Median Tumour Mutational Burden Research Articles

Tumour mutational burden and survival with molecularly matched therapy

BackgroundThe impact of tumour mutational burden (TMB) on outcome with molecularly matched therapy is unknown. Higher TMB could predict resistance to molecularly matched therapy through co-occurring driver mutations. MethodsOne hundred and four patients with advanced cancers underwent molecular profiling in the DKTK-MASTER program. Fifty-five patients received systemic therapy excluding immunotherapy. Patients with molecularly matched (n = 35) or non-molecularly informed therapy (n = 20) were analysed for TMB and survival. Results were validated in an independent cohort of patients receiving molecularly matched (n = 68) or non-molecularly informed therapy (n = 40). Co-occurring driver mutations and TMB were analysed in the exploratory cohort and The Cancer Genome Atlas (TCGA) datasets. ResultsPatients were stratified by the median TMB of 1.67 mutations per Megabase (mut/Mb) of 35 patients receiving molecularly matched therapy into TMB-high or TMB-low groups. Median overall survival (4 months [95% CI, 3.3–7.6] versus 12.8 months [95% CI, 10-not reached], p < 0.001) and progression-free survival (1.8 months [95% CI, 1.1–3.7] versus 7.9 months [95% CI, 2.8–17.0], p = 0.003) were significantly shorter in the TMB-high group compared to the TMB-low group. In the validation cohort, shorter OS and PFS were identified in the TMB-high group (TMB cut-off of 4 mut/Mb) treated with molecularly matched therapy. No differences were observed in patients receiving non-molecularly informed systemic therapy. A significant correlation between co-occurring driver mutations and TMB (n = 104, r = 0.78 [95% CI, 0.68–0.85], p < 0.001) was found in the exploratory cohort as well as the majority (24/33) of TCGA studies. ConclusionA high TMB was associated with unfavourable outcome in patients receiving molecularly matched therapy, indicating untargeted resistance pathways. Therefore, TMB should be further investigated as a predictive biomarker in precision oncology programs.

Genomic profiling of ovarian clear cell carcinoma in Chinese patients reveals potential prognostic biomarkers for survival

Introduction Ovarian clear cell carcinoma (OCCC) has distinct clinical and molecular features and heterogeneous prognosis. Insights into the somatic genomic abnormalities of OCCC provide the basis for deeper understanding and potential therapeutic avenues. Herein, we performed extensive genomic profiling in Chinese patients to illustrate the mutation landscape and genetic prognostic biomarkers of OCCC. Patients and methods We used targeted DNA sequencing on 61 OCCC cases with a panel of 520 cancer-related genes. Correlations between clinicopathological features and survival were evaluated. Nomogram-based models were constructed to predict progress-free survival (PFS). Results We detected 763 somatic mutations spanning 286 genes. The most frequent genetic alterations, ARID1A (49%) and PIK3CA (48%), were concurrently mutated. Comprehensive copy number alterations (CNAs) were identified in chromosomes 20q13.2 and 8q. Most (73.7%) patients harboured potentially targetable driver mutations. The mean and median tumour mutational burden were 7.0 and 3.0 mutations/Mb, respectively. Microsatellite instability (high) was identified in 8.2% of patients. Mutation of the base-excision repair pathway was significantly higher in patients of stage II/III/IV. ATM mutation was associated with platinum sensitivity (p < .05). Survival analysis identified chr8q CNAs in all patients, PIK3CA mutations in stage I patients and SWI/SNF complex (ARID1A and SMARCA4) mutations in stage II/III/IV patients as potential prognosticators (p < .05). Integration of genetic alterations (SWI/SNF complex mutations, ATM mutations and chr8q CNAs) improved the performance of a nomogram based on tumour stage and residual disease (concordance index 0.75 vs. 0.70, p < .05). Conclusions We described somatic genomic alterations in Chinese OCCC patients and observed different genomic alterations between stage I and stage II/III/IV tumours. Genetic factors may supplement clinical factors in nomogram modelling for PFS prediction. Key Messages We performed extensive genomic profiling in a well-annotated cohort of 61 Chinese ovarian clear cell carcinoma (OCCC) patients. PIK3CA mutations were associated with worse overall survival (OS) in stage I OCCC, and SWI/SNF gene mutations were associated with improved OS in stage II/III/IV disease. We propose an easy-to-use nomogram using clinical factors (tumour stage and residual disease) and genetic alterations (SWI/SNF complex mutations, ATM mutations and chr8q CNAs) to predict the progress-free survival (PFS) of OCCC.

Elucidating the genomic and transcriptomic landscape of L858R vs ex19del EGFR-mutated NSCLC.

