Text for supplement: Supplementary methods, supplementary figure legends

Background The present prospective study aimed at determining the impact of cell-free tumor DNA (ct-DNA), CA125 and HE4 from blood and ascites for quantification of tumor burden in patients with advanced high-grade serous epithelial ovarian cancer (EOC). Methods Genomic DNA was extracted from tumor FFPE and ct-DNA from plasma before surgery and on subsequent post-surgical days. Extracted DNA was subjected to hybrid-capture based next generation sequencing. Blood and ascites were sampled before surgery and on subsequent post-surgical days. 20 patients (10 undergoing complete resection (TR0), 10 undergoing incomplete resection (TR>0)) were included. Results The minor allele frequency (MAF) of TP53 mutations in ct-DNA of all patients with TR0 decreased significantly, compared to only one patient with TR>0. It was not possible to distinguish between patients with TR0 and patients with TR>0, using CA125 and HE4 from blood and ascites. Conclusions Based upon the present findings, ct-DNA assessment in patients with high-grade serous EOC might help to better determine disease burden compared to standard tumor markers. Further studies should prospectively evaluate whether this enhancement of accuracy can help to optimize management of patients with EOC.

EGFR-inhibitors are effective in EGFR-mutant NSCLC, but resistance invariably develops. Various mechanisms of resistance are described, including upregulation of immune-related factors. CD73 is an immunoinhibitory protein promoting tumor growth and metastasizing, and has been proposed to be modulated by EGFR-TKI treatment. We aimed at evaluating CD73 and related immune marker expression at various time points during sequenced EGFR-TKI treatment, including osimertinib, to identify potential biomarker combinations / signatures for CD73-based therapeutic opportunities in EGFR-resistant tumors.

Abstract Background T-DXd (Enhertu®) is an FDA-approved antibody-drug conjugate (ADC) targeting HER2. T-DXd has shown anti-tumor activity, not only in patients with HER2-overexpressing (IHC3+/2+ ISH+) breast cancer (BC) but also in patients with BC with low HER2 expression (IHC1+/2+ ISH−). Current HER2 protein expression assessment is based on manual pathologist scoring that classifies tumors by the percentage of tumor cells with highest intensity and completeness of staining. A critical need exists for more objective and quantitative methods to assess HER2 expression, specifically to better identify patients with low-level expression if T-DXd proves to be efficacious in this patient population. Methods We used deep learning (DL)-based image analysis (IA) to generate a novel HER2 Quantitative Continuous Score (QCS). Data analytic techniques determined optimal HER2 QCS for the J101 trial (NCT02564900) of 151 patients with varying HER2 expression levels (1+, 2+, 3+). HER2 QCS consists of DL models to detect membrane, cytoplasm, and nuclei of all tumor cells. QCS was extensively trained using pathologists’ annotations, and the performance was validated on unseen data to ensure its generalization and robustness. QCS was blindly applied to J101 data. The optical density (OD; level of brown stain intensity) was computed on detected membrane to derive features that could be linked to survival prediction. QCS features were selected to maximize ORR in positive group, minimize ORR in negative group maintaining while high prevalence in the positive group. Results Analytical validation showed high correlation between QCS from automatically detected membranes and QCS from those annotated by pathologists (R=0.993). This is in the same range as correlation between three pathologists (R=0.995). HER2 QCS was largely consistent with pathologist HER2 scoring as well but showed broad quantitative overlap between IHC and ISH categories. HER2 QCS showed a direct linear relationship between ORR and increased HER2 expression across the entire assay range. In the HER2-low population (n = 65), for whom HER2-targeting therapies are not currently approved, 42% of patients responded to T-DXd, with a median PFS (mPFS) of 11 mo. Using HER2 QCS, we were able to further stratify this population into a subgroup of QCS-high patients (above a staining intensity cut-off determined by IA), with response and mPFS increased to 53% (95% CI: 36%-68%) and 14.5 mo (95% CI: 10.9 mo-NR) respectively, while the QCS-low group only showed ORR of 24% (95% CI: 9%-45%) and mPFS of 8.6 mo (95% CI: 4.2 mo-NR). Generally, best-performing QCS cutoffs were driven by most tumor cells expressing a minimal amount of HER2, in contrast to current clinical guidelines that are driven by a minority of cells expressing higher levels of HER2. We also examined spatial heterogeneity by characterizing cells as either bearing membrane stain above a determined OD threshold (positive cell) or lying within certain distances from a positive cell. We observed similar efficacy with best performing-cutoffs, again, being found when a minimal level of HER2 expression (OD) was examined. Conclusions Taken together, these data establish a first proof-of-concept demonstrating that use of HER2 QCS can potentially enhance prediction of patient outcome with T-DXd by increasing sensitivity and specificity of response, especially in the HER2-low population. The ability to identify patients in the HER2-low group who could benefit from T-DXd is critical for its use in a patient population with a high unmet need that would otherwise not be treated with anti-HER2 therapy. Further clinical verification and validation is ongoing. Citation Format: Mark Gustavson, Susanne Haneder, Andreas Spitzmueller, Ansh Kapil, Katrin Schneider, Fabiola Cecchi, Sriram Sridhar, Guenter Schmidt, Sotirios Lakis, Regina Teichert, Anatoliy Shumilov, Ana Hidalgo-Sastre, Magdalena Wienken, Hadassah Sade, J. Carl Barrett, Danielle Carroll. Novel approach to HER2 quantification: Digital pathology coupled with AI-based image and data analysis delivers objective and quantitative HER2 expression analysis for enrichment of responders to trastuzumab deruxtecan (T-DXd; DS-8201), specifically in HER2-low patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD6-01.

