Wild-type Cases Research Articles

IDH1 and IDH2 Driven Intrahepatic Cholangiocarcinoma (IHCC): A comprehensive genomic and immune profiling study.

4009 Background: IDH1/2 genetic aberrations (GA) occur in 20% of IHCC cases and may be specifically targeted by IDH inhibitors. The genomic and immunologic profile of IHCC with IDH1/2 GA remains undefined. IDH1 mutations impair DNA damage repair (DDR), loss of heterozygosity (LOH) and may represent a biomarker for DDR in these patients. Methods: Comprehensive genomic profiling (CGP) was performed in 3,067 cases of advanced stage IHCC using a hybrid capture-based FDA-approved assay. Tumor mutational burden (TMB) was determined on 0.8 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 95 loci. PD-L1 expression in tumor cells (Dako 22C3) was measured by immunohistochemistry (IHC). Genomic LOH (gLOH) was assessed for samples meeting quality criteria. Densities of tumor associated immune cells and immune-checkpoint markers expressed in epithelial malignant cells and in the tumor microenvironment of 100 surgical samples from 96 patients were evaluated by IHC using digital image analysis of 14 markers (CD3, CD4, CD8, CD68, PD1,PD-L1, B7-H4, B7-H3, IDO1, ICOS, VISTA, OX40, TIM3, LAG3). Tissue microarrays were generated for multiplex immune panel analysis. A p value < 0.05 was considered statistically significant. Results: 426 (14%) of IHCC were IDH1+ and 125 (4%) were IDH2+ (Table) and were mutually exclusive. All IDH1 GA occurred at the R132 locus and included R132C (69%) and R132L/G/S/H/F (16%/7%/4%/3%/ < 1%) and 119Q ( < 1%) and IDH2 GA at R172 (94.4%) and R140 (6.6%). IDH1+ and IDH2+ IHCC had fewer co-occurring targetable GA than IDH1/2 wildtype (WT, IDHwt) cases including FGFR2 rearrangements (RE) (P <.0001), ERBB2 (P =.0009) and BRAF (P =.04). Median gLOH were not significantly different between IDH1+ IDH2+ IHCC vs. IDHwt IHCC (p = 0.37). Potential biomarkers of immune checkpoint inhibition (ICI) response including MSI High, TMB > 10 mut/Mb, and PD-L1 positivity were more frequent in IDHwt IHCC than IDH1+ IHCC. 29/96 (30%) surgical patients were positive for IDH1/2 mutation though mutational status did not confer a statistically significant difference in the 14 immune biomarker panel expression. Conclusions: CGP reveals significant differences in GA between IDH1+, IDH2+ and IDHwt IHCC consistent with IDH1 and IDH2 being driver oncogenes for IHCC. Immune biomarker expression and gLOH did not differ significantly between IDH mutated and WT cases.[Table: see text]

AcornHRD: A novel tool to identify homologous recombination deficiency events in whole genome sequencing data.

e15078 Background: Recently, homologous recombination deficiency (HRD) scores are associated with the efficacy of Poly‐(ADP‐Ribose)‐Polymerase (PARP) inhibition and platinum-based chemotherapy in a variety of cancers. Evaluating HRD level in patients with cancers is becoming far more important and influential, so far, there is no standard method to be used in clinical. In this study, we developed an algorithm to detect HRD from next-generation sequencing (NGS) for finding additional patients may potentially benefit from target therapy. Methods: Forty-eight patients were enrolled, including breast cancer, ovarian cancer, prostatic cancer. Fifteen cell lines with breast cancer and endometrial carcinoma were collected from Cobioer biosciences co., LTD. Forty-eight Formalin-fixed, paraffin embedded (FFPE) samples and 15 cell lines were performed by DNA extracting. We developed an HRD score algorithm, termed as AcornHRD algorithm. HRD score was analyzed by whole-genome sequencing, and GATK mutect2 software was used to detect BRCA1/2mutation by deep sequencing. Results: BRCA1/2 deleterious mutations were observed in 20 patients (41.7%). HRD was explained by deficiencies in 17 patients (85.0%) with BRCA mutation, whereas eight HRD-high tumors were non- BRCA related (28.6%). Among BRCA wild-type patients, the corresponding percentage of HRD positive patients in breast cancer, ovarian cancer and prostate cancer were 36.3%, 37.5% and 11.1%, respectively. Similar results were also verified in the cell line datasets. The findings showed that 100% (3/3) BRCA1/2 deficient cell lines are also HRD-high. Furthermore, HRD scores were highly correlated with standard results in the cell line datasets. Conclusions: We here report the NGS-based HRD scores to distinguish similarly well between BRCA mutant and BRCA wild-type cases in a cohort of Chinese population. AcornHRD scores were highly associated with BRCA1/2 deficiency. AcornHRD algorithm can be a useful tool to detect HRD events in clinical settings.

Recurrent Somatic MAP2K1 Mutations in Papillary Thyroid Cancer and Colorectal Cancer.