8530 Background: Epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) is heterogeneous and L858R derive less benefit from osimertinib than ex19del in both the metastatic and adjuvant setting, which remains poorly understood. We sought to examine the genomic and transcriptomic features of L858R vs ex19del. Methods: Consecutive patients with AJCC7 Stage IA-IIIA EGFR-mutated NSCLC diagnosed 1/1/2010 – 31/12/2019 who underwent surgery at National Cancer Centre Singapore were included. Patients who received neoadjuvant therapy were excluded. Fresh frozen tumour and normal samples were subject to whole exome sequencing (WES) at 400X and 100X coverage respectively, with approximately 50 million paired-end reads for RNA-seq per sample. Wilcoxon and Fisher’s exact test were used for association analysis. Results: A total of 203 patients were included. Median age at diagnosis was 66, 66.0% (134/203) were females and 84.2% (171/203) never-smokers. Stage IA comprised 44.3% (90/203), IB 28.6% (58/203), II 15.8% (32/203) and IIIA 11.3% (23/203). Median tumour mutational burden (TMB) was 1.3 mutations/megabase (range 0.3 – 44.3). Ex19del represented 46.3% (94/203) and L858R 41.9% (85/203). Whole genome doubling (WGD) was found in 70.0% (142/203) and was more common in TP53-mutated compared to TP53-wildtype (81.1% vs. 63.6%, p=0.01). TP53 mutations were more common in stage II/IIIA tumours compared with stage IB and IA (50.9% vs 32.8% vs 30.0%, p=0.035). Comparing ex19del and L858R, there was no difference in stage distribution (p=0.337), proportion of TMB≥10 (1.1% vs 5.9%, p=0.103), number of cancer co-driver mutations (p=0.174), TP53 mutations (39.4% vs 32.9%, p=0.437) and WGD (69.1% vs 76.5%, p=0.316). L858R had a higher incidence of smoking mutational signature (median 0.35 vs 0.28, p=0.018) compared to ex19del despite similar smoking history (15.3% vs 12.8%, p=0.67), whereas APOBEC mutational signature was higher in ex19del (median 0.06 vs 0.04, p=0.015). L858R tumors were associated with a higher incidence of co-mutations in RBM10 (21.2% vs 6.38%, p=0.004), RNF213 (10.6% vs 1.06%, p=0.007) and amplification of NTHL1 (15.3% vs 2.13%, p=0.001) and AXIN1 (17.6% vs 3.19%, p=0.002) compared to ex19del. Transcriptomic subtype differed significantly by EGFR mutation with a higher representation of TRU subtype among L858R than ex19del (51.0% vs 35.3%, p=0.010), while GEP score was similar (median 0.189 vs 0.325, p=0.122). Conclusions: L858R have distinct co-mutations and copy number alterations compared to ex19del, in addition to a higher representation of smoking mutational signature and TRU subtype. TP53 co-mutations are more frequently observed in higher stage tumours and are associated with WGD. Our findings highlight the molecular heterogeneity of resected EGFR-mutated NSCLC, which could contribute to the differential outcomes to adjuvant osimertinib between ex19del and L858R.

Comprehensive genomic landscape in pancreatic cancer using whole-exome sequencing.

e16307 Background: Pancreatic ductal adenocarcinoma is one of the fatal malignancies in the world, mostly as a result of the absence of early detection and specific treatment solutions. Consequently, identifying mutational profiles and molecular biomarker are essential for increasing the viability of precision therapy for pancreatic cancer. Methods: We gathered blood and tumour tissue samples from 47 Chinese pancreatic cancer patients and used whole-exome sequencing (WES) to evaluate the genetic landscape. Results: Our results showed the most frequently somatic alteration genes were KRAS (74.5%), TP53 (51.1%), SMAD4 (17%), ARID1A (12.8%), CDKN2A (12.8%), TENM4 (10.6%), TTN (8.5%), RNF43 (8.5%), FLG (8.5%) and GAS6 (6.4%) in Chinese pancreatic ductal adenocarcinoma patients. We also found that three deleterious germline mutations ( ATM c.4852C &gt; T/p. R1618*, WRN c.1105C &gt; T/p. R369*, PALB2 c.2760dupA/p. Q921Tfs*7) and two novel fusions ( BRCA1-RPRML, MIR943 (intergenic)- FGFR3). When compared to the Cancer Genome Atlas (TCGA) database, there is a greater mutation frequency of TENM4 (10.6% vs. 1.6%, p= 0.01), GAS6 (6.4% vs. 0.5%, p= 0.035), MMP17 (6.4% vs. 0.5%, p= 0.035), ITM2B (6.4% vs. 0.5%, p= 0.035) and USP7 (6.4% vs. 0.5%, p= 0.035) as well as a reduced mutation frequency of SMAD4 (17.0% vs. 31.5%, p= 0.075) and CDKN2A (12.8% vs. 47.3%, p&lt; 0.001) were observed in the Chinese cohort. Among the 41 individual examined for PD-L1 expression, 15 (36.6%) had positive PD-L1 expression. The median tumour mutational burden (TMB) was found to be 12 muts (range, 0-124). The TMB index was higher in patients with mutant-type KRAS MUT/ TP53 MUT ( p&lt; 0.001), CDKN2A ( p= 0.547), or SMAD4 ( p= 0.064) compared to patients with wild-type KRAS/ TP53, CDKN2A, or SMAD4. Conclusions: We exhibited real-world genetic traits and new alterations in Chinese individuals with cancer of pancreas, which might have interesting implications for future individualized therapy and medication development.