Salivary gland carcinomas represent a heterogeneous group of poorly characterized head and neck tumors. The purpose of this study was to evaluate ALK gene and protein aberrations in a large, well-characterized cohort of these tumors. A total of 182 salivary gland carcinomas were tested for anaplastic lymphoma kinase (ALK) positivity by immunohistochemistry (IHC) using the cut-off of 10% positive cells. ALK positive tumors were subjected to FISH analysis and followed by hybrid capture–based next generation sequencing (NGS). Of the 182 tumors, 8 were ALK positive by IHC. Further analysis using hybrid capture NGS analysis revealed a novel MYO18A (Exon1-40)- ALK (exon 20-29) gene fusion in one case of intraductal carcinoma. Additional genomic analyses resulted in the detection of inactivating mutations in BRAF and TP53 , as well as amplifications of ERBB2 and ALK . ALK rearrangements are a rare entity in salivary gland carcinomas. We identified a potentially targetable novel ALK fusion in an intraductal carcinoma of minor salivary glands.

Background/Aim: Ovarian cancer (OVCA) is characterized by genomic/molecular intra-patient heterogeneity (IPH). Tissue histology and morphological features are surrogates of the underlying genomic/molecular contexture. We assessed the morphological IPH of OVCA tumor compartments and of lymphocytic infiltrates in multiple matched samples per patient. Materials and Methods: We examined 294 hematoxylin & eosin (H&E) OVCA tumor whole sections from 70 treatment-naïve patients who had undergone cytoreductive surgery. We assessed morphological subtypes as immunoreactive (IR), solid – proliferative (SD), papilloglandular (PG), and mesenchymal transition (MT); subtype load per patient; stromal tumor-infiltrating lymphocyte (sTIL) density as average per sample; and, as maximal sTIL values (max-TILs) among all samples per patient, ovaries and implants. Results: Among all 294 tumor sections, the most frequent primary morphological subtype was PG (n=150, 51.0%), followed by MT (71, 24.1%), SD (48, 16.3%) and IR (15, 5.1%). Subtype combinations were observed in 67/294 sections (22.8%) and IPH in 48/70 patients (68.6%). PG prevailed in ovaries (p<0.001), SD and MT in implants (p=0.023 and p<0.001, respectively). sTILs were higher in SD compared to non-SD (p=0.019) and lower in PG, respectively (p<0.001). sTIL density was higher in implants than in ovaries (p<0.001). Higher max-TILs were associated with stage IV disease (p=0.043), upper abdominal dissemination (p=0.024), endometrioid histology (p=0.013), and grade 3 tumors (p=0.021). Favorable prognosticators were higher max-TILs per patient (PFS, OS) and higher SD-load (PFS). Conclusion: Clinically relevant morphological and host immune-response IPH appear to be the norm in OVCA. This may complicate efforts to decipher sensitivity of the tumor to certain treatment modalities from a single pre-operative biopsy.