Mitogen-activated protein kinase kinase 1 (MAP2K1) is a dual specificity protein kinase that phosphorylates both threonine and tyrosine residues in ERK. MAP2K1 mutations have been identified in several cancers. However, their role in Middle Eastern papillary thyroid cancer (PTC) and colorectal cancer (CRC) is lacking. In this study, we evaluated the prevalence of MAP2K1 mutations in a large cohort of Middle Eastern PTC and CRC using whole-exome and Sanger sequencing technology. In the discovery cohort of 100 PTC and 100 CRC cases (comprising 50 MAPK mutant and 50 MAPK wildtype cases each), we found one MAP2K1 mutation each in PTC and CRC, both of which were MAPK wildtype. We further analyzed 286 PTC and 289 CRC MAPK wildtype cases and found three MAP2K1 mutant PTC cases and two MAP2K1 mutant CRC cases. Thus, the overall prevalence of MAP2K1 mutation in MAPK wildtype cases was 1.1% (4/336) in PTC and 0.9% (3/339) in CRC. Histopathologically, three of the four MAP2K1 mutant PTC cases were follicular variant and all four tumors were unifocal with absence of extra-thyroidal extension. All the three CRC cases harboring MAP2K1 mutation were of older age (> 50 years) and had moderately differentiated stage II/III tumors located in the left colon. In conclusion, this is the first comprehensive report of MAP2K1 somatic mutations prevalence in PTC and CRC from this ethnicity. The mutually exclusive nature of MAP2K1 and MAPK mutations suggests that each of these mutation may function as an initiating mutation driving tumorigenesis through MAPK signaling pathway.

A rare variant in EZH2 is associated with prostate cancer risk.

Prostate cancer (PrCa) is highly heritable, and although rare variants contribute significantly to PrCa risk, few have been identified to date. Herein, whole-genome sequencing was performed in a large PrCa family featuring multiple affected relatives spanning several generations. A rare, predicted splice site EZH2 variant, rs78589034 (G > A), was identified as segregating with disease in all but two individuals in the family, one of whom was affected with lymphoma and bowel cancer and a female relative. This variant was significantly associated with disease risk in combined familial and sporadic PrCa datasets (n=1551; odds ratio [OR]=3.55, P=1.20 × 10-5 ). Transcriptome analysis was performed on prostate tumour needle biopsies available for two rare variant carriers and two wild-type cases. Although no allele-dependent differences were detected in EZH2 transcripts, a distinct differential gene expression signature was observed when comparing prostate tissue from the rare variant carriers with the wild-type samples. The gene expression signature comprised known downstream targets of EZH2 and included the top-ranked genes, DUSP1, FOS, JUNB and EGR1, which were subsequently validated by qPCR. These data provide evidence that rs78589034 is associated with increased PrCa risk in Tasmanian men and further, that this variant may be associated with perturbed EZH2 function in prostate tissue. Disrupted EZH2 function is a driver of tumourigenesis in several cancers, including prostate, and is of significant interest as a therapeutic target.

MYB rearrangements and over-expression in T-cell acute lymphoblastic leukemia.

We investigated MYB rearrangements (MYB-R) and the levels of MYB expression, in 331 pediatric and adult patients with T-cell acute lymphoblastic leukemia (T-ALL). MYB-R were detected in 17 cases and consisted of MYB tandem duplication (tdup) (= 14) or T cell receptor beta locus (TRB)-MYB (= 3). As previously reported, TRB-MYB was found only in children (1.6%) while MYB tdup occurred in both age groups, although it was slightly more frequent in children (5.2% vs 2.8%). Shared features of MYB-R T-ALL were a non-early T-cell precursor (ETP) phenotype, a high incidence of NOTCH1/FBXW7 mutations (81%) and CDKN2AB deletions (70.5%). Moreover, they mainly belonged to HOXA (=8), NKX2-1/2-2/TLX1 (=4), and TLX3 (=3) homeobox-related subgroups. Overall, MYB-R cases had significantly higher levels of MYB expression than MYB wild type (MYB-wt) cases, although high levels of MYB were detected in ~ 30% of MYB-wt T-ALL. Consistent with the transcriptional regulatory networks, cases with high MYB expression were significantly enriched within the TAL/LMO subgroup (P = .017). Interestingly, analysis of paired diagnosis/remission samples demonstrated that a high MYB expression was restricted to the leukemic clone. Our study has indicated that different mechanisms underlie MYB deregulation in 30%-40% of T-ALL and highlighted that, MYB has potential as predictive/prognostic marker and/or target for tailored therapy.

High discrepancy in thrombotic events in non-small cell lung cancer patients with different genomic alterations.