Abstract 2225: Describing the molecular landscape of extramedullary multiple myeloma using whole genome sequencing: Insights into pathology and therapeutic targets

Abstract Background: Extramedullary disease (EMD) affects 30% of multiple myeloma (MM) patients, predicting poor overall survival due to aggressive disease kinetics and multi-drug resistance. Understanding EMD genomics may inform targeted treatment approaches. Methods: We obtained EMD samples from 15 patients (n=8 fresh and n=7 formalin-fixed, paraffin embedded). Second EMD biopsies were taken in n=3, with n=1 having 3 sequential EMD biopsies. 8 patients had peripheral blood plasma (Streck DNA tubes) for circulating tumour DNA (ctDNA). Germline variants were excluded using buccal swab DNA. DNA was extracted using QIAGEN DNeasy kit prior to whole genome sequencing (WGS) (30x; xGen PRISM library preparation; Illumina Novaseq). Bioinformatics identified short nucleotide variants (SNVs), copy number variations (CNVs) and structural variants (SV) using the Broad Institute GATK best practice pipeline, CNVpytor and Manta, respectively. Droplet digital PCR was used to confirm SNV and CNV findings, and to explore the presence of mutations in ctDNA. Results: The median age at diagnosis of MM was 52 years (n=4 primary EMD, n=11 secondary EMD). 46% were hyperdiploid. Driver mutations (DM) in the MAPK pathway were seen in 80%, primarily at codon 61 of NRAS and KRAS (n=5, n=3); only two non-p.Q61 mutations were seen (NRAS p.G13R and KRAS p.A146V). Three patients had activating BRAF mutations (two p.V600E, one p.G469A). Variant allele frequencies (VAF) of DM suggested clonal rather than subclonal level. A quarter of patients (26.6%) had loss of function TP53 mutations. In those with no MAPK DM identified, the median whole genome SNV was 77,142 with a tumour mutational burden (TMB) of 15 mutations/Mb, compared to 20,543 and 3.2 in DM patients. The median SNV and TMB increased with relapse or progression of EMD. CNV/SV analysis identified gain(3q), gain(1q), del(1p) and monosomy 13/del(13q) in 93%, 86%, 46% and 73% respectively. Gains of BRAF (66%) and MYC (53%) and loss of TP53 (40%) were frequent. Secondary translocations were seen in 40% of patients, involving MYC, FGFR3, CCND2 and CCND3 in 20%, 13%, 6% and 6%, respectively, and partnered with IGH, IGL and TXNDC5. The majority of SV had not been previously identified in these patients. There were increased numbers of CNV/SV with relapsed EMD after therapy, with persistence of the MAPK DM and previously seen CNV/SV. Liquid biopsies show MAPK DM are present in ctDNA at the time of EMD biopsy. The same DM are detectable in the blood prior to development of EMD, suggesting DM clones arise early in disease course. The VAF of DM in ctDNA mirrors PET responses to therapy. Conclusion: MAPK DM were frequent in EMD, and detectable in ctDNA, suggesting roles for both MAPK-targeted therapies and for ctDNA as a biomarker in these patients. A high TMB was identified in patients lacking a MAPK driver mutation; immunotherapy should be considered in this subgroup. Citation Format: Nicholas E. Bingham, Jaynish Shah, Antonia Reale, Daniel Wong, Tiffany Khong, Sridurga Mithraprabhu, Andrew Spencer. Describing the molecular landscape of extramedullary multiple myeloma using whole genome sequencing: Insights into pathology and therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2225.

Tumour mutational burden as a biomarker in patients with mismatch repair deficient/microsatellite instability-high metastatic colorectal cancer treated with immune checkpoint inhibitors

