhibitor (CTx+ICI) therapy has become the preferred 1st line treatment in patients with metastatic NSCLC without oncogenic driven mutations. However, the optimal subsequent 2nd line treatment is not defined and several alternatives exist. The purpose of this analysis was to evaluate the efficacy of 2nd line docetaxel plus ramucirumab (D+R) initiated after failure of 1st line CTx+ICI. Methods Retrospective data were collected during routine care from German thoracic oncology centers. Only patients who had received at least one course of 2nd line D+R were included. ORR, PFS, OS and numbers of courses of D+R were investigated with PFS after initiation of D+R being the primary endpoint. Results Seventy-seven patients met the inclusion criteria. 2nd line treatment with D+R achieved an ORR and DCR of 32.5% and 62.4%, respectively. Median PFS for 2nd line therapy was 3.9 months with a DOR of 6.4 months. Median OS of 15.5 and 7.5 months were observed from the start of 1st line therapy and 2nd line treatment, respectively. No unexpected toxicities occurred. Presence of KRAS mutations was associated with significantly worse median PFS to D+R (2.8 vs. 4.5 months in wild-type cases; P=0.021) and was an independent predictor of inferior PFS in multivariate analysis. Conclusions D+R is an effective and safe 2nd line treatment after failure of 1st line CTx+ICI irrespective of NSCLC histology. However, patients with a KRAS mutation did not benefit from D+R in terms of PFS and will require further investigations.

Biphenotypic sinonasal sarcoma (BSNS) is a rare, low grade spindle cell sarcoma, recently recognized in the WHO classification of head and neck tumors, which is characterized by a dual myogenic and neural differentiation and recurrent gene fusions, often involving PAX3-MAML3, and less commonly PAX3 fusions with other partners such as NCOA1, NCOA2, or WWTR1. Yet, in about 4% of tumors no gene rearrangements are identified. Herein, we describe a RREB1-MKL2 fusion in a BSNS lesion occurring in a 73-year-old female patient with a right maxillo-ethmoidal angle lesion. The polypoid, moderately cellular tumor with infiltrative submucosal growth was composed of fascicles of relatively bland spindle cells embedded in a loose collagenous matrix. The tumor cells showed moderate amounts of eosinophilic cytoplasm with indistinct borders and uniform, pale, ovoid to slender nuclei. The slowly proliferating neoplastic cells co-expressed smooth muscle actin and S100, and showed focal nuclear positivity for s-catenin, while lacking staining for cytokeratins, desmin, myogenin, caldesmon, glial fibrillary acid protein, and SOX-10. Molecular analysis by targeted RNA-based next-generation sequencing identified an in-frame fusion between exon 8 of RREB1 and exon 11 of MKL2, a genetic event that was reported to be a molecular hallmark of ectomesenchymal chondromyxoid tumor. Gene rearrangements in both genes were independently verified by fluorescence in situ hybridization (FISH). To evaluate its recurrent potential an additional group of 15 fusion negative BSNS were tested for abnormalities in RREB1 and MKL2 genes by FISH, but no additional positive cases were identified. This article is protected by copyright. All rights reserved.

Background Mutations in DNA damage repair genes, in particular genes involved in homology-directed repair, define a subgroup of men with prostate cancer with a more unfavorable prognosis but a therapeutic vulnerability to PARP inhibition. In current practice, mutational testing of prostate cancer patients is commonly done late i.e., when the tumor is castration resistant. In addition, most sequencing panels do not include TP53, one of the most crucial tumor suppressor genes in human cancer. In this proof-of-concept study, we sought to extend the clinical use of these molecular markers by exploring the early prognostic impact of mutations in TP53 and DNA damage repair genes in men with primary, nonmetastatic prostate cancer undergoing radical prostatectomy (RPX). Methods Tumor specimens from a cohort of 68 RPX patients with intermediate (n = 11, 16.2%) or high-risk (n = 57, 83.8%) disease were analyzed by targeted next generation sequencing using a 37 DNA damage repair and checkpoint gene panel including TP53. Sequencing results were correlated to clinicopathologic variables as well as PSA persistence or time to PSA failure. In addition, the distribution of TP53 and DNA damage repair gene mutations was analyzed in three large publicly available datasets (TCGA, MSKCC and SU2C). Results Of 68 primary prostate cancers analyzed, 23 (33.8%) were found to harbor a mutation in either TP53 (n = 12, 17.6%) or a DNA damage repair gene (n = 11, 16.2%). The vast majority of these mutations (22 of 23, 95.7%) were detected in primary tumors from patients with high-risk features. These mutations were mutually exclusive in our cohort and additional data mining suggests an enrichment of DNA damage repair gene mutations in TP53 wild-type tumors. Mutations in either TP53 or a DNA damage repair gene were associated with a significantly worse prognosis after RPX. Importantly, the presence of TP53/DNA damage repair gene mutations was an independent risk factor for PSA failure or PSA persistence in multivariate Cox regression models. Conclusion TP53 or DNA damage repair gene mutations are frequently detected in primary prostate cancer with high-risk features and define a subgroup of patients with an increased risk for PSA failure or persistence after RPX. The significant adverse impact of these alterations on patient prognosis may be exploited to identify men with prostate cancer who may benefit from a more intensified treatment.