BackgroundAcute complications, such as venous thromboembolism (VTE), are common in patients with advanced severe lung cancers. However, current VTE risk scores cannot adequately identify high-risk patients with non-small cell lung cancer (NSCLC). The study proposed to elucidated the incidence of thromboembolism (TE) in patients with different oncogenic aberrations and the impact of these aberrations on the efficacy of targeted therapy in patients with NSCLC.MethodsA systemic review was conducted in Web of Science, PubMed, Embase and the Cochrane Library to evaluate the incidence of TE in different molecular subtypes of NSCLC. Data from patients diagnosed of advanced NSCLC who harboring anaplastic lymphoma kinase (ALK) or ROS proto-oncogene 1 receptor tyrosine kinase (ROS1) rearrangements since 2016 to 2019 were also retrospectively collected. A meta-analysis with random-effects model, sensitivity analysis and publication bias were performed. The principal summary measure was incidence of thrombotic events in NSCLC patients. And the efficacy of tyrosine kinase inhibitor (TKI) therapy was compared between the two subgroups.ResultsA total of 5,767 cases from 20 studies were included in the analysis of the incidence of thrombosis in patients with different oncogenic alterations. The pooled analysis showed a higher risk of thrombosis in ROS1-fusion types (41%, 95% CI: 35–47%) and ALK-fusion types (30%, 95% CI: 24–37%) than in EGFR-mutation (12%, 95% CI: 8–17%), KRAS-mutation (25%, 95% CI: 13–50%), and wild-type (14%, 95% CI: 10–20%) cases. A high prevalence of thrombosis (ALK: 24.4%; ROS1: 32.6%) was observed in the Shanghai Pulmonary Hospital (SPH) cohort of 224 patients with ALK or ROS1 fusion. Furthermore, patients with embolism had significantly shorter progression-free survival (PFS) after TKI therapy than those without embolism, both in the ALK+ cohort (5.6 vs. 12.9 months, P<0.0001) and in the ROS1+ cohort (9.6 vs. 17.6 months, P=0.0481).ConclusionsNSCLC patients with ALK/ROS1 rearrangements are more likely to develop thrombosis than patients with other oncogenic alterations. Thrombosis may also be associated with an inferior response and PFS after TKI therapy.

Long-Term Follow-Up, Association between CARD15/NOD2 Polymorphisms, and Clinical Disease Behavior in Crohn’s Disease Surgical Patients

Background CARD15/NOD2 is the most significant genetic susceptibility in Crohn's disease (CD) even though a relationship between the different polymorphisms and clinical phenotype has not been described yet. The study is aimed at analyzing, in a group of CD patients undergoing surgery, the relationship between CARD15/NOD2 polymorphisms and the clinical CD behavior after a long-term follow-up, in order to identify potential clinical biomarkers of prognosis. Methods 191 surgical CD patients were prospectively characterized both for the main single nucleotide polymorphisms of CARD15/NOD2 and for many other environmental risk factors connected with the severe disease form. After a mean follow-up of 7.3 years, the correlations between clinical features and CD natural history were analyzed. Results CARD15/NOD2 polymorphisms were significantly associated with younger age at diagnosis compared to wild type cases (p < 0.05). Moreover, patients carrying a 3020insC polymorphism presented a larger Δ between diagnosis and surgery (p = 0.0344). Patients carrying an hz881 and a 3020insC exhibited, respectively, a lower rate of responsiveness to azathioprine (p = 0.012), but no difference was found in biologic therapy. Finally, the risk of surgical recurrence was significantly associated, respectively, to age at diagnosis, to familial CD history, to diagnostic delay, to arthritis, and to the presence of perioperative complications. Conclusions 3020insC CARD15 polymorphism is associated with an earlier CD onset, and age at CD diagnosis < 27 years was confirmed to have a detrimental effect on its clinical course. In addition, the familiarity seems to be connected with a more aggressive postoperative course. Finally, for the first time, we have observed a lower rate of responsiveness to azathioprine in patients carrying an hz881 and a 3020insC.

Clinical Value of EGFR Copy Number Gain Determined by Amplicon-Based Targeted Next Generation Sequencing in Patients with EGFR-Mutated NSCLC.

BackgroundThe clinical relevance of epidermal growth factor receptor (EGFR) copy number gain in patients with EGFR mutated advanced non-small cell lung cancer on first-line tyrosine kinase inhibitor treatment has not been fully elucidated.ObjectiveWe aimed to estimate EGFR copy number gain using amplicon-based next generation sequencing data and explored its prognostic value.Patients and MethodsNext generation sequencing data were obtained for 1566 patients with non-small cell lung cancer. EGFR copy number gain was defined based on an increase in EGFR read counts relative to internal reference amplicons and normal controls in combination with a modified z-score ≥ 3.5. Clinical follow-up data were available for 60 patients treated with first-line EGFR-tyrosine kinase inhibitors.ResultsSpecificity and sensitivity of next generation sequencing-based EGFR copy number estimations were above 90%. EGFR copy number gain was observed in 27.9% of EGFR mutant cases and in 7.4% of EGFR wild-type cases. EGFR gain was not associated with progression-free survival but showed a significant effect on overall survival with an adjusted hazard ratio of 3.14 (95% confidence interval 1.46–6.78, p = 0.003). Besides EGFR copy number gain, osimertinib in second or subsequent lines of treatment and the presence of T790M at relapse revealed significant effects in a multivariate analysis with adjusted hazard ratio of 0.43 (95% confidence interval 0.20–0.91, p = 0.028) and 0.24 (95% confidence interval 0.1–0.59, p = 0.001), respectively.ConclusionsPre-treatment EGFR copy number gain determined by amplicon-based next generation sequencing data predicts worse overall survival in EGFR-mutated patients treated with first-line EGFR-tyrosine kinase inhibitors. T790M at relapse and subsequent treatment with osimertinib predict longer overall survival.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11523-021-00798-2.