BackgroundImmune checkpoint inhibitors (ICIs) are the standard treatment in patients with mismatch repair deficient (dMMR)/microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC). Tumour mutational burden (TMB) is a promising biomarker for the prediction of treatment outcomes. Patients and methodsWe screened 203 patients with dMMR/MSI-H mCRC treated with an anti-PD-(L)1 (anti-Programmed-Death-(Ligand)1) plus or minus an anti-Cytotoxic T-Lymphocyte Antigen 4 (anti-CTLA-4) agent at three Italian Academic Centers. TMB was tested by Foundation One Next Generation Sequencing assay and correlated with clinical outcomes, in the overall population and according to ICI regimen. ResultsWe included 110 patients with dMMR/MSI-H mCRC. Eighty patients received anti-PD-(L)1 monotherapy and 30 received anti-CTLA-4 combinations. Median TMB was 49 mut/Mb (range: 8–251 mut/Mb). The optimal prognostic cut-off for progression-free survival (PFS) stratification was 23 mut/Mb. Patients with TMB ≤23 mut/Mb had significantly worse PFS (adjusted Hazard Ratio [aHR] = 4.26, 95% confidence interval [CI]:1.85–9.82, p = 0.001) and overall survival (OS) (aHR = 5.14, 95% CI: 1.76–14.98, p = 0.003). Using a cut-off optimised for predicting treatment outcome, anti-CTLA-4 combination was associated with a significant PFS/OS benefit versus anti-PD-(L)1 monotherapy in patients with TMB>40 mut/Mb (2-year PFS: 100.0% versus 70.7%, p = 0.002; 2-year OS: 100.0% versus 76.0%, p = 0.025), but not in those with TMB ≤40 mut/Mb (2-year PFS: 59.7% versus 68.6%, p = 0.888; 2-year OS: 80.0% versus 81.0%, p = 0.949). ConclusionPatients with dMMR/MSI-H mCRC and relatively lower TMB value displayed early disease progression when receiving ICIs, whereas patients with the highest TMB values may obtain the maximal benefit from intensified anti-CTLA-4/PD-1 combination.

Comprehensive assessment of actionable genomic alterations in primary colorectal carcinoma using targeted next-generation sequencing

BackgroundThe clinical utility of comprehensive genomic profiling (CGP) for guiding treatment has gradually become the standard-of-care procedure for colorectal carcinoma (CRC). Here, we comprehensively assess emerging targeted therapy biomarkers using CGP in primary CRC.MethodsA total of 575 primary CRCs were sequenced by ACTOnco® assay for genomic alterations, tumour mutational burden (TMB), and microsatellite instability (MSI).ResultsEighteen percent of patients were detected as MSI-High (MSI-H), and the remaining cases were classified as microsatellite stable (MSS). Driver mutation prevalence in MSS CRCs were APC (74%), TP53 (67%), KRAS (47%), PIK3CA (21%) and BRAF (13%). The median TMBs for MSI-H and MSS patients were 37.8 mutations per mega base (mut/Mb) and 3.9 mut/Mb, respectively. Forty-seven percent of MSI-H CRC harboured at least one loss-of-function mutations in genes that may hamper immune checkpoint blockade. Among MSS RAS/RAF wild-type CRCs, 59% had at least one actionable mutation that may compromise the efficacy of anti-EGFR therapy. For late-stage CRC, 51% of patients are eligible for standard care actionability and the remaining 49% could be enrolled in clinical trials with investigational drugs.ConclusionsThis study highlights the essential role of CGP for identifying rational targeted therapy options in CRC.

Clinicopathological and molecular profile of grade 3 gastroenteropancreatic neuroendocrine neoplasms.

The 2019Word Health Organization (WHO) subclassified grade 3 (G3) gastroenteropancreatic (GEP) neuroendocrine neoplasms (NEN) into neuroendocrine carcinoma (NEC) or tumours (G3NET) based on morphology and proliferation. Yet, few data exist on molecular profiles for G3NEN. We compared clinicopathological and molecular characteristics of these two groups. We retrospectively reviewed consecutive G3 GEP NEN patients and had their tumour tissues reviewed, reclassified as per the WHO 2019, and analyzed by a next-generation sequencing (NGS) panel. Between 2000 and 2019, 43 patients had pathology revision: 29 (67%) were NEC and 14 (33%) were G3NET, with a 23% change in diagnosis. Median overal survival for G3NET and NEC patients was 55.6 and 11.9months, respectively (hazard ratio=2.78 [95% confidence interval=1.09-7.11], p=.042), which was confirmed by an adjusted analysis (hazard ratio=2.90NEC vs. G3NET; p=.03). NGS was performed in 32 cases: 21NEC and 11 G3NET. Mutations in RB1 and PTEN were exclusively encountered in NEC. Median tumour mutational burden was 5 (0-67) mutations per megabase in NEC and 4.5 (0-9) among G3NET. Microsatellite instability was found in 3 (14.3%) NEC cases. In conclusion, pathology revision is essential to estimate prognosis and therapeutic plan. G3 GEP NEN generally harbour low tumor mutation burden and fewer actionable mutations, but 14% of NEC cases were microsatellite unstable and could benefit from immune checkpoint inhibitors.

