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Homologous repair deficiency (HRD) is present in many cancer types at variable prevalence and can indicate response to platinum-based chemotherapy and PARP inhibition. We developed a tumor classification system based on the loss of function of genes in the homologous recombination repair (HRR) pathway. To this end, somatic and germline alterations in BRCA1/2 and 140 other HRR genes were included and assessed for the impact on gene function. Additionally, information on the allelic hit type and on BRCA1 promoter hypermethylation was included. The HRDsum score including LOH, LST, and TAI was calculated for 8847 tumors of the TCGA cohort starting from genotyping data and for the subcohort of ovarian cancer also starting from WES data. Pan-cancer, deleterious BRCA1/2 alterations were detected in 4% of the tumors, while 18% of the tumors were HRD-positive (HRDsum ≥ 42). Across 33 cancer types, both BRCA1/2 alterations and HRD-positivity were most prevalent in ovarian cancer (20% and 69%). Pan-cancer, tumors with biallelic deleterious alterations in BRCA1/2 were separated strongly from tumors without relevant alterations (AUC = 0.89), while separation for tumors with monoallelic deleterious BRCA1/2 alterations was weak (AUC = 0.53). Tumors with biallelic deleterious alterations in other HHR genes were separated moderately from tumors without relevant alterations (AUC = 0.63), while separation for tumors with such monoallelic alterations was weaker (AUC = 0.57). In ovarian cancer, HRDsum scores calculated from WES data correlated strongly with HRDsum scores calculated from genotyping data (R = 0.87) and were slightly (4%) higher. We comprehensively analyzed HRD scores and their association with mutations in HRR genes in common cancer types. Our study identifies important parameters influencing HRD measurement and argues for an integration of HRDsum score with specific mutational profiles.

Ki-67 has been clinically validated for risk assessment in breast cancer, but the analytical validation and cutpoint-definition remain a challenge. Intraclass correlation coefficients (ICCs) are a statistical parameter for Ki-67 interobserver performance. However, the maximum degree of variance among pathologists allowed for meaningful biomarker results has not been defined. Different amounts of variance were added to central pathology Ki-67 data (n = 9069) from three cohorts (IBCSGVIII + IX, BIG1-98, GeparTrio) by simulation of 4500 evaluations for each cohort, which were grouped by ICCs, ranging from excellent (ICC = 0.9) to poor concordance (ICC = 0.1). Endpoints were disease-free survival (DFS) and pathological complete response (pCR, GeparTrio). Ki-67 was a significant continuous prognostic marker for DFS over a wide range of cutpoints between 8% and 30% in all three cohorts. In our modelling approach, Ki-67 was a stable prognostic marker despite increased interpathologist variance. Even for a poor ICC of 0.5, one or more significant Ki-67 cutoffs were detected in 86.8% (GeparTrio), 92.4% (IBCSGVIII + IX) and 100% of analyses (BIG1-98). Similarly, in GeparTrio, even with an extremely low ICC of 0.2, 99.6% of analyses were significant for pCR. Our study shows that Ki-67 is a continuous marker which is extremely robust to pathologist variation. Even if only 50% of variance is attributable to true Ki-67-based proliferation (ICC = 0.5), this information is sufficient to obtain statistically significant differences in clinical cohorts. This stable performance explains the observation that many Ki-67 studies achieve significant results despite relevant interobserver variance and points to a high clinical validity of this biomarker. For clinical decisions based on analysis of individual patient data, ongoing efforts to further reduce interobserver variability, including ring trials and standardized guidelines as well as image analysis approaches, should be continued.

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.

Background Pre-operative treatment planning in head and neck squamous cell carcinoma (HNSCC) is mainly dictated by clinical staging, which has major shortcomings. Histologic grading is irrelevant due to its lack of prognostic impact. Recently, a novel grading termed Cellular Dissociation Grade (CDG) based on Tumour Budding and Cell Nest Size was shown to be highly prognostic for resected HNSCC. We aimed to probe the predictive and prognostic impact of CDG in the pre-operative biopsies of HNSCC. Methods We evaluated CDG in n = 160 pre-therapeutic biopsies from patients who received standardised treatment following German guidelines, and correlated the results with pre- and post-therapeutic staging data and clinical outcome. Results Pre-operative CDG was highly predictive of post-operative tumour stage, including the prediction of occult lymph node metastasis. Uni- and multivariate analysis revealed CDG to be an independent prognosticator of overall, disease-specific and disease-free survival (p Conclusions CDG is a strong outcome predictor in the pre-treatment scenario of HNSCC and identifies patients with nodal-negative disease. CDG is a purely histology-based prognosticator in the pre-therapeutic setting that supplements clinical staging and may aide therapeutic stratification of HNSCC patients.