Abstract PS5-04: Therapeutic considerations in microsatellite instability high (MSI- H) breast cancers (BC) identified by comprehensive genomic profiling (CGP)

Abstract Background: Non-colorectal MSI-H tumors are increasingly identified by CGP. Rare types such as MSI-H BC remain poorly defined with an evidence gap on how to optimally sequence or combine with standard of care treatment. MSI can be measured by either IHC, PCR, or CGP and can be caused by both sporadic and germline variants within different tumor types. Prior studies in BC have shown evidence of dMMR by IHC cases MSS based on PCR. This could be due to intra-tumor heterogeneity, specific microsatellite loci evaluated, or penetrance of germline, somatic, or epigenetic alterations. Published data suggests carriers of germline pathogenic MMR variants have a BC risk equivalent to the normal population and currently germline testing is recommended only for BRCA. Currently in advanced BC, standard tumor biomarker testing includes IHC, PCR, and FISH; however, with increasing use of CGP we demonstrate additional actionable biomarkers as well as potential germline variants in MSI-H BC. Methods: DNA was extracted and hybrid capture CGP was performed on 29,160 BC cases. TMB was determined on 0.8-1.2 Mb of DNA and MSI status on 95-114 loci. Genomic LOH was also evaluated. Comparative analysis was done with 101 MSI-H BC, 841 MSS BC and 4,988 non-breast MSI-H cancers. Histological subtype was obtained from the pathology along with orthogonal testing for ER/PR/HER2 status. Somatic-germline-zygosity (SGZ) status was predicted using a published research use algorithm. Select case reports with clinical outcomes will be presented. Results: We identified 101 (0.35% of total) MSI-H BC cases: 29 ER+/HER2-, 5 HER2+, 29 TNBC, and 28 unknown. Amongst BC cases with known subtype, TNBC was enriched for MSI-H vs MSS (53.4 vs 35.8%, p=0.005). The median TMB in MSI-H BC (26.1 mut/Mb, IQR 17.4;42.8) was significantly lower than that of MSI-H colon (46.1mut/MB) and higher than that of MSI-H uterine tumors (22.6mut/Mb) in our comparison group (p&amp;lt;0.001 for both, Kruskal-Wallis test). Pathogenic variants in an MMR gene were found in 61.4% of MSI-H BC with MLH1 loss being the most common (13.6%) and much higher vs. the non-breast MSI-H cohort (2.4%, p&amp;lt;0.0001). Germline mutations in MMR genes in BC are rare yet 5/52 MMR short variants identified in 101 MSI-H BCs were predicted to be germline, 34 somatic, and 13 could not be determined. We identified 21 MSI-H BC patients with a total of 25 pathogenic BRCA1/2 alterations of which 4 were likely germline, 10 were homozygous, and were enriched in TNBC. These were mainly frameshift mutations, including BRCA2 T3033fs* in 5/18 (28%) cases; however, 7/25 were deletions, rearrangements, or nonsense mutations. Median gLOH was significantly higher in BRCA altered (19.7%) compared to BRCA wild-type MSI-H BC cases (9.6%) (p=0.007, Wilcox test). Additional potentially targetable biomarkers included 26 CDx eligible PIK3CA mutations, 11 ERBB2 activating point mutations in the TKD or ECD domain, 1 FGFR2 rearrangement, and 6 AKT1 E17K mutations. Four cases also had concurrent (CD274) PD-L1 amplifications. Conclusion: MSI-H BC is rare but CGP can identify additional therapeutic options for rational combination with targeted therapies such as PI3K, PARP, and HER2 inhibitors. BRCA alterations may be of germline or somatic origin and they may be targetable, as demonstrated by gLOH, rather than passenger mutations. Further characterization of these tumors and comparison to both MSS BC and non-breast MSI-H tumor types, combined with treatment outcomes, can provide insights on rationale combinations and/or sequencing of therapeutic agents. Citation Format: Kimberly McGregor, Natalie Danzinger, Jeffrey S. Ross, Kyle Gowen, Alexa B. Schrock, Garrett M. Frampton, Dean C. Pavlick, Jan W. Davis, Carl R. Gray, Jeffrey M. Venstrom. Therapeutic considerations in microsatellite instability high (MSI- H) breast cancers (BC) identified by comprehensive genomic profiling (CGP) [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 PS5-04.