Tumour mutational burden predicts resistance to EGFR/BRAF blockade in BRAF-mutated microsatellite stable metastatic colorectal cancer

AimTo unveil genomic and immunohistochemical expression profiles associated with primary resistance to EGFR/BRAF targeted therapy in patients with BRAF-mutated and microsatellite stable (MSS) metastatic colorectal cancer. Experimental designIn this multicenter case-control study on patients treated with EGFR/BRAF ± MEK blockade, we compared a primary resistance cohort (N = 20; RECISTv1.1 PD/SD, and progression-free survival [PFS] <16 weeks) versus a sensitive one (N = 19; RECISTv1.1 PR/CR, and PFS ≥16 weeks) in terms of clinical and genomic/expression data by means of comprehensive genomic profiling, tumour mutational burden (TMB), BRAF-mutant transcriptional subtypes (BM) classification and PTEN expression. ResultsLeft-sided tumours (28% of the total) were enriched in the sensitive versus resistant cohort (53% versus 10%, P = 0.010). Genomic alterations in the PIK3CA/MTOR pathway, BM1 status and PTEN loss were similarly distributed among patients with resistant and sensitive tumours. Amplification of CCND1-3 genes were found only in patients with primary resistance (20% versus 0%, P = 0.106). TMB and prevalence of intermediate TMB (TMB-I 6–20 mutations/Mb) were higher in the resistant versus sensitive cohort (median TMB: 6 [IQR, 3–7.29] versus 3 [IQR, 1.26–3.5]; P = 0.013; TMB-I/H: 60% versus 11%; P = 0.001). Patients with TMB-I had shorter PFS (3.3 versus 5.9 months; HR = 2.19, 95%CI, 1.07–4.47, P = 0.031) and overall survival (6.3 versus 10.5 months; HR = 2.22, 95%CI, 1.02–4.81, P = 0.044). ConclusionDespite the small sample size, the association of a relatively higher TMB with limited benefit from EGFR/BRAF blockade in patients with MSS and BRAF-mutated mCRC deserves prospective validation.

1053P Tumour mutational burden (TMB) assessment using next generation sequencing (NGS) for the prediction of complete response (CR) to immunotherapy (IO) in metastatic melanoma

TMB is an emerging biomarker of IO response, however in the setting of a wide variation in median TMB values between cancer subtypes, histology specific thresholds predicting response remain unclear. The TruSight Oncology 500® (TSO 500) is a comprehensive 523 gene NGS assay and is highly concordant with whole exome sequencing in TMB assessment. Thresholds predicting meaningful response to IO such as CR in metastatic melanoma using this panel are needed.

PATH-09. NEXT-GENERATION SEQUENCING OF GLIOBLASTOMA MULTIFORME ‘EXTREME RESPONDERS’

Abstract BACKGROUND Glioblastoma Multiforme (GBM) is the most common primary brain among adults. Despite steady advances in treatment, the outcome remains universally lethal. Median overall survival in a population-based study is approximately 10 to 12 months. Prognostic factors in GBM patients include age, performance score, extent of surgical resection, IDH1 mutation status and MGMT methylation status. Survival &amp;gt; 2 years is rare; for the purpose of this study we have defined these patients ‘extreme responders.’ This study aims to identify genomic signatures and gene alterations that may have contributed to ‘extreme response’ to standard adjuvant chemoradiotherapy. METHODS A total of 12 out of 141 primary GBM patients were identified as ‘extreme responders’ as part of a retrospective chart review between 2005 - 2016. Of the 12 suitable for NGS testing, 9 were analyzed using the Foundation One CDx® (next-generation sequencing platform) which interrogates 324 genes, microsatellite status and tumour mutational burden (TMB). Baseline demographics, clinical characteristics, and NGS results were compared. RESULTS Of the 9 patients tested, over half were female (5, 55%) and median age at diagnosis was 53 years. Gross total resection was performed at diagnosis in 3 cases. MGMT status was available for 5 patients and was methylated in 3. NGS results revealed: MSI stable in all patients, median TMB 3 muts/Mb and most common gene alterations were TP53 (5/9); ATRX (4/9); EGFR (3/9); CDKN2A/B (3/9); IDH1(3/9); PIK3R1(3/9) and PTEN (3/9). CONCLUSION Acknowledging the fact that our sample size was small and no control group was tested, our study infers a higher incidence of IDH1, TP53 and ATRX mutations which are more common in secondary glioblastomas and this may be a contributing factor towards longer survival in our cohort. We did not identify any universal molecular marker predictive of improved prognosis or exquisite sensitivity to chemoradiotherapy.