Abstract Small cell lung cancer (SCLC) patient-derived xenografts (PDX) can be generated from biopsies or circulating tumor cells (CTC), though scarcity of tissue and low efficiency of tumor growth have previously limited these approaches. Applying an established clinical–translational pipeline for tissue collection and an automated microfluidic platform for CTC enrichment, we generated 17 biopsy-derived PDXs and 17 CTC-derived PDXs in a 2-year timeframe, at 89% and 38% efficiency, respectively. Whole-exome sequencing showed that somatic alterations are stably maintained between patient tumors and PDXs. Early-passage PDXs maintain the genomic and transcriptional profiles of the founder PDX. In vivo treatment with etoposide and platinum (EP) in 30 PDX models demonstrated greater sensitivity in PDXs from EP-naïve patients, and resistance to EP corresponded to increased expression of a MYC gene signature. Finally, serial CTC-derived PDXs generated from an individual patient at multiple time points accurately recapitulated the evolving drug sensitivities of that patient's disease. Collectively, this work highlights the translational potential of this strategy. Significance: Effective translational research utilizing SCLC PDX models requires both efficient generation of models from patients and fidelity of those models in representing patient tumor characteristics. We present approaches for efficient generation of PDXs from both biopsies and CTCs, and demonstrate that these models capture the mutational landscape and functional features of the donor tumors. Cancer Discov; 8(5); 600–15. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 517

This clinical trial assessed the efficacy and toxicity of panitumumab combined with oxaliplatin and capecitabine as first-line treatment in KRAS exon 2 wild-type metastatic colorectal cancer (mCRC) patients. Patients with exon 2 KRAS wild-type mCRC received panitumumab 9 mg/Kg, oxaliplatin 130 mg/m2, and capecitabine 2000 mg/m2 repeated every 3 weeks. The primary endpoint was objective response rate (ORR, minimum 42 responses). We retrospectively assessed mutations in genes implicated in CRC with massively parallel sequencing; ERBB2 and EGFR amplification with fluorescence in situ hybridization, and tumor-infiltrating lymphocyte density. Among 78 patients enrolled, 45 (57.7%) completed 6 cycles. Most common grade 3–4 toxicities were skin rash (19.2%), diarrhea (18%), and neuropathy (6.4%). Among 5 (6.4%) potentially treatment-related deaths, 2 (2.6%) were characterized toxic. Objective response occurred in 43 (55.1%) of the patients (complete 6.4% and partial response 48.7%; stable 17.9% and progressive disease 7.7%), while 3.8% were non-evaluable and 15% discontinued their treatment early. Additional mutations in KRAS/NRAS/BRAF were found in 11/62 assessable (18%) tumors. After 51 months median follow-up, median progression-free (PFS) was 8.1 and overall survival 20.2 months, independently of KRAS/NRAS/BRAF or PI3K-pathway mutation status. Patients with TP53 mutations (n = 34; 55%), as well as those with left colon primary tumors (n = 66; 85%), had significantly better PFS, also confirmed in multivariate analysis. Although the clinical trial met its primary endpoint, according to the current standards, the efficacy and tolerability of the drug combination are considered insufficient. Extended genotyping yielded interesting results regarding the significance of TP53 mutations.