Prognostic Assessment of BRAF Mutation in Preoperative Thyroid Fine-Needle Aspiration Specimens

We investigate the potential role of BRAF testing in guiding surgical intervention in papillary thyroid carcinoma (PTC). Thyroid fine-needle aspiration (FNA) cases with available BRAF result and follow-up thyroidectomy for PTC were included in the study. Cytology and surgical diagnoses were correlated with BRAF status. There were 151 cases of thyroid FNA specimens with BRAF testing (70 mutant and 81 wild-type BRAF) and histologically confirmed unilateral, unifocal PTCs. There were no differences in age, sex, tumor size, or lymphovascular invasion on thyroidectomy specimens between mutant and wild-type BRAF cases. BRAF mutation was significantly associated with cytology diagnosis (P < .001), PTC subtype (P < .001), extrathyroidal extension (ETE) (P = .006), and higher tumor (T) stage (P = .04). However, an analysis within the histologic subtypes of PTC revealed no significant association between BRAF mutation and ETE or higher T stage. There was also no difference in central (P = .847) or lateral (p = 1) neck lymph node (LN) metastasis. BRAF mutation identified in thyroid FNA specimens correlates with histologic subtypes but is not an independent factor for predicting PTC biological behavior and should not be used to guide the extent of LN dissection.

Clinical and genomic characterization of patients diagnosed with the provisional entity acute myeloid leukemia with BCR-ABL1, a Swedish population-based study.

Acute myeloid leukemia (AML) with t(9;22)(q34;q11), also known as AML with BCR-ABL1, is a rare, provisional entity in the WHO 2016 classification and is considered a high-risk disease according to the European LeukemiaNet 2017 risk stratification. We here present a retrospective, population-based study of this disease entity from the Swedish Acute Leukemia Registry. By strict clinical inclusion criteria we aimed to identify genetic markers further distinguishing AML with t(9;22) as a separate entity. Twenty-five patients were identified and next-generation sequencing using a 54-gene panel was performed in 21 cases. Interestingly, no mutations were found in NPM1, FLT3, or DNMT3A, three frequently mutated genes in AML. Instead, RUNX1 was the most commonly mutated gene, with aberrations present in 38% of the cases compared to around 10% in de novo AML. Additional mutations were identified in genes involved in RNA splicing (SRSF2, SF3B1) and chromatin regulation (ASXL1, STAG2, BCOR, BCORL1). Less frequently, mutations were found in IDH2, NRAS, TET2, and TP53. The mutational landscape exhibited a similar pattern as recently described in patients with chronic myeloid leukemia (CML) in myeloid blast crisis (BC). Despite the concomitant presence of BCR-ABL1 and RUNX1 mutations in our cohort, both features of high-risk AML, the RUNX1-mutated cases showed a superior overall survival compared to RUNX1 wildtype cases. Our results suggest that the molecular characteristics of AML with t(9;22)/BCR-ABL1 and CML in myeloid BC are similar and do not support a distinction of the two disease entities based on their underlying molecular alterations.

How Far Are We from the Rapid Prediction of Drug Resistance Arising Due to Kinase Mutations?

In all living organisms, protein kinases regulate various cell signaling events through phosphorylation. The phosphorylation occurs upon transferring an ATP’s terminal phosphate to a target residue. Because of the central role of protein kinases in several proliferative pathways, point mutations occurring within the kinase’s ATP-binding site can lead to a constitutively active enzyme, and ultimately, to cancer. A select set of these point mutations can also make the enzyme drug resistant toward the available kinase inhibitors. Because of technical and economical limitations, rapid experimental exploration of the impact of these mutations remains to be a challenge. This underscores the importance of kinase–ligand binding affinity prediction tools that are poised to measure the efficacy of inhibitors in the presence of kinase mutations. To this end, here, we compare the performances of six web-based scoring tools (DSX-ONLINE, KDEEP, HADDOCK2.2, PDBePISA, Pose&Rank, and PRODIGY-LIG) in assessing the impact of kinase mutations on their interactions with their inhibitors. This assessment is carried out on a new structure-based BINDKIN benchmark we compiled. BINDKIN contains wild-type and mutant structure pairs of kinase–inhibitor complexes, together with their corresponding experimental binding affinities (in the form of IC50, Kd, and Ki). The performance of various web servers over BINDKIN shows that they cannot predict the binding affinities (ΔGs) of wild-type and mutant cases directly. Still, they could catch whether a mutation improves or worsens the ligand binding (ΔΔGs) where the highest Pearson’s R correlation coefficient is reached by DSX-ONLINE over the Ki dataset. When homology models are used instead of Ki-associated crystal structures, DSX-ONLINE loses its predictive capacity. These results highlight that there is room to improve the available scoring functions to estimate the impact of protein kinase point mutations on inhibitor binding. The BINDKIN benchmark with all related results is freely accessible online (https://github.com/CSB-KaracaLab/BINDKIN).

Assessment of Mortality and Hospital Admissions Associated with Confirmed Infection with SARS-CoV-2 Variant of Concern VOC-202012/01 (B.1.1.7) a Matched Cohort and Time-to-Event Analysis

Background: The emergence of VOC202012/01 in England, known as B.1.1.7 or informally as the ‘UK variant’, has coincided with rapid increases in the number of PCR-confirmed positive cases in areas where the variant has been concentrated. Methods: To assess whether infection with SARS-CoV-2 variant VOC202012/01 is associated with more severe clinical outcomes compared to wild-type infection, genomically sequenced and confirmed variant and wild-type cases were linked to routine healthcare and surveillance datasets. Two statistical analyses were conducted to compare the risk of hospital admission and death within 28 days of test between variant and wild-type cases: a case-control study and an adjusted Cox proportional hazards model. Differences in severity of disease were assessed by comparing hospital admission and mortality, including length of hospitalisation and time to death.Results: Of 63,609 genomically sequenced COVID-19 cases tested in England between October and December 2020 6,038 were variant cases. In the matched cohort analysis 2,821 variant cases were matched to 2,821 to wild-type cases. In the time to event analysis we observed a 34% increased risk in hospitalisation associated with the variant compared to wild-type cases, however, no significant difference in the risk of mortality was observed. Conclusion: We found evidence of increased risk of hospitalisation after adjusting for key confounders, suggesting increase infection severity associated with this variant. Follow-up studies are needed to assess potential longer-term differences in the clinical outcomes of people infected with the VOC-202012/01 variant.