PD-12 Clinical, pathological and molecular profiles of G3 neuroendocrine tumors and neuroendocrine carcinomas

In 2019, WHO subclassified grade 3 (G3) gastroenteropancreatic (GEP) neuroendocrine neoplasms (NEN) into neuroendocrine carcinoma (NEC) or tumors (G3 NET). Yet, few data exist on clinical and molecular profiles for G3 NEN. We aimed to compare the clinicopathological and molecular characteristics of G3 GEP NET and GEP NEC. We retrospectively collected data of all G3 GEP NEN patients (pts) diagnosed and treated at our cancer center from 2000 to 2019. Cases with available tumor tissues were reviewed and reclassified according to the WHO 2019 classification. Formalin-fixed, paraffin-embedded NEN tissues had DNA extracted for genomic profiling through next-generation sequencing (NGS), which evaluated mutations profiles, tumor mutation burden (TMB) and the presence of microsatellite instability. Survival from treatment start was the primary endpoint. Seventy-seven pts were included: median age at the diagnosis was 56 (26-91) years and 62% (48/77) were males. The most common primary sites were gastric (27%, 21/77), pancreas (23%, 18/77) and unknown primary NEN (23%, 18/77). Metastatic disease was present in 69% at diagnosis and 91% developed metastasis. Out of 77, 43 (56%) had pathology revision: 29 cases were NEC (67%) and 14 were reclassified as G3 NET (33%), with a change in diagnosis of 23%. In a median follow-up of 44 months, median OS in non-pancreatic digestive G3 NET, pancreatic G3 NET, and GEP NEC were 23.7, 55.6 and 10.8 months, respectively (non-pancreatic G3 NET vs NEC, p=0.01). Median OS in G3 NET with ki67 > 55%, NEC 55% was 19, 25 and 8 months, respectively. In Cox regression multivariate analysis, only cell differentiation sustained as a risk of death (HR 3.14 NEC vs G3 NET; P = 0.025). NGS was performed in 42% (32/77) cases: 21 NEC and 11 G3 NET. Median tumour mutational burden was 5.67 (0 – 66.82) mutations per megabase among NEC and 4.52 (0 – 8.83) among G3 NET. Tumors were microsatellite unstable in 3 (14.3%) NEC cases but zero among G3 NET pts. The most commonly mutated genes in G3 NET were TP53 (N=3; 27.3%), CDKN2A (N=3; 27.3%) and KRAS (2/11, 18.2%) and TP53 (N=15; 71.4%), Rb1 (N=7; 33.3%), PTEN (N=6; 28.6%) and APC (N=4; 19%) among NEC. Non-pancreatic digestive G3 NET is associated with increased survival compared to NEC. Pathology revision is essential to estimate prognosis and consequently therapeutic plan, as some of G3 NET can be treated as G2 NET. While G3 GEP NEN generally harbours low TMB and fewer actionable mutations, 14% of NEC cases were microsatellite unstable and could benefit from immune checkpoint inhibitors.

Tumour immune characterization in adenoid cystic carcinoma identifies prognostic and immunotherapeutically relevant messenger RNA signatures.

e15191 Background: Here, we profiled genetic hallmarks of Chinese adenoid cystic carcinoma (ACC) patients and immune cells infiltration levels by gene expression-based computational methods to investigate the correlation with immunotherapy. Methods: 54 patients with pathologically confirmed ACC admitted from January 2016 to October 2019 were included. Mutation and expression profiles were obtained by whole-exome sequencing (WES) and RNA-seq, respectively. A nomogram that integrated clinicopathological features with the immune signature to predict recurrence or distant metastasis probability was constructed by multivariate Cox regression. Results: As expected, we observed MYB fusion and somatic mutations in our cohort (57%, 31/54), strengthening the role of these aberrations as critical events in ACC. Mutations in NOTCH1 were also found in our cohort (19%, 10/54). Median tumour mutational burden (TMB) of these patients is 0.85 Muts/Mbp (0-11 Muts/Mbp). Analysis of WES data showed that all patients were microsatellite instability (MSI)-negative, and 17 of them were confirmed by conventional MSI-PCR testing. Lack of PD-L1 expression is 96.0% (n = 46/48) at a tumour proportion score 0% and 22.9% (11/48) of cases showed more than 10% CD8+ prevalence. We compared cancer types using an immune infiltration score (IIS) and an antigen presenting machinery expression (APM) score and find that ACC is among the lowest for both scores. Statistically significant correlations were found between IIS and distant metastasis-free survival (univariate analysis, HR = 2.64, 95% confidence interval [CI], 1.134 to 6.145, p = 0.023). Patients with higher IIS had shorter distant metastasis-free survival time (log-rank p = 0.020), and patients with higher T cell infiltration score (TIS) had shorter locoregional recurrence-free survival time (log-rank p = 0.050). In addition, we investigated the pretreatment immune profile of five ACC patients treated with PD-1 checkpoint inhibitors, and found that both TIS and APM were elevated in responding patient (with a partial response to camrelizumab) whereas they were in the lowest for patients with progressive disease on camrelizumab or pembrolizumab. Conclusions: Our study highlights the immune infiltration patterns and unravels mRNA signatures with prognostic utility and immunotherapeutic biomarker potential in ACC.