Background Tumor-infiltrating lymphocytes (TILs) have been identified as prognostic parameter in breast cancer. Objectives The aim of this review article is to provide an overview on the clinical and analytical validation of TILs in breast cancer. Materials and methods Summary of international guidelines of the TIL working group as well as clinical and translational studies. Results, conclusions Breast carcinomas with a high TIL level have an improved response to neoadjuvant chemotherapy. Triple-negative and HER2-positive carcinomas with increased TIL levels have improved survival. TILs are a new prognostic biomarker for routine histopathological diagnosis.

In Germany it is required by law that basically every type of physician needs to be capable of executing a correct external post-mortem examination of a corpse. In recent years, numerous investigations on external post-mortem examinations repeatedly reported systematic mistakes and erroneous procedures in various clinical and medicolegal case groups. Accordingly, the completion of death certificates is frequently performed incorrectly. As one of the typical unnatural death cases, decedents dying from fatal head trauma (FHT) represent a special autopsy case group, which is expected to be correctly recognized during the primary external post-mortem examination because the external injuries are mostly obvious.The present study aimed at investigating the quality of the external post-mortem examination in medicolegal FHT cases by means of comparison of death certificates and autopsy reports from a 10-year period.In a retrospective study design all autopsy cases from the Institute of Legal Medicine of the University Hospital Münster in the years 2006-2015 (n = 3611) were analyzed as to the presence of FHT. A total of 328 cases with FHT and the concomitant presence of a death certificate filled out before the autopsy were identified. Subsequently, the cause of death according to the death certificate was compared with the cause of death according to the autopsy. The degree of agreement was classified into six different categories from I to VI. While category I represented a complete lack of agreement, category VI was assigned to cases with full agreement.In 58.5% of the cases (category VI) FHT was identified correctly during the external post-mortem examination. In 1.5% of the cases, a completely different cause of death was determined during the external post-mortem examination (category I). In 19.2% of the cases, no cause of death or the statement "unclear" was given as the cause of death in the death certificate (categories II and III). Cross-analyses and intuitive heatmap visualization were generated to identify case constellations with an increased risk for discrepancies. These analyses revealed that among all discrepant cases (categories I-V), falls were found significantly more often than in the nondiscrepant cases (p 0.01), especially falls of women older than 57 years (median age of women) or falls considered as accidents by the examiner. In addition, traffic-associated FHT of men older than 44.5 years (median age of men) was identified more frequently in the external post-mortem examination.Despite the fact that FHT should be a cause of death that is comparably easy to identify during external post-mortem examination, more than one third of the cases were not sufficiently recognized. Therefore, special attention must still be paid to certain case constellations during the external post-mortem examination. Typical examples of such cases are burned bodies, cases of advanced putrefaction and falls.

Precision oncology is obtaining a central role in the therapy of malignant diseases. The indication for targeted therapy is based on the identification of molecular targets for which next-generation sequencing (NGS) is commonly used nowadays. All approved predictive biomarkers and molecular targets, including gene fusions and copy number alterations, can be identified depending on panel design and method applied. Some clinical scenarios, however, may require more holistic genomic approaches, such as whole-genome/whole-exome and transcriptome analysis, which must be embedded in a clinical trial. Here, key aspects and applications of each method are summarized and discussed.