Simultaneous analysis of ALK, RET, and ROS1 gene fusions by NanoString in Brazilian lung adenocarcinoma patients.

BackgroundGene fusions have been successfully employed as therapeutic targets for lung adenocarcinoma. However, tissue availability for molecular testing of multiples alterations is frequently unfeasible. We aimed to detect the presence of ALK, RET, and ROS1 rearrangements by a RNA-based single assay in Brazilian lung adenocarcinomas and to associate with clinicopathological features and genetic ancestry.MethodsFrom a FFPE series of 444 molecularly characterized lung adenocarcinomas, 253 EGFR/KRAS wild-type cases were eligible for gene rearrangement analysis. Following RNA isolation, ALK, RET, and ROS1 rearrangements were simultaneously analyzed employing the ElementsXT Custom panel (NanoString Technologies). Rearrangements were further associated with clinicopathological features and genetic ancestry of the patients.ResultsThe NanoString platform was performed in subset of 142 cases. Gene fusion results were conclusive for 94.4% (n=134) cases (failure rate =5.6%). ALK rearrangements were observed in 21 out of 134 cases, and associated with younger, never smokers, metastatic disease, and metastases in the central nervous system. RET and ROS1 fusions were detected in two and one out of 134 cases, respectively. Genetic ancestry was not associated with gene fusions. Overall, considering all cases for which a molecular analysis was conclusive (EGFR/KRAS/ALK/RET/ROS1), ALK fusions frequency was observed in 6.5% (21/325), RET in 0.6% (2/325), and ROS1 in 0.3% (1/325).ConclusionsThis study successfully used a RNA-based single assay for the simultaneous analysis of ALK, RET, and ROS1 fusions employing routine biopsies from Brazilian patients lung adenocarcinoma allowing an extensive molecular testing for actionable rearrangements contributing to guide clinical strategies.

H3K27M and TERT promoter mutations are poor prognostic factors in surgical cases of adult thalamic high-grade glioma.

BackgroundThalamic high-grade gliomas (HGGs) are rare tumors with a dismal prognosis. H3K27M and telomerase reverse transcriptase promoter (TERTp) mutations reportedly contribute to poor prognoses in HGG cases. We investigated the outcomes of surgically treated adult thalamic HGGs to evaluate the prognostic significance of H3K27M and TERTp mutations.MethodsWe retrospectively analyzed 25 adult patients with thalamic HGG who underwent maximum surgical resection from January 1997 to March 2020. The histological and molecular characteristics, progression-free survival (PFS), and overall survival (OS) of the patients were compared. For molecular characteristics, target sequencing was used to determine the H3F3A, HIST1H3B, and TERTp mutations.ResultsH3K27M mutations were detected in 12/25 (48.0%) patients. TERTp mutations were not detected in H3K27M-mutant gliomas but were detected in 8/13 (61.5%) of H3 wild-type gliomas. Although it was not significant, H3K27M-mutant gliomas tended to have a shorter PFS (6.7 vs 13.1 months; P = .2928) and OS (22.8 vs 24.4 months; P = .2875) than H3 wild-type gliomas. Moreover, the prognosis of TERTp-mutant gliomas was as poor as that of H3K27M-mutant gliomas. Contrary, 5 gliomas harboring both H3 and TERTp wild-type showed a better median PFS (59.2 vs 6.4 months; P = .0456) and OS (71.8 vs 24.4 months; P = .1168) than those harboring H3K27M or TERTp mutations.Conclusions TERTp-mutant gliomas included in the H3 wild-type glioma group limited patient survival as they exhibited an aggressive course similar to H3K27M-mutant gliomas. Comprehensive molecular work-up for the H3 wild-type cases may further confirm this finding.

RAS Mutational Status in Advanced Colorectal Adenocarcinoma Treated With Anti-angiogenics: Preliminary Experience With Liquid Biopsy.

To determinate molecular changes in the downstream epidermal growth factor receptor signaling pathway using serial liquid biopsies in patients with metastatic colorectal tumors (mCRC) under anti-angiogenic treatment. Determination of RAS mutation in primary tissue samples from colorectal tumors was performed in the 23 patients included in the study at diagnosis using quantitative-polymerase chain reaction. Sequential mutations were studied in circulating tumor (ct) DNA obtained from plasma samples. Twenty-three patients with RAS-mutated primary tumors were included. In the first ctDNA determination, 17 of these patients were found to have wild-type RAS status. Remarkably, three out of these 17 wild-type cases changed to RAS-mutated in subsequent ctDNA assays. Serial liquid biopsies in patients with mCRC might be a useful tool for identifying changes in the RAS mutation status in patients who had undergone previous anti-angiogenic therapy. The understanding of these changes might help to better define the landscape of mCRC and be the path to future randomized studies.