Der post-operative Tumorrest (TR) ist der wichtigste Prognosefaktor der Therapie des high- grade serösen Ovarialkarzinom (HGSOC). Die Beurteilung des TR ist jedoch anfällig für subjektive Einschätzungen. Ein objektives Maß wäre hilfreich.

The aim of this study was to investigate the prognostic value of the Hedgehog (Gli, Patched-1, Shh, Smo) and Notch (Jag1, Notch2, Notch3) pathway members, in comparison to a panel of proteins (ER, PgR, HER2/neu, Ki67, p53, p16, PTEN and MMR) previously suggested to be involved in the pathogenesis of endometrial cancer, in association with clinical outcome and standard clinicopathological characteristics. A total of 204 patients with histological diagnosis of endometrial cancer treated from 2004 to 2013 were included. The evaluation of protein expression was assessed by immunohistochemistry. Univariate analysis showed that higher Ki67 labeling, expression of PTEN, p16, Notch2 and Notch3 proteins, as well as MMR proficiency were associated with increased relapse and mortality rate. Additionally, Patched-1 protein expression was associated with worse DFS, while p53 overexpression was associated with worse OS. In multivariate analyses, patients with MMR proficient tumors had more than double risk for death than patients with MMR deficient (MMRd) tumors (adjusted HR = 2.19, 95% CI 1.05-4.58, p = 0.036). Jag1 positivity conferred reduced mortality risk (HR = 0.48, 95% CI 0.23-0.97, p = 0.042). However, as shown by hierarchical clustering, patients fared better when their tumors expressed high Jag1 protein in the absence of Notch2 and Notch3, while they fared worse when all three proteins were highly expressed. Patched-1 positivity conferred higher risk for relapse (HR = 2.04, 95% CI 1.05-3.96, p = 0.036). Aberrant expression of key components of the Notch and Hedgehog signaling pathways, as well as MMRd may serve as independent prognostic factors for recurrence and survival in patients with endometrial cancer.

Biliary tract cancer (BTC) represents a heterogeneous disease with dismal outcome. Herein, we examined genotype and angiogenesis features in BTC. We applied genotyping (Sanger, qPCR, 101-gene panel NGS), mRNA relative quantification methods, and β-catenin immunohistochemistry in 84 FFPE BTC (55 gallbladder [GBC], 14 intrahepatic [ICC], 15 extrahepatic [ECC] carcinomas). We identified 541 mutations in 68 (81%) tumors. Top mutated genes were CTNNB1 (36%); PTEN (33%); TP53 (31%); PIK3R1 (29%); PIK3CA (13%); BRCA2 and KRAS (12%); BRCA1 (11%). Six GBCs were hypermutated [hm] displaying a distinct mutational pattern. Mutations in TP53 and PI3K, Wnt and RAS components were prevalent among non-hypermutated tumors. All hmGBCs carried mutations in BRCA2 and other homologous recombination repair (HRR) genes, in PD1, but not in CTNNB1 and KRAS. None of the pathogenic BRCA2 p.D2723G and BRCA1 p.Q563* and c.5266dupC was present at frequencies expected for germline mutations. We observed copy gains (>6 copies) in EGFR (9% of informative tumors), PRKAR1A (7%), PIK3CA (6%), ERBB2 (5%) and MET (4%). TP53 mutations were prevalent in GBC (P<0.001) and PRKAR1A copy gains in ICC (P=0.007). PTEN was frequently co-mutated with CTNNB1 (P<0.001). Unrelated to CTNNB1 mutations, nuclear β-catenin was detected in 45% of tumors, among them in 5/6 hmGBC. We observed strong mRNA expression correlation of the two neuropilins (NRP1 and NRP2) with each other (Spearman's rho 0.59) and with the endothelin receptor (NRP2 rho 0.66; NRP2 rho 0.51), and between VEGFA and its receptors (FLT1 rho 0.49; KDR rho 0.45). All PIK3CA mutated tumors expressed endothelin 1 mRNA (P=0.010). Most tumors expressing nuclear β-catenin were negative for VEGFC (P=0.009) and FLT4 (P=0.002) mRNA expression. In conclusion, we confirmed the presence of known genomic aberrations in BTC and different genotypes between BTC subsets. Novel findings are the coexistence of PI3K and WNT pathway gene alterations in BTC, their association with angiogenesis, and the hypermutated GBCs with HRR gene mutations, all of which may be considered for new treatment options in this difficult to treat disease.