A retrospective analysis of 66 colorectal cancer cases from Guy’s and St Thomas’ (GSTT) Molecular Tumour Board

Abstract Background The utility of next generation sequencing (NGS) is rapidly evolving within our medical practice with various applications. The 100K Genomes Project was established to sequence 100 000 genomes from NHS patients with cancer or rare diseases, and to date actionable findings have been found for approximately 50% of cancer cases. Data from the 100K Genomes Project were reviewed at Guy’s Hospital Molecular Tumour Board (MTB). Methods Whole genome sequencing (WGS) was performed for the 100K Genomes Project and Genomics England using an Illumina platform. Results Between December 2016 and August 2018, 66 patients with colorectal cancer (CRC) were treated with surgery at GSTT. Median age was 68.5 (52-95). Majority were left sided (69%) vs right sided (30%) adenocarcinomas. TNM status was 1.5% / 47% / 37.9% / 13.6% for stage I/II/III/IV respectively. Analyses were performed in FFPE (35.8%) and frozen tissue (64.2%). Median estimated tumour purity was 53.5% (20-100%). Median tumour mutational burden (TMB) was 5.92 (2.94-173.26). Mutational signatures 6, 10 and 15 were found for 8 patients and were associated with high TMB. A total of 557 Domain 1 (actionable) variants (range 1-68) were identified for this cohort. After review in the MTB, at least one clinically relevant actionable variant was identified for 59.7% of the patients; KRAS 37.3%, NRAS 6%, BRAF 11.9%, TP53 70.1%, PIK3CA 35.8%, ATM 17.9%, RICTOR 14.9%, APC and PTEN 11.9% each and mTOR 8.6%. Somatic BRCA1 and BRCA2 variants were found in 7.6% and 12.1% respectively. Further validation was recommended for 9 patients (13.4%). DPYD alterations were identified in 11.9%, but none of the clinically relevant DPYD mutations related to 5FU toxicity were discovered for 2 of the patients that were tested. 9.4% were identified with potentially pathogenic germline variants (7.6% with RAD51D, RAD51C, MUTYH, MSH6 and PMS2 class 4 & 5 variants) and referral to Clinical Genetics was recommended. Conclusions GSTT MTB identified actionable mutations in more than half patients in the CRC cohort, out of whom 14% had alterations that made them eligible for clinical trials. Over 9% had potentially pathogenic germline variants. Our data from this small cohort resemble data in the literature from other MTBs. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

1965P - Comparison of molecular profiles between primary tumour and matched metastasis in non-small cell lung cancer

1965P - Comparison of molecular profiles between primary tumour and matched metastasis in non-small cell lung cancer

913PD - APACHE: An open label, randomized, phase II study of durvalumab (Durva), alone or in combination with tremelimumab (Treme), in patients (pts) with refractory germ cell tumours (GCT): Results from the expanded combination therapy cohort

BackgroundInterim results from APACHE (NCT03081923) have been presented, with the monotherapy arm stopped for futility (Necchi, Eur Urol 2018). Updated results with the expanded combination therapy cohort are presented. MethodsApache is an open-label, randomized, 3-stage, phase 2 study. Pts who have failed ≥2 chemotherapy (CT) regimens received Durva, 1.5gr q4w, x 13 cycles (arm A) or Durva plus with Treme, 75mg q4w, x 4 cycles, followed by Durva alone x total 13 cycles (arm B). The primary endpoint is the modified objective response-rate (mORR=RECIST 1.1 complete or partial response [PR] or stable disease [SD]+STM reduction >10%). H0: mORR rate ≤10%, H1: mORR ≥25% (α and β=10%). The total sample size of 120 pts is split into 3 stages: in stage 1, each arm is terminated whenever no response is observed in 11 pts/arm. In stage 2, 29 additional pts/arm are enrolled. Biomarker analyses include: PD-L1 expression on immune cells (Ventana SP142) and comprehensive genomic profiling (CGP) with FoundationOne assay. ResultsFrom 02/17-11/18, 29 pts were enrolled (11 arm A and 18 arm B). 22 had gonadal and 7 extragonadal GCT, 19 had received >/=3 prior CT regimens. Median tumour mutational burden (TMB) was 4 mutations (mut)/mb, 1pt showed microsatellite instability in arm A. 10 pts (34.5%) experienced any-Grade AEs, without differences between arms. In the expanded arm B, 2 responses (11.1%, 1 RECIST-PR in seminoma and 1 SD with STM reduction in nonseminoma) and 2 SD were observed. PD occurred regardless of PD-L1 expression and TMB. After median follow-up of 16.3 months, 12-month OS in arm A was 30% (95%CI: 7.2-57.7) and in arm B was 29.6% (95%CI: 10.2-52.2). Further CT post-PD was administered in 8 pts (27.6%), but was mostly ineffective (7/8 SD/PD). ConclusionsDespite objective responses were rare with IO, long-term OS results are encouraging in very highly pre-treated GCT pts, suggesting a role for maintenance approach in earlier setting (2nd-line CT), and privileging Durva+Treme due to more responses. Conventional IO biomarkers did not allow for patient selection, and further research is warranted. Clinical trial identificationNCT03081923. Legal entity responsible for the studyAstraZeneca. FundingAstraZeneca. DisclosureS.M. Ali: Full / Part-time employment: Foundation Medicine. J. Chung: Full / Part-time employment: Foundation Medicine. J.S. Ross: Full / Part-time employment: Foundation Medicine. A. Necchi: Honoraria (institution), Advisory / Consultancy: Merck; Honoraria (institution), Advisory / Consultancy: AstraZeneca; Honoraria (institution), Advisory / Consultancy, Spouse / Financial dependant: Bayer; Advisory / Consultancy: BMS; Advisory / Consultancy: Clovis; Honoraria (institution), Advisory / Consultancy: Janssen; Honoraria (institution), Advisory / Consultancy: Roche. All other authors have declared no conflicts of interest.