Abstract PO-046: LKB1 loss-associated changes in histone acetylation are potential therapeutic targets in lung adenocarcinoma

Abstract Loss of liver kinase B (LKB1, encoded by STK11) has been observed in about one-third of lung adenocarcinomas (ADCs) and can cause the inactivation of its major target AMP-activated protein kinase (AMPK) and subsequently, activation of the mammalian target of rapamycin (mTOR) pathway. In addition to its inhibitory effects on tumor growth and proliferation, AMPK may affect acetyl-CoA homeostasis and induces histone acetylation through regulating the activity and nuclear translocation of acyl-CoA synthetase short-chain family member 2 (ACSS2) and ATP citrate lyase (ACLY). Nuclear and/or cytoplasmic expression of LKB1/AMPK/mTOR pathway markers (LKB1, p-AMPKα, p-S6), enzymes involved in acetate and lipid metabolism (p-ACC, ACSS2, ACLY), acetyl-histone H3 (ac-H3), and immune regulatory proteins (PD-L1, COX-2) was assessed in lung ADCs (N=100) by immunohistochemistry. The expression of mRNAs coding for proteins involved in antitumor immunity was also analyzed in STK11-mutant and wild-type cases with using TCGA data. Associations with the clinicopathological parameters as well as the antiproliferative effects of mTOR and metabolic inhibitors (rapamycin, metformin, phenformin, and ACSS2 inhibitor) and a histone deacetylase (HDAC) inhibitor (valproic acid) were also studied in ADC cell lines harboring KRAS and/or STK11 mutations. In cases with low or no LKB1 expression, we observed significantly lower expression of p-AMPKα, p-ACC, ac-H3, ACSS2, and ACLY (P &amp;lt; .01). PD-L1 expression was also slightly decreased in these samples. The expression of most of the studied mRNAs, particularly those involved in innate immunity, was also lower in STK11 mutant cases. In our in vitro studies, rapamycin had a significant antiproliferative effect on KRAS- and/or STK11-mutant cell lines with high mTOR activity. The ACSS2 inhibitor and valproic acid were not effective as monotherapies, however, valproic acid significantly increased the antitumoral effects of rapamycin in STK11-mutant cells similarly to phenformin. Our results suggest that histone acetylation levels are lower in ADCs with LKB1 loss, perhaps by lower expression and decreased nuclear translocation of ACSS2 and ACLY. HDAC inhibitors may have the benefit to maintain histone acetylation in STK11-mutant ADCs, therefore, restoring the expression of tumor suppressors or proteins involved in antitumor immunity, which are silenced in cancer cells. Simultaneous inhibition of mTOR and HDACs may exhibit significant antitumor properties, especially in cancer cells with STK11 mutation and high mTOR activity. Supported by the Hungarian Respiratory Foundation, National Bionics Program of National Research, Development and Innovation Fund of Hungary (ED_17-1-2017-0009), and grants of the Hungarian National Research, Development and Innovation Office (NKFI-FK-128404). Citation Format: Ildiko Krencz, Daniel Sztankovics, Titanilla Danko, Gabor Petovari, Eniko Vetlenyi, Regina Raffay, Judit Moldvay, Judit Papay, Anna Sebestyen. LKB1 loss-associated changes in histone acetylation are potential therapeutic targets in lung adenocarcinoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-046.

BRAFV600E mutation impinges on gut microbial markers defining novel biomarkers for serrated colorectal cancer effective therapies

BackgroundColorectal cancer (CRC) harboring BRAFV600E mutation exhibits low response to conventional therapy and poorest prognosis. Due to the emerging correlation between gut microbiota and CRC carcinogenesis, we investigated in serrated BRAFV600E cases the existence of a peculiar fecal microbial fingerprint and specific bacterial markers, which might represent a tool for the development of more effective clinical strategies.MethodsBy injecting human CRC stem-like cells isolated from BRAFV600E patients in immunocompromised mice, we described a new xenogeneic model of this subtype of CRC. By performing bacterial 16S rRNA sequencing, the fecal microbiota profile was then investigated either in CRC-carrying mice or in a cohort of human CRC subjects. The microbial communities’ functional profile was also predicted. Data were compared with Mann-Whitney U, Welch’s t-test for unequal variances and Kruskal-Wallis test with Benjamini–Hochberg false discovery rate (FDR) correction, extracted as potential BRAF class biomarkers and selected as model features. The obtained mean test prediction scores were subjected to Receiver Operating characteristic (ROC) analysis. To discriminate the BRAF status, a Random Forest classifier (RF) was employed.ResultsA specific microbial signature distinctive for BRAF status emerged, being the BRAF-mutated cases closer to healthy controls than BRAF wild-type counterpart. In agreement, a considerable score of correlation was also pointed out between bacteria abundance from BRAF-mutated cases and the level of markers distinctive of BRAFV600E pathway, including those involved in inflammation, innate immune response and epithelial-mesenchymal transition. We provide evidence that two candidate bacterial markers, Prevotella enoeca and Ruthenibacterium lactatiformans, more abundant in BRAFV600E and BRAF wild-type subjects respectively, emerged as single factors with the best performance in distinguishing BRAF status (AUROC = 0.72 and 0.74, respectively, 95% confidence interval). Furthermore, the combination of the 10 differentially represented microorganisms between the two groups improved performance in discriminating serrated CRC driven by BRAF mutation from BRAF wild-type CRC cases (AUROC = 0.85, 95% confidence interval, 0.69–1.01).ConclusionOverall, our results suggest that BRAFV600E mutation itself drives a distinctive gut microbiota signature and provide predictive CRC-associated bacterial biomarkers able to discriminate BRAF status in CRC patients and, thus, useful to devise non-invasive patient-selective diagnostic strategies and patient-tailored optimized therapies.