Background: Salivary gland carcinomas represent a heterogeneous group of poorly characterized head and neck tumors. The purpose of this study was to evaluate ALK gene and protein aberrations in a large, wellcharacterized cohort of these tumors. Materials and methods: 182 salivary gland carcinomas were tested for ALK positivity by immunohistochemistry using the cut-off of 10% positive cells. ALK positive tumors were subjected to FISH analysis and followed by hybrid capture based next generation sequencing (NGS). Results: Of the 182 tumors, 8 were ALK positive. Further analysis showed ALK amplification in one case of intraductal carcinoma/low grade cribriform cystadenocarcinoma (LGCCC). Hybrid capture NGS analysis revealed a novel MYO18A (Exon1-40)-ALK (exon 20-29) gene fusion. Additional genomic analyses resulted in the detection of inactivating mutations in BRAF and TP53, as well as amplifications of ERBB2. Conclusion: We identified a potentially targetable novel ALK fusion in an intraductal carcinoma/LGCCCC of minor salivary glands.

BACKGROUND:Tissue genomic heterogeneity (t-HET) in patients with epithelial ovarian cancer (OVCA) is related to tissue plasticity, i.e., flexibility to adapt to adverse molecular environments. Here, we interrogated the presence and clinical relevance of OVCA t-HET. METHODS:We applied high-depth (>2000×) sequencing on 297 paraffin tissue samples (fallopian tubes, ovaries, intra-abdominal metastases) from 71 treatment-naïve patients who subsequently received first-line platinum-based chemotherapy. Based on tissue mutation patterns, we distinguished tissue genotypes into: no mutation (33/297 samples; 11.1%), stable (173; 58.2%) and unstable (91; 30.7%). We profiled genotypes per patient and assessed t-HET in 69 patients. Predicted pathogenic mutations refer to germline and/or tissues. RESULTS:Among all 71 patients, 46 (64.8%) had pathogenic BRCA1 mutations and 15 (21.7%) had BRCA1/2 disruption (i.e., pathogenic mutations with position-LOH). We classified 29 patients with t-HET (42%), all with pathogenic BRCA1; t-HET was observed in 64% with such mutations (p < 0.001). As opposed to non-t-HET, matched tissues in t-HET shared pathogenic BRCA1 (p < 0.001) but not BRCA2 and TP53. Germline BRCA1 mutations in tissues exhibited position-LOH; heterozygous status; or, partial loss of the inherited allele accompanied by additional clonal mutations. Patients with t-HET had worse outcome (log-rank p = 0.048 [progression-free]; p = 0.037 [overall survival]), including 12/15 patients with disrupted BRCA1/2 and 3 BRCA1 carriers with partial germline loss in tissues. CONCLUSIONS:Pathogenic BRCA1 mutations appear necessary but may not be sufficient for the establishment of t-HET. t-HET may be associated with worse outcome, including in patients with disrupted BRCA1/2, which is usually considered as a favourable marker. OVCA t-HET may need to be addressed for treatment decisions.