108P - Biological difference of tumour mutational burden (TMB) and microsatellite instability (MSI) status in patients (pts) with somatic vs germline BRCA1/2-mutated advanced gastrointestinal (GI) cancers using cell-free DNA (cfDNA) sequencing analysis in the GOZILA study

108P - Biological difference of tumour mutational burden (TMB) and microsatellite instability (MSI) status in patients (pts) with somatic vs germline BRCA1/2-mutated advanced gastrointestinal (GI) cancers using cell-free DNA (cfDNA) sequencing analysis in the GOZILA study

1040P - Mutational landscapes and tumour mutational burden expression in endometrial cancer

BackgroundEndometrial cancer (EC) is one of the most common gynaecological tumours. Tumour mutational burden (TMB) has emerged as a promising predictor to evaluate efficacy to immunotherapy in several kinds of solid tumours. However, the relationship between TMB and genetic features of EC remains unclear. MethodsTotal 50 EC patients including 41 endometrioid adenocarcinoma, 6 uterine serous adenocarcinoma, 1 uterine clear cell carcinoma, 1 endometrial squamous cell carcinoma and 1 endometrial adenosquamous carcinoma were enrolled in this study. The ECs had been classified as FIGO I (n=14), II (n=6), III (n=12), IV (n=16) and not available (n=2). FFPE tumour and matched blood samples were collected from patients for NGS-based targeted panel sequencing (450 genes). Genomic alterations and TMB were assessed. ResultsThe 50 patients had a median age of 56 years (range, 32-73 years) with a median TMB of 3.8 muts/Mb (interquartile range (IQR), 1.5-13.7 muts/Mb). We found recurrent mutations, including PTEN (64%), PIK3CA (44%), ARID1A (40%), PIK3R1 (36%), TP53 (32%), CHD4 (20%), and KRAS (20%). FGFR2 mutations were occurred in 7 (14%) patients. The most frequently mutated genes in early-stage (FIGO I and II) were PTEN (85%), ARID1A (50%), PIK3R1 (50%), PIK3CA (40%), LRP1B (30%), and CHD4 (30%), while the most common mutations in advanced-stage (FIGO III and IV) were PTEN (46%), PIK3CA (43%), TP53 (43%), ARID1A (36%), PIK3R1 (29%), and KRAS (21%). We also found that all POLE mutations (5/5) occurred in early-stage. Mutations of PIK3R1, CHD4, CTCF, SETD2, PPP2R1A, NF1, BRCA2, ARID1B and POLE were associated with TMB-high (TMB-H, TMB≥10muts/Mb) (P<0.05 for all). Importantly, all POLE mutations occurred in EC with TMB-H, including two cases of EC with ultrahigh TMB (TMB > 100 muts/Mb). At least one actionable mutation was identified in 86% (43/50) patients. ConclusionsPI3K signaling pathway genes, PTEN, PIK3CA and PIK3R1 were most frequently mutated in EC. 26% patients (13/50) had TMB-H. POLE mutations likely occurred in early stage and were related with TMB-H, which may provide potential targets for immunotherapy of EC. Legal entity responsible for the studyThe authors. FundingNational Natural Science Foundation of China (Youth fund project, no. 81602267). DisclosureY. Zhang: Full / Part-time employment: OrigiMed. S. Zhang: Full / Part-time employment: OrigiMed. S. Zhao: Full / Part-time employment: OrigiMed. F. Guo: Full / Part-time employment: OrigiMed. F. Pang: Full / Part-time employment: OrigiMed. L. Zhang: Full / Part-time employment: OrigiMed. X. Dong: Full / Part-time employment: OrigiMed. K. Wang: Full / Part-time employment: OrigiMed. All other authors have declared no conflicts of interest.