Background: Epidermal growth factor receptor-mutated (EGFR+) non-small-cell lung cancer (NSCLC) patients failing tyrosine kinase inhibitors (TKI) can benefit from next-line targeted therapies, but implementation is challenging. Methods: EGFR+ NSCLC patients treated with first/second-generation (1G/2G) TKI at our institution with a last follow-up after osimertinib approval (February 2016), were analyzed retrospectively, and the results compared with published data under osimertinib. Results: A total of 207 patients received erlotinib (37%), gefitinib (16%) or afatinib (47%). The median age was 66 years, with a predominance of female (70%), never/light-smokers (69%). T790M testing was performed in 174/202 progressive cases (86%), positive in 93/174 (53%), and followed by osimertinib in 87/93 (94%). Among the 135 deceased patients, 94 (70%) received subsequent systemic treatment (43% chemotherapy, 39% osimertinib), while 30% died without, either before (4%) or after progression, due to rapid clinical deterioration (22%), patient refusal of further therapy (2%), or severe competing illness (2%). Lack of subsequent treatment was significantly (4.5x, p Conclusion: Osimertinib after 1G/2G TKI failure prolongs survival, but approximately 15% and 30% of patients forego molecular retesting and subsequent treatment, respectively, mainly due to rapid clinical deterioration. This is an important remediable obstacle to sequential TKI treatment for EGFR+ NSCLC. It pertains also to other actionable resistance mechanisms emerging under 1G/2G inhibitors or osimertinib, whose rate for lack of next-line therapy is similar (approximately 35% in the FLAURA/AURA3 trials), and highlights the need for closer monitoring alongside broader profiling of TKI-treated EGFR+ NSCLC in the future.

Journal of medical internet research 23(2), e23436 (2021). doi:10.2196/23436, Published by Healthcare World, Richmond, Va.

With this external quality assessment (EQA) scheme, we aim to investigate the diagnostic performance of the currently available methods for the detection of ALK alterations in non-small cell lung cancer on a national scale, namely, in situ hybridization (ISH), immunohistochemistry (IHC), and RNA/DNA sequencing (NGS). The EQA scheme cohort consisted of ten specimens, including four ALK positive and six ALK negative samples, which were thoroughly pretested using IHC, ISH, and RNA/DNA NGS. Unstained tumor sections were provided to the 57 participants, and the results were retrieved via an online questionnaire. ISH was used by 29, IHC by 38, and RNA/DNA sequencing by 19 participants. Twenty-eight institutions (97%) passed the ring trial using ISH, 33 (87%) by using IHC, and 18 (95%) by using NGS. The highest sensitivity and interrater agreement (Fleiss ‘ kappa) was observed for RNA/DNA sequencing (99%, 0.975), followed by ISH (94%, 0.898) and IHC (92%, 0.888). However, the proportion of samples that were not evaluable due to bad tissue quality was also higher for RNA/DNA sequencing (4%) compared with ISH (0.7%) and IHC (0.5%). While all three methods produced reliable results between the different institutions, the highest sensitivity and concordance were observed for RNA/DNA sequencing. These findings encourage the broad implementation of this method in routine diagnostic, although the application might be limited by technical capacity, economical restrictions, and tissue quality of formalin-fixed samples. Supplementary Information The online version contains supplementary material available at 10.1007/s00428-021-03106-5.