e23146 Background: The importance and clinical use of liquid biopsies to identify somatic, targetable alterations in the plasma of cancer patients is steadily increasing. However, their concordance with alterations identified in cancer specimens tested in routine diagnostics is not being monitored. In a cohort of non-squamous cell lung cancer patients, our aim was to systematically compare alterations found by a massively parallel sequencing liquid biopsy assay covering 39 cancer related genes (NEOliquid) with alterations identified by routine diagnostics in a certified central pathology laboratory. Methods: Routine Diagnostics: DNA mutational analysis was performed using cobas or Sanger-Sequencing. Rearrangements were identified using immunohistochemistry or FISH. NEOliquid assay: Cell-free DNA from plasma was subjected to hybrid-capture based next-generation sequencing to detect point mutations, small insertions and deletions, copy number alterations and genomic translocations in 39 clinically relevant genes in a single assay. To evaluate and compare the performance of liquid biopsies, we applied the NEOliquid tests on blood samples of 82 non-squamous NSCLC patients and correlated them with results of routine diagnostics of matched tissue samples. Results: Routine diagnostics for lung cancer related oncogenes (EGFR, ALK, ROS1, KRAS, BRAF) identified a total of 50 somatic alterations of which 37 were point mutations, 11 InDels and 2 gene fusions. NEOliquid analysis of the corresponding plasma sample revealed a total of 34 alterations, including 21 targetable driver alterations. Overall, the concordance of mutation calls between routine diagnostic testing and NEOliquid was 97.6%, with a sensitivity of > 70% and a specificity of 100%. Importantly, NEOliquid identified additional clinically relevant genomic alterations, not covered by routine testing. Conclusions: We have shown high concordance of genomic alterations identified from liquid biopsies and tumor tissue specimens. The excellent specificity of actionable alterations identified by NEOliquid offers analytical performance for routine diagnostic use.

Biomarker changes between primary (PT) and metastatic tumor (MT) site may be significant in individualizing treatment strategies and can result from actual clonal evolution, biomarker conversion, or technical limitations of diagnostic tests.This study explored biomarker conversion during breast cancer (BC) progression in 67 patients with different tumor subtypes and metastatic sites via mRNA quantification and subsequently analyzed the concordance between real-time qPCR and immunohistochemistry (IHC). Immunostaining for estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki-67 was performed on formalin-fixed, paraffin-embedded PT and MT tissue sections. RT-qPCR was performed using a multiplex RT-qPCR kit for ESR1, PGR, ERBB2, and MKI67 and the reference genes B2M and CALM2.Subsequent measurement of tumor biomarker mRNA expression to detect conversion revealed significant decreases in ESR1 and PGR mRNA and MKI67 upregulation (all p < 0.001) in MT compared to PT of all tumor subtypes and ERBB2 upregulation in MT from triple-negative PT patients (p = 0.023). Furthermore, ERBB2 mRNA was upregulated in MT brain biopsies, particularly those from triple-negative PTs (p = 0.023). High concordance between RT-qPCR and IHC was observed for ER/ESR1 (81%(κ 0.51) in PT and 84%(κ 0.34) in MT, PR/PGR (70%(κ 0.10) in PT and 78% (κ -0.32) in MT), and for HER2/ERBB2 (100% in PT and 89% in MT). Discordance between mRNA biomarker assessments of PT and MT resulting from receptor conversion calls for dynamic monitoring of BC tumor biomarkers. Overall, RT-qPCR assessment of BC target genes and their mRNA expression is highly concordant with IHC protein analysis in both primary and metastatic tumor.