Background: Phosphatidylinositol 3-kinase mutations (PIK3CA) are common in breast cancer (BC). Mutations are predominantly found in hot-spots located in the helical and kinase domains (exons 9 and 20). We recently demonstrated that PIK3CA mutations predict lower pathological complete response (pCR) to double blockade with trastuzumab/lapatinib in HER2+ve primary BC. Methods: We evaluated PIK3CA mutations in 293/403 HER2+ve tumors of participants of the neoadjuvant GeparSepto (G7) study (Untch et al. SABCS 2014). The G7 study investigated the effect of exchanging paclitaxel for nab-paclitaxel prior to EC. All patients received trastuzumab and pertuzumab. The G7 study showed a significantly higher pCR rate in patients receiving nab-paclitaxel. HER2, hormone receptors (HR), Ki67 and tumor infiltrating lymphocytes (TILs) were centrally assessed prior to randomization. PIK3CA mutations in exons 9 and 20 were evaluated in formalin-fixed, paraffin embedded core biopsies taken before therapy using deep targeted massive parallel sequencing with a minimum coverage of 500 and a mean coverage of 6520 and 6346 per amplicon, (exon9 and exon 20). Only non-synonymous mutations in the coding region that were called at variant allele frequency ≥10% were taken into consideration. Only cases with a tumor cell content of ≥20% were included. Results: In the G7 study, 396 patients with HER2+ve BC have been randomized from 06/2012 to 01/2014 and started treatment. From these 293 could be sequenced. Median age in the analyzed cohort was 49 years (range 22-75); most tumors were cT1-2 (89.9%); cN0 (54.4%); ductal invasive (88.7%), grade 3 (53.9%), HR+ve (69.6%), Ki67>20% (69.3%), LPBC-negative (83.2%). Overall, 22.2% of the tumors were found to have a PIK3CA mutation, 20.1% in HR+ve and 27.0% in HR-ve. Overall, the pCR rate was significantly lower in the PIK3CA mutant tumors compared to the wild type (wt) group (47.7% vs. 66.7%; p=0.009). This effect was seen both in the HR+ve (43.9% vs. 61.3%; p=0.052) and the HR-ve population (54.2% vs. 80.0%; p=0.029). There was also a significant difference in pCR according to PIK3CA mutation status dependant on the taxane. In the nab-paclitaxel group, pCR rates were significantly lower in patients with PIK3CA mutations compared to those without PIK3CA mutations (38.7% vs. 72.0%; p=0.001), whereas in the paclitaxel group, there was no significant difference between patients with and without a PIK3CA mutation (55.9% vs. 60.9%; p=0.690). The respective interaction could be demonstrated in univariate (p=0.039) as well as multivariate regression analysis (p=0.010) after adjusting for known baseline factors. Conclusion: Patients with PIK3CA mutant HER2+ve BC have a significantly lower pCR rate compared to patients with wt tumors. In contrast to the results with double anti-HER2 blockade consisting of trastuzumab/lapatinib, the effect was evident irrespective of the HR status. In addition, PIK3CA mutation status was significantly associated with higher pCR following nab-paclitaxel. The project has partly been funded within the EU-FP7 project RESPONSIFY No 278659 and the German Cancer Consortium (DKTK). Citation Format: Loibl S, Budczies J, Weichert W, Furlanetto J, Stenzinger A, Pfarr N, von Minckwitz G, Jackisch C, Schneeweiss A, Fasching P, Schmatloch S, Aktas B, Nekljudova V, Weber K, Untch M, Denkert C. PIK3CA mutations predict resistance to trastuzumab/pertuzumab and nab-paclitaxel in primary HER2-positive breast cancer – Massive parallel sequencing analysis of 293 pretherapeutic core biopsies of the GeparSepto study. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-03.

Tumor-infiltrating lymphocytes (TILs) have been identified as prognostic parameter in breast cancer.The aim of this review article is to provide an overview on the clinical and analytical validation of TILs in breast cancer.Summary of international guidelines of the TIL working group as well as clinical and translational studies.Breast carcinomas with a high TIL level have an improved response to neoadjuvant chemotherapy. Triple-negative and HER2-positive carcinomas with increased TIL levels have improved survival. TILs are a new prognostic biomarker for routine histopathological diagnosis.

Tyrosine kinase inhibitors (TKI) have improved prognosis in metastatic anaplastic lymphoma kinase (ALK)-driven lung adenocarcinoma, but patient outcomes vary widely. We retrospectively analyzed the clinical course of all cases with assessable baseline TP53 status and/or ALK fusion variant treated at our institutions (n = 102). TP53 mutations were present in 17/87 (20%) and the echinoderm microtubule-associated protein-like 4 (EML4)-ALK variant 3 (V3) in 41/92 (45%) patients. The number of metastatic sites at diagnosis was affected more by the presence of V3 than by TP53 mutations, and highest with both factors (mean 5.3, p0.001). Under treatment with ALK TKI, progression-free survival (PFS) was shorter with either TP53 mutations or V3, while double positive cases appeared to have an even higher risk (hazard ratio [HR] = 2.9, p = 0.015). The negative effect of V3 on PFS of TKI-treated patients was strong already in the first line (HR = 2.5, p = 0.037) and decreased subsequently, whereas a trend for PFS impairment under first-line TKI by TP53 mutations became stronger and statistically significant only when considering all treatment lines together. Overall survival was impaired more by TP53 mutations (HR = 4.9, p = 0.003) than by V3 (HR = 2.4, p = 0.018), while patients with TP53 mutated V3-driven tumors carried the highest risk of death (HR = 9.1, p = 0.02). Thus, TP53 mutations and V3 are independently associated with enhanced metastatic spread, shorter TKI responses and inferior overall survival in ALK

Purpose Rapidly evolving genomics technologies, in particular comprehensive next-generation sequencing (NGS), have led to exponential growth in the understanding of cancer biology, shifting oncology toward personalized treatment strategies. However, comprehensive NGS approaches, such as whole-exome sequencing, have limitations that are related to the technology itself as well as to the input source. Hence, clinical implementation of comprehensive NGS in a quality-controlled diagnostic workflow requires both the standardization of sequencing procedures and continuous validation of sequencing results by orthogonal methods in an ongoing program to enable the determination of key test parameters and continuous improvement of NGS and bioinformatics pipelines. Patients and Methods We present validation data on 220 patients who were enrolled between 2013 and 2016 in a multi-institutional, genomics-guided precision oncology program (Molecularly Aided Stratification for Tumor Eradication Research) of the National Center for Tumor Diseases Heidelberg and the German Cancer Consortium. Results More than 90% of clinically actionable genomic alterations identified by combined whole-exome sequencing and transcriptome sequencing were successfully validated, with varying frequencies of discordant results across different types of alterations (fusions, 3.7%; single-nucleotide variants, 2.6%; amplifications, 1.1%; overexpression, 0.9%; deletions, 0.6%). The implementation of new computational methods for NGS data analysis led to a substantial improvement of gene fusion calling over time. Conclusion Collectively, these data demonstrate the value of a rigorous validation program that partners with comprehensive NGS to successfully implement and continuously improve cancer precision medicine in a clinical setting.