Frequency of MYD88 L256P mutation and its correlation with clinico-hematological profile in mature B-cell neoplasm

Objective/backgroundB-cell neoplasms are clonal tumors of B cells at various stages of maturation, including diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic lymphoma (CLL), Burkitt lymphoma (BL), lymphoplasmacytic lymphoma (LPL)/Waldenström’s macroglobulinemia (WM), splenic marginal zone lymphoma (SMZL), nodal marginal zone lymphoma (NMZL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and hairy cell leukemia (HCL). In this study, we analyzed the frequency of MYD88 L265P mutation and its correlation with clinico-hematological profile in mature B-cell neoplasms. MethodsA total of 110 consecutive cases of B-cell neoplasms showing peripheral blood and/or bone marrow infiltration were included. MYD88 L265P mutation was detected by polymerase chain reaction amplification of exon 5 of MYD88 gene, followed by restriction fragment length polymorphism analysis. ResultsAmong the 110 cases, the major group was of CLL (54.5%, n = 60), followed by HCL. Other cases included MCL, LPL, DLBCL, SMZL, NMZL, FL, and BL. MYD88 L265P mutation was seen in 21 (19.1%) cases of B-cell neoplasm, whereas 89 (80.9%) cases were negative for MYD88 L265P mutation. It was most commonly seen in LPL/WM cases followed by HCL, SMZL, CLL, and MCL cases. No case of DLBCL, FL, and BL showed MYD88 L265P mutation. Statistically significant difference was seen for hemoglobin level in CLL cases, with MYD88 L265P mutated cases showing higher mean hemoglobin levels than MYD88 wild-type cases (p = .001). For other parameters, no statistically significant difference was noted between mutated and unmutated cases. ConclusionMYD88 L265P mutation is seen in various B-cell neoplasms; it is most commonly seen in LPL/WM cases but not specific for it.

CTNI-06. SINGLE-CENTER REAL-WORLD ANALYSIS ON CEREBROSPINAL FLUID IN PATIENTS WITH LEPTOMENINGEAL METASTASES OF NON-SMALL CELL LUNG CANCER

Abstract OBJECTIVE To investigate the clinical values of real-world cerebrospinal fluid (CSF) examination in the diagnosis and prognosis of leptomeningeal metastases (LM) in the patients with non-small cell lung cancer (NSCLC). METHODS The clinical manifestations of patients diagnosed with NSCLC-LM in Guangdong Sanjiu Brain Hospital from 2010 until 2019 and the results from the routine, biochemical and cytologic examinations of their CSF were analyzed retrospectively. Moreover, the effects of different indicators on the prognosis of patients were determined by survival analysis. RESULTS Fifty-eight of the 80 patients underwent CSF examination, and the median values of the initial CSF pressure measured by lumbar puncture was 255mmH2O (60-350mmH2O). Tumor cells were found by the cytologic examination of CSF in 56 of the 58 cases (96.6%). The median levels of protein, glucose, chloride, lactate dehydrogenase (LDH) and carcinoembryonic antigen (CEA) in CSF were 0.4g/L (0–8.3g/L), 2.7mmol/L (0–11.9mmol/L), 120.7mmol/L (0–140.5mmol/L), 21.3U/L (0–221.5U/L) and 31.4ng/ml (0–784.2ng/ml), respectively. Sixteen patients underwent gene detection, and the results showed that there were 4 cases of wild type and 12 cases of mutation type. The survival analysis revealed that the median overall survival (mOS) after being diagnosed with LM, patients in LDH≤37U/L group and those in LDH &amp;gt;37U/L group were 8.0 months, 23.7 months and 4.6 months (P=0.039), respectively, which indicated that the LDH level in CSF was the independent prognostic factor for the survival. CONCLUSION Using the cytologic examination of CSF in patients with LM to detect tumor cells is conducive to the early diagnosis of LM. The high CSF pressure in patients with LM is one of the reasons for the neurologic signs and symptoms. The gene mutations found in the CSF in some patients can be used to guide the further systematic treatment. The LDH level in CSF is the independent prognostic factor affecting the survival.