Abstract Background - aim: Germline mutation carriers in BRCA1 and related genes frequently develop triple-negative breast carcinomas (TNBC) that presumably preserve the inherited mutation. Herein, we examined the clinicopathologic features and prognostic impact of paired germline and tumor genotypes in this disease. Methods: We compared baseline germline and paraffin tumor genotype data (next generation [NGS] and Sanger sequencing) from 194 patients with operable TNBC who were treated with anthracyclines-taxanes based chemotherapy within adjuvant trials by our Group. Because tissue NGS did not target BRCA-related genes, we additionally interrogated the presence of germline mutations in tumor and non-cancerous breast tissues, in carriers with available tissue material (n=33). We validated all NGS germline mutations with Sanger sequencing and ensured matched germline/tumor identity with microsatellite testing. Results: We identified 50 (26%) germline mutation carriers, 39 in BRCA1, 4 in BRCA2 and the rest in RAD51C, BARD1, RAD50, NBN, MRE11A and BRIP1. BRCA1 carriers were younger as compared to non-BRCA1 carriers (median 42y.o. vs. 56y.o, respectively; p=0.020), premenopausal (p=0.014), and had lower nodal status (p<0.001). Somatic mutations were found in 136 tumors, TP53 in 113 (81%). The germline mutant allele was lost in 14/33 (42%) tumor and matched normal tissues (mut-LOH). Loss affected 12 BRCA1 mutations, among them the common pathogenic p.Q1756fsX74 (c.5266dupC) and p.G1738R (c.5212G>A), and 2 BRCA2 mutations. In one such tumor, the germline mutation was replaced by a somatic BRCA1 (p.Q1811X [c.5431C>T]). Tumors with mut-LOH were more often TP53 mutant as compared to those with preserved germline mutations, which had mutations in other genes as well (p=0.034). Overall though, TP53 mutations were not associated with germline status. Germline mutations and tumor TP53 mutations were not individually associated with patient disease-free survival (DFS) but interacted with each other: in non-carriers, TP53 status did not affect outcome; in carriers, those with mutant TP53 had unfavorable DFS compared to those with wild-type TP53 tumors (interaction p, multivariate analysis: 0.053). Conclusions: Germline BRCA-related TNBC may be distinguished into two groups with potentially different biological characteristics, those with preserved and lost inherited mutation. The interaction between germline status and tumor TP53 mutations may have treatment implications and seems worthy pursuing in larger studies. Combined germline/tumor genotype testing may be needed for TNBC patient classification, especially in the context of clinical trials. Citation Format: Vassiliki Kotoula, Florentia Fostira Fostira, Kyriaki Papadopoulou, Paraskevi Apostolou, Eleftheria Tsolaki, Georgios Lazaridis, Kyriaki Manoussou, Flora Zagouri Zagouri, Dimitrios Pectasides, Ioannis Vlachos, Ioannis Tikas, Sotiris Lakis, Irene Konstantopoulou, George Pentheroudakis, Helen Gogas, Pavlos Papakostas, Christos Christodoulou, Drakoulis Yannoukakos, George Fountzilas. The fate of germline BRCA related mutations in breast tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1755. doi:10.1158/1538-7445.AM2017-1755

The use of immune checkpoint inhibitors has shown promise in lung cancer as well as several other tumor types. However, two of their limitations are the overall relatively low response rate as well as the lack of powerful predictors for response. Recently, several studies have shown that stratification of patients according to the load of somatic mutations can provide predicitive information for either ‘mono’ or ‘combinational’ immunotherapy. Here, we describe the use of a hybrid-capture based next-generation sequencing assay, NEOplus, to determine the somatic mutational load in clinical samples. NEOplus is a hybrid-capture based next-generation sequencing assay that covers clinically relevant genomic alterations in a panel of more than 90 tumor-associated genes, including point mutations, small insertions and deletions, copy number alterations and gene fusions. First, the correlation between number of mutations observed in the NEOplus exonic territory and response to immune checkpoint inhibitors was analyzed in silico based on published data. Second, tumors of multiple histological types with available outcome data were subjected to NEOplus analysis to assess mutational load. We were able to correlate the amount of mutations detected within the exonic territory covered by the NEOplus assay and clinical response to immune checkpoint inhibitors. Furthermore, we confirmed that hybrid-capture based next-generation sequencing assays, covering sub-fractions of the exome, can reliably detect somatic mutation load in diagnostic patient samples. We show correlations between mutational load and patient response to immunotherapy and are able to indicate how to stratify patients who would most likely benefit from treatment.