Background: Ki67 has been suggested as a marker for diagnosis of luminal A and B breast carcinomas. Interestingly, on one hand a multitude of studies have described significant results for Ki67 as a prognostic marker, while on the other hand the analytical validation and standardization of this marker has been a challenge. The best parameter for Ki67 interobserver performance is the interclass correlation coefficient (ICC). ICC values between 0.59 and 0.92 have been reported. Recently a minimum ICC of 0.8 has been suggested as a goal for the international ring trial and as a prerequisite for introduction of Ki67 into clinical practice. However, this suggested ICC is not derived from analysis of data, and the amount of pathologist variance that is allowed for meaningful biomarker results is still not defined. Methods: This study is based on a total of 9069 tumor samples from three large clinical cohorts (IBCSG VIII+IX, BIG1-98, and GeparTrio). In a systematic modeling approach, we introduced different amounts of variance to previously generated central pathology Ki67 datasets by simulation of a total of 1800 different pathologist evaluations for each study cohort. These evaluations were grouped into groups with defined ICCs, ranging from very good concordance (ICC=0.9) to extremely poor concordance (ICC=0.1). For each of the simulated pathologist evaluations, all possible Ki67 cutoffs were systematically evaluated using the web-based software Cutoff Finder (http://molpath.charite.de/cutoff/). As endpoints, we used DFS for all three study cohorts as well as pCR for the neoadjuvant cohort. Results: For the neoadjuvant GeparTrio study, the different groups with ICCs of 0.8, 0.6 and 0.4 showed a very similar performance resulting in significant analyses for prediction of pCR across a wide range of cutoffs. The odd ratios for pCR were slightly lower with lower ICC. Even with an extremely low ICC of 0.2, 99% of the analyses had one or more significant cutpoints. The survival endpoint DFS was shown to be very stable despite increased interpathologist variance in all three clinical cohorts. Even with a poor ICC of 0.4, the majority of cutpoints were significant for DFS. For IBCSG VIII+IX 85% of the analyses with an ICC of 0.4 had one or more significant cutpoints for Ki67. In the large BIG 1-98 dataset (n=6090) even an ICC of 0.2 resulted in one or more significant DFS cutpoints in 100% of the analyses. Comparable results were obtained if the analysis was restricted to luminal tumors. Conclusion: Our results suggest that Ki67 is extremely robust to pathologist variation. Even if less than 40% of the variance is attributable to true Ki67-based proliferation (ICC Citation Format: Denkert C, Budczies J, Regan M, Loibl S, Dell'Orto P, von Minckwitz G, Mastropasqua M, Mehta K, Müller V, Kammler R, Pfitzner BM, Fasching PA, Viale G. Systematic analysis and modulation of Ki67 interobserver variance in 9069 patients from three clinical trials – How much pathologist concordance is needed for meaningful biomarker results?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-02.

