Sample size required for prognostic genes analysis in colorectal cancer

The identification of prognostic genes can help in the clinical management of non-small cell lung cancer (NSCLC). However, there is little overlap in the prognostic genes identified in different NSCLC studies. One reason for this may be the inadequate sample size. Here, the effect of sample size on prognostic genes analysis was investigated based on 515 stage II/III NSCLC cases from two cohorts detected by whole-exome sequencing. Prognostic genes analysis was repeatedly performed 100 times for each sample size level using random resampling methods. In stage II lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) cases from the TCGA Pan-Lung Cancer cohort, the number of statistically significant prognostic genes first increased with sample size in a power law, then fluctuated steadily, and finally decreased slightly. The power law growth curves were also observed in stage III LUAD and LUSC cases from the TCGA Pan-Lung Cancer cohort and stage III Chinese LUAD cases from the OncoSG cohort. The correlation R2 of the fitted power law growth curves were all greater than 0.99. In addition, at the sample size level where the number of prognostic genes peaked, the mean proportion of true prognostic genes in patients with stage II LUAD and LUSC was 28.32% and 23.12%, which could partly explain the little overlap in prognostic genes between reports. In conclusion, the number of prognostic genes takes a power law growth with the sample size in NSCLC, independent of histopathological subtype, race, and stage. These results also show how sample size affects the reliability of prognostic genes and will aid trial design for genomic mutation-based prognostic studies in NSCLC.

Background: African Americans (AAs) have the highest incidence and mortality rates of colorectal cancer (CRC) cases in the United States, with higher levels of early-onset and right-sided CRC, yet there are little data on carcinogenesis in AA CRC. Copy number variations (CNVs) affect large portions of the cancer genome, and they provide information about mechanisms of cancer initiation and progression. Given the unique clinical-pathological features of AA CRC, we hypothesized that different mechanisms operate in colorectal carcinogenesis in AAs. To test this hypothesis, we evaluated CNVs in AA CRC as compared to publically available data on CRC from the TCGA. Methods: Tumor DNA samples from 46 AA CRC cases were available through the Chicago Colorectal Cancer Consortium. Determinations of CNVs were performed using the Affymetrix Cytoscan HD platform, which provides data from 2.67 million copy number probes and 750,000 genotype probes for analysis of loss of heterozygosity (LOH) with and without copy number change. Whole chromosome-arm gains and losses were defined as the gain or loss of >50% of probes on an arm. Gains and losses were tabulated and compared to data from the TCGA. Statistical analyses of arm losses and gains were performed with Fisher exact tests, adjusting for multiple testing using a Bonferroni correction. Statistical analysis of total chromosome number alterations was performed with the chi-square test. Results: Overall, significantly fewer chromosome-arm gains and losses occurred in AA CRC cases than in TCGA CRC cases with 10% of gains and losses in AA and 34% of gains and losses from TCGA (p < 0.0001). More specifically, we found chromosome-arm gains occurred significantly less frequently for 8p (28% to 0%), 13q (56% to 26%), and 20 p and q (58% to 17% and 72% to 39%, respectively). Similarly, we found chromosome-arm losses occurred significantly less frequently for 1p (19% to 0%), 5q (17% to 0%), 8p (50% to 20%), 14q (30% to 0%), 17p (56% to 24%), 18p and q (61% to 26% and 66% to 28%, respectively), 20p (32% to 7%), and 21q (22% to 2%). All adjusted p values were less than 0.05 for these comparisons. No chromosome-arm gains or losses were significantly more frequent in AA CRC cases than in TCGA cases. The difference in chromosome-arm gain and loss was not the result of segmental gain or loss. However, analysis of genotype data revealed that copy-number neutral LOH in AA CRC cases substantially compensates for the lower frequency of chromosome-arm losses and focal amplifications for the lower frequency of gains. For example, in AAs, for chromosomal region 17p where TP53 localizes, 24% of cases exhibited 17p loss and 26% exhibited copy-number neutral LOH, for a total of 50% TP53 loss. For chromosomal region 13q where CDK8 localizes, 26% of cases exhibited 13q gain and 28% exhibited focal gains around CDK8, for a total of 54% CDK8 gain. The total frequency of tumor-suppressor gene losses and oncogene gains was similar in AA CRC and TCGA cases. Conclusion: These data show that, although the major carcinogenesis genes (as identified by LOH and gene amplification) appear to be similar in AA and TCGA CRC cases, the mechanisms of genomic instability that generate these genetic changes are different. Chromosome-arm losses are more frequent in TCGA CRC cases whereas AA CRC is driven by homologous recombination and segregation resulting in copy-number neutral LOH. Chromosome-arm gains are more frequent in TCGA CRC cases whereas AA CRC is driven by focal amplification, which could result from unequal sister chromatid exchange. Thus, these CNV differences could reflect variation in the regulation of fundamental genomic integrity mechanisms in AAs. More studies are needed to confirm these results and elucidate the mechanisms of carcinogenesis in AA CRC. This abstract is also presented as Poster A62. Citation Format: Gaius J. Augustus, Rosa Xicola, Arthur R. Brothman, Xavier Llor, Nathan A. Ellis. Homologous recombination drives African American colorectal carcinogenesis. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr PR02.