Introduction: Tumor mutational burden (TMB) is an emerging biomarker to identify patients more likely to benefit from immuno-oncologic therapy. Therefore, academia- and industry-driven endeavors are underway to establish mostly panel-based sequencing solutions. Aside from various unsettled technical aspects, tumor biology itself might play an important role in determining a tumor’s TMB. Intratumor heterogeneity (ITH) is frequent in pulmonary adenocarcinoma (ADC) and seems to be a key factor regarding response failure and resistance development during therapy. In addition, ITH might contribute to inconsistent distribution of a biomarker and therefore an assay’s readout might depend on the region of tumor which is submitted for testing. Therefore, the assessment of ITH is an essential step in the evaluation of new diagnostic applications. Experimental Set-up: In order to investigate potential spatial differences in the TMB of a tumor, we analyzed a comprehensively morpho-molecularly characterized cohort of pulmonary ADC by sequencing 3-4 sections per tumor including lymph node metastases. So far 10 tumors were included in the ongoing analysis. None of the selected tumors had an oncogenic EGFR mutation, six were KRAS positive and four had no known driver mutation. TMB was determined applying the TruSightTM Oncology 500 targeted sequencing panel (TSO 500), covering 500 genes, with a NextSeq 500 sequencing system (both Illumina Inc., San Diego, CA, USA). Results Summary: The current sequencing analysis using the TSO 500 panel covered an average genomic coding region of 1.26 Mbp with a mean read depth of x658. The average TMB of all samples was 8.55 mut/Mbp (range 0.79 - 18.95). Comparing different regions within a given tumor, we detected a considerable variance of TMB (± 5 mut/Mbp) in about half of the analyzed cases, with an overall mean absolute deviation of 2.2 and a maximum difference of 12.63 mut/Mbp. However, in some samples an in depth evaluation of morpho-molecular characteristics revealed several factors impairing TMB read out. Aside from technical issues like insufficient depth of coverage in combination with unadjusted automated variant calling, sample characteristics like tumor cell content had a prominent influence on TMB values. Often, tumor regions with lower TMB turned out to have lower tumor cell content, which led to decreased allele frequencies of tumor specific mutations and subsequently compromised mutation detection. Conclusions: Our data demonstrate spatial heterogeneity of TMB in pulmonary ADC, which can impact clinical decision making and therapy. However, spatial genetic heterogeneity reflecting tumor biology needs to be carefully dissected from technical conditions resulting in pseudo-heterogeneity. Citation Format: Volker Endris, Michael Allgäuer, Jan Budczies, Jonas Leichsenring, Anna-Lena Volckmar, Martina Kirchner, Olaf Neumann, Regine Brandt, Eugen Rempel, Carolin Plöger, Moritz von Winterfeld, Alexander Harms, Mark Kriegsmann, Roland Penzel, Peter Schirmacher, Daniel Kazdal, Albrecht Stenzinger. Spatial heterogeneity of tumor mutational burden (TMB) counts in pulmonary adenocarcinoma: Separating biological effects from technical artifacts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4101.

e13026 Background: Cancer of unknown primary (CUP) denotes a malignancy with histologically confirmed metastatic spread while the primary tumor remains elusive. Given the typically dismal response to empiric chemotherapy, the deciphering of molecular profiles holds a particular promise for targeted therapy. Here, we address the prognostic implications of mutations and copy number variations (CNVs) detected in tumor tissue in the context of a comprehensive clinical risk assessment. Methods: We performed targeted panel sequencing in a series of 252 CUP patients. Median age was 60.3 years and 59.1% were female. 71.2% of patients had unfavorable CUP. Lymph nodes, bone marrow, lung, peritoneum and pleura were the most frequent metastatic sites. Among histologic subtypes, adeno- (74.7%) and squamous cell carcinomas (13.7%) prevailed. DNA was extracted from micro-dissected formalin-fixed, paraffin-embedded tissue samples. For library preparation mostly multiplex PCR-based Ion Torrent AmpliSeq technology (Life Technologies) with Oncomine comprehensive assays v1 and v3 was used. Results: The prevailing genetic alterations were deletions in tumor suppressor genes TP53 (49.6%), CDKN2A (19.0%) and NOTCH1 (14.1%) as well as oncogenic activation of KRAS (23.4%), FGFR4 (14.9%) and PIK3CA (10.7%). KRAS activation was predominantly found in adenocarcinomas (p = 0.01) and PIK3CA activation in squamous cell carcinomas (p = 0.03). Regarding prognosis, male sex, high ECOG score, unfavorable CUP subtype, a higher number of involved organs as well as RAS activation predicted decreased event-free and overall survival in multivariate analysis. Deletions in CDKN2A were a poor prognostic factor regarding overall survival. Markedly, TP53 mutations did not significantly influence prognosis. Since July 2015, 17/198 (8.6%) patients received a recommendation for molecularly guided therapy. However, due to lack of coverage by insurers or rapid clinical deterioration only 7 of these patients (3.5%) actually received targeted treatment. Conclusions: Besides offering targets for therapy, targeted panel sequencing in CUP is prognostically relevant, with RAS activation and CDKNA2 deletion emerging as independent risk factors in a comprehensive assessment with clinical risk factors.

2632 Background: Tumor mutational burden (TMB) is an emerging biomarker in immuno-oncology (IO). Panel sequencing (PS) is a promising approach for its implementation in clinical practice. Methods: We analyzed TMB as predictor of IO response in a multi-cancer cohort published by Miao et. al. The performance of three large panels (Illumina TSO500, Qiagen TMB [QIAseq] and Oncomine TMB [OTMB]) and two small panels (Illumina TST170 and Oncomine Comprehensive Assay [OCAv3]) was compared to WES by in silico simulations. Separation of responders (PR/CR) from non-responders (PD) was analyzed in the multi-cancer cohort (n = 193), in the lung cancer (n = 36) and in the melanoma (n = 125) subcohort. We also simulated PS in the TCGA pan-cancer cohort. Results: In lung cancer, TMB was strongly predictive for IO response (area under ROC curve [AUC] 0.78-0.94). WES performed (borderline)-significantly better than PS for all five panels (OCAv3: p = 0.011, TST170: p = 0.01, QIAseq: p = 0.048, OTMB: p = 0.063, TSO500: p = 0.11). For the cut-point of 199 mutations, misclassification rates compared to WES (16.7%) were borderline-significantly higher for the small panels OCAv3 (33.3%, p = 0.087) and TST170 (36.1%, p = 0.054), but not for the large panels. In melanoma, TMB was moderately predictive (AUC 0.58-0.63) and WES performed (borderline)-significantly better than the OCAv3, TST170, QIAseq and TSO500 panels. In the multi-cancer cohort, WES did not perform better than PS. TBM estimates from PS include an inherent fuzziness originating from restriction to a limited part of the coding sequence. Based on a random mutation model, we derived a mathematical formula for the coefficient of variation (CV) of TMB: The CV decreases inversely proportional with both the square root of the TMB level and with the square root of the panel size. We showed that the mathematical law is valid for real-word mutation data. Conclusions: Small panels (size < 1 Mpb) performed imprecise in diagnostic TMB estimation. Even using the largest commercially available panels it can be challenging to capture the full predictive information of TMB. The detrimental effect of small panel size can be addressed by using larger panels, but halving the CV of TMB necessitates quadruplication of the panel size.

Metabolomics, the newest of the omics sciences that also include genomics, transcriptomics and proteomics, has matured into a reliable high-throughput technology. Gas chromatography combined with time-of-flight mass spectrometry (GC-TOFMS) is a suitable method to analyze the central metabolism in fresh frozen tumor tissue samples. Bioinformatics methods, including the PROFILE clustering developed by us, permit integrated analysis and fast interpretation of metabolomics data in the context of enzymatic reactions and metabolic pathways. The metabolome analyses of three solid tumor types presented here, together with the results of other authors, show that metabolites are suitable as biomarkers and provide diverse options for translation into the clinical setting.

Background:Currently, breast cancer during pregnancy (BCP) is not believed to be biologically different from breast cancer unrelated to pregnancy based on limited datasets mainly obtained by immunohistochemistry. However, some groups report that BCP patients have an inferior survival compared to young non-pregnant breast cancer patients. The largest analysis based on the BCP registry by the German Breast Group (GBG) revealed however, no difference between pregnant and non-pregnant breast cancer patients, indicating that treatment rather than biology might be the reason for the inferior survival reported by others. Methods: The BCP study (GBG 29/BIG 03-02) is a multicentre observational study for breast cancer during pregnancy. In tumour tissue collected within this study from pregnant M0 patients we investigated the following genes: AKT1, ATM, BRAF, CBFB, CCND1, CDH1, CDKN2A, CTCF, EGFR, ERBB2, ESR1, FGFR2, GATA3, KRAS, MAP2K4, MAP3K1, MDM2, MED12, MYC, PIK3CA, PIK3R1, PTEN, RB1, RUNX1, and TP53 by massive parallel sequencing (MPS). This included patients with all molecular subtypes: HR+/-, HER2+/-. Sequencing was done on an IonTorrent Proton using a custom designed Breast Cancer Panel (BCPv2). This panel comprises 236 amplicons split into two primer pools and covers hotspot regions of 138 exons of the 25 genes. To test the hypothesis that breast cancer diagnosed during pregnancy is biologically not different from breast cancer diagnosed in young non-pregnant women, we compared the molecular profiles obtained, with genetic data from M0 patients not known to be pregnant from TCGA with age Results: Material from 141 patients from the BCP study was available from which ultimately 109 fully evaluable MPS datasets could be obtained. In the TCGA data set 114 breast cancer patients Exact matching by variables age, HR, HER2 and grade yielded 40 patients from both datasets. In these subcohorts, still divergent mutational rates for TP53 and PIK3CA between pregnant and non-pregnant women were noted, however, the differences failed to reach statistical significance. Conclusions: Overall the mutational landscapes do not seem to be overtly different between pregnant patients and no-pregnant controls, although slight imbalances in mutational rates occurred, which might be partly explained by a selection bias and a small sample size after matching. Further comparisons using other datasets, looking into survival and regarding copy number variation are currently conducted. This research is been funded by the German Cancer Consortium-DKTK and the BANSS Foundation. Citation Format: Loibl S, Pfarr N, Weber K, Neunhöffer T, Villegas S, Stenzinger A, Furlanetto J, Aktas B, Budczies J, Marmé F, Kahmann L, Denkert C, Weichert W. Comparison of the mutational landscape of breast cancer during pregnancy and non-pregnant controls [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-03-09.

Cancer medicine relies on the paradigm that cancer is an organ- and tissue-specific disease, which is the basis for classifying tumors. With the extensive genomic information now available on tumors it is possible to conduct analyses to reveal common genetic features across cancer types and to explore whether the established anatomy-based tumor classification is actually reflected on the genetic level, which might provide important guides to new therapeutic directions. Here, we have conducted an extensive analysis of the genetic similarity of tumors from 14 major cancer entities using somatic mutation data from 4,796 cases available through The Cancer Genome Atlas (TCGA) based on all available genes as well as different cancer-related gene sets. Our analysis provides a systematic account of the genetic similarity network for major cancer types and shows that in about 43% of the cases on average, tumors of a particular anatomic site are genetically more similar to tumors from different organs and tissues (trans-similarity) than to tumors of the same origin (self-similarity). The observed similarities exist not only for carcinomas from different sites but are also present among neoplasms from different tissue origin, such as melanoma, acute myeloid leukemia, and glioblastoma. The current WHO cancer classification is therefore reflected on the genetic level by only about 57% of the tumors. These results provide a rationale to reconsider organ- and tissue-specificity in cancer and contribute to the discussion about whether personalized therapies targeting specific genetic alterations may be transferred to cancers from other anatomic sites with similar genetic properties.

The tunneling of Gaussian wave packets has been investigated by numerically solving the one-dimensional Schr\"odinger equation. The shape of wave packets interacting with a square barrier has been monitored for various values of the barrier width, height and initial width of the wave packet. Compared to the case of free propagation, the maximum of a tunneled wave packet exhibits a shift, which can be interpreted as an enhanced velocity during tunneling., Comment: 6 pages, 3 figures

The Larmor clock which was proposed to determine tunneling times of particles with spin through barriers is studied. Tunneling of wave packets is numerically derived from the time-dependent Schr\"odinger-Pauli equation. The Larmor clock often does not proceed unidirectionally in time. The asymptotic readings are well defined. For wide wave packets the asymptotic Larmor times agree with the stationary-state predictions; deviations are seen for narrow wave packets. B\"uttiker's dwell time is investigated and its connection with the Larmor times is confirmed in the case of wave packets. \textcopyright{} 1996 The American Physical Society.

We have developed a microfluidics based sampling system for tissue analytics. The proof-of-concept of the sampling system was demonstrated by extracting lipid samples from tissue biopsies. The sample collection system consists of a disposable silicon based multiport microneedle integrated with polymer microfluidics. The polymethyl methacrylate polymer microfluidic chip has a 10 ll sample reservoir and actuation membranes for liquid pumping. A special automated robotic system was developed to control the positioning of the needle and the sampling procedure on preselected spots on the tissue. Real breast cancer tissue samples were used to test the feasibility of the sampling system. We successfully measured indicative cancer biomarkers from the tissue surface. Phosphatidylcholine and phosphoethanolamine were extracted from the tissue membrane with methyl tert-butyl ether solvent and detected by mass spectrometry. In the future, this tool could be used in characterization of preoperative biopsies and tumour tissues removed during surgery.

The color-flavor transformation, an identity that connects two integrals, each of which is over one of a dual pair of Lie groups acting in the fermionic Fock space, is extended to the case of the special unitary group. Using this extension, a toy model of lattice QCD is studied: N_f species of spinless fermions interacting with strongly coupled SU(N_c) lattice gauge fields in 1+1 dimensions. The color-flavor transformed theory is expressed in terms of gauge singlets, the meson fields, organized into sectors distinguished by the distribution of baryonic flux. A comprehensive analytical and numerical search is made for saddle-point configurations of the meson fields, with various topological charges, in the vacuum and single-baryon sectors. Two definitions of the static baryon on the square lattice, straight and zigzag, are investigated. The masses of the baryonic states are estimated using the saddle-point approximation for large N_c., LateX, 53 pages, 13 figures

10514 Background: Methods of analytical chemistry allow a global view on metabolic changes occurring during the malignant transformation. Methods: Metabolite profiling of 369 fresh-frozen breast ti...

10526 Background: We have recently developed a standardized system and defined cutoffs for assessment of molecular tumor subtype based on RNA expression of ESR1 and HER2 in FFPE tissue. (Muller et ...

10511 Background: The polyadenosine diphosphate [ADP]-ribose polymerase-1 (PARP-1) plays a key role in the genomic stability, transcription, chromatin remodelling and apoptosis. Increased expressio...

10514 Background: Novel biomarkers for prediction of a pathological complete response (pCR) in neoadjuvant chemotherapy for breast cancer are needed. Thymosin beta 15 is a small peptide with essential functions in the regulation of actin polymerization and immune response, as shown in various cancer cell lines. Methods: TMSB15A isoform mRNA expression in fresh-frozen preoperative biopsies of patients from the neoadjuvant GeparTrio trial (TAC; n = 55) as well as in cultured tumor cells was determined by Affymetrix array analysis. Kinetic RT-PCR was used to investigate mRNA expression in formalin-fixed, paraffin-embedded (FFPE) biopsies from the GeparTrio (n = 257) and the GeparQuattro trial (neoadjuvant EC-Doc or EC-DocX; n = 392). Results: TMSB15A mRNA expression was identified as a positive predictor for pCR in frozen tumor tissue from GeparTrio by a ROC analysis (p = 0.0042, auc = 0.824). In FFPE tissue from GeparTrio, TMSB15A mRNA expression again showed significant predictive impact: OR = 7.583, 95% C...

Background:Luminal breast cancers (LBC), defined as positive for estrogen receptor (ER) and negative for HER2, are of variable prognosis. We hypothesize that those showing potential sensitivity to neoadjuvant chemotherapy (NACT) are those with an unfavorable prognosis unless they achieve a pathological complete response (pCR = no invasive residual in breast and nodes). We therefore aimed to develop a Neoadjuvant Luminal Response Score (NLRS), identifying luminal tumors with a high chance for a pCR.Patients and methods:We examined mRNA expression of 57 candidate genes and 3 normalization genes by kinetic RT-PCR in FFPE tissues from pre-therapeutic core biopsies from 380 patients with LBC and cT2-T4 disease either receiving NACT with 6x docetaxel/ doxorubicin/ cyclophosphamide in GeparTrio (n=148, training cohort) or epirubicin/ cyclophosphamide followed by Docetaxel (+simultaneous or sequential capecitabine) in GeparQuattro (n=232, validation cohort). Samples were classified as LBC by mRNA expression analysis of ESR1 and Her2 using pre-defined cut-off values. The continuous NLRS was generated by linear combination of expression values of 3 out of 57 genes.The first pre-specified primary objective of training part was to demonstrate that the percentage of patients with LBC with a pCR is higher in NLRS+ tumors than in all LBC. The second pre-specified primary objective for validation part was to demonstrate that the NLRS score is able to identify the pCR cases in the LBC with a sensitivity of ≥80% (upper confidence interval [CI] limit) and a positive predictive value (PPV) of ≥20%.Results:In the GeparTrio training cohort, the NLRS was predictive of pCR by logistic regression analysis (AUC=0.88). Using a cutoff defined on the basis of the ROC curve, the NLRS was able to predict pCR with a sensitivity of 82%, a specificity of 77%. While the pCR rate in all LBC was only 8.2%, this rate increased to 24% in the group of 57 patients with a NLRS above the cutoff. The pCR rate in 91 patients with a NLRS below the cutoff was 1.1%.When patients of all 3 treatment arms of the GeparQuattro validation cohort were analyzed together, AUC was 0.69 (p=0.004). Using the pre-defined cutoff, sensitivity was 52% (95% CI: 33-71%), specificity 74% (69-79%) and PPV 16%. However, if only patients treated without capecitabine containing NACT (to be more comparable to GeparTrio) (N=68) were included, AUC was 0.72 and sensitivity increased to 63 (23-89)% and PPV to 21%, passing the pre-defined limits for positive validation.Conclusion:Our study provides proof-of-principle that it is possible to develop and validate predictive signatures for response to NACT based on pretherapeutic FFPE biopsies. The 3-gene NLRS is a first validated gene signature selecting LBC with a high chance for pCR to NACT. The results are a basis for further development of predictive signatures. A prospective evaluation will be performed in the PREDICT study, a substudy to GeparQuinto.Supported by a BMBF grant. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2132.

11054 Background: Preclinical investigations suggest an involvement of the immune system in chemotherapy response; however, this contribution has not been validated in prospective clinical trials so far. Methods: We investigated the inflammatory infiltrate in a total of 1058 pretherapeutic breast cancer core biopsies from two neoadjuvant anthracycline-taxane-based studies. As a training cohort, we used 218 core biopsies from the GeparDuo study. The results were validated in a completely independent cohort of 840 samples from the GeparTrio study. In addition, molecular parameters of lymphocyte recruitment and activation in breast cancer tissue were evaluated by kinetic PCR. Results: While the overall rate of pathological complete response (pCR) in the GeparDuo cohort was 12.8%, it was significantly increased to 42.7% in the tumors with more than 60% intratumoral or stromal lymphocytes. In a multivariate regression analysis, the percentage of intratumoral lymphocytes (iTu-Ly) was an independent significant parameter for pCR (P = 0.013) in the GeparDuo cohort. In the validation set from the GeparTrio trial, the inflammatory infiltrate was a strong predictor of pCR in univariate (P < 0.0005) and multivariate logistic regression (P < 0.0005). For tumors with 60% iTu-Ly the pCR rate was 40% with an odds ratio (OR) of 8.5 (4.0–18.2) which were similar to those values in hormone receptor negative samples (pCR: 36%; OR: 6.0 [4.0–9.1]). The expression of inflammatory marker genes and chemokines related to B and T infiltration in tumor tissue was significantly linked to the presence of a lymphocytic infiltrate and mRNA expression data showed a significant association of T cell markers with a pCR (P < 0.003). Conclusions: An increased lymphocytic inflammatory infiltrate in breast cancer is a new independent predictor of response to anthracyclin-taxane neoadjuvant chemotherapy and might be helpful to identify patients with benefit from cytotoxic chemotherapy. [Table: see text]