Purpose: The most frequent cause of hereditary colorectal cancer (CRC) is Lynch syndrome, accounting for 3% of all CRC cases. The underlying germline predisposition is a mutation in any of the four mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2), while microsatellite instability (MSI) is a hallmark of these tumors. However, CRC cases with MSI, no MLH1 somatic hypermethylation and absence of MMR germline mutation account for up to 60% of cases of MMR-defective tumors. They have been termed Lynch-like syndrome (LLS) and treatment management decisions are complicated due to unconfirmed suspicions of hereditary cancer. The aim of the present study was to investigate whether LLS cases in patients under the age of 40 carry potentially pathogenic germline variants in new genes for CRC predisposition causing a MMR-defective tumor phenotype. Methods: We performed whole-exome sequencing (WES) in the germline and tumoral DNA of 16 early-onset LLS patients. We filtered for recessive germline variants in genes involved in DNA repair. An exhaustive functional evaluation for 2 MCM8 genetic variants detected in one patient was performed. The CRISPR/Cas9 system was used to generate MCM8ko clones in a cell model (DLD-1). Site-directed mutagenesis produced the genetic variants for ectopic expression in the MCM8ko model. Functional characterization included the comet assay, which detected DNA damage induced by oxaliplatin in the transfected cells. MCM8ko cells were evaluated for MSI after 30, 60 and 90 days of culture, and mutational signatures were assessed with SigProfiler. Results: The MCM8 variants c.351_354delAAAG (p.Lys118GlufsTer5, p.K118fs) and c.414A>G (p.Ile138Met, p.I138M) were identified in one early-onset LLS patient with biallelic somatic inactivation of MLH1. Manual evaluation of WES data showed that germline MCM8 variants were in trans. In the comet assay, both MCM8 variants revealed higher sensitivity to oxaliplatin and more DNA damage compared to the wild-type control. One of the MCM8KO clones showed MSI after 30 days of sub-culturing. After 120 days, MCM8KO clones had acquired a significant contribution of the SBS20 mutational signature, which is associated with defective MMR DNA. Conclusions: We postulate that MCM8 is a new germline CRC predisposing gene in early-onset LLS, which may be involved in both MMR and double-strand break repair. We further provide evidence that in some LLS CRC cases, especially in young patients, the biallelic somatic MMR inactivation may be caused by new CRC germline predisposing genes. Additional mutational screening is warranted for such cases. Citation Format: Mariano Golubicki, Laia Bonjoch, José G. Acuña-Ochoa, Marcos Diaz-Gay, Jennifer Muñoz, Miriam Cuatrecasas, Teresa Ocaña, Juan Robbio, Enrique L. Roca, Antoni Castells, Fracesc Balaguer, Marina Antelo, Sergi Castellvi-Bel. Germline biallelic mutations in MCM8 are associated with early-onset Lynch-like syndrome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2366.

BackgroundThere is already evidence that the faecal immunochemical test (FIT) is a useful tool for the diagnosis of colorectal cancer (CRC) that helps to identify symptomatic patients requiring early colonoscopy. Although the recommendation to use FIT is widely accepted, there are no data concerning whether this strategy improves patient survival.The objective was to assess whether the survival is higher if CRC patients have been first diagnosed by FIT (as compared with the rest of patients with CRC).MethodsWe identified all cases of CRC diagnosed between 2009 and 2016 in Donostialdea (Spain), excluding all the CRC detected in population screening. We focused on symptomatic patients. One thousand five hundred twenty-seven cases of CRC were divided into two groups based on the route to diagnosis: group 1: individuals who tested positive in a FIT during the year before diagnosis, and group 2: others.Survival was assessed by Kaplan-Meier estimation, and with the log-rank test. A Cox regression model was used to adjust for differences between groups due to other variables associated with survival.ResultsOne thousand nine hundred sixty-seven cases of invasive CRC were identified, of which 22.4% were detected in population screening. Of the 1527 cases diagnosed in symptomatic patients, 317 patients had undergone a FIT in the year before the diagnosis of CRC. In 279 cases(18.3%), the result had been positive and this was the first step towards their CRC diagnosis (group 1). Group 2 was composed of the 1248 cases of CRC (81.7%). Considering these cases, 1210 patients with CRC did not undergo any FIT while 38 patients presented a negative result in the year before the diagnosis. The rate of early-stage disease (stage I or II) was higher in group 1 (51.3% vs 45.5% in group 2) (p = 0.04). Furthermore, the 3-year survival was longer in group 1 (72% vs 59% in group 2) (HR 1.50; 95% CI 1.22–1.84).The variables independently associated with worse survival were: group 2, age > 70 years and stage at the moment of diagnosis.ConclusionsThe use of FIT as a diagnostic strategy in symptomatic patients may improve survival in CRC. Nonetheless,FIT is still not widely used in our region.

Colon Cancer Screening Models: Lessons and Challenges

Using the binomial distribution, the effect of sample size on the variability among sample test results when sampling a lot with 1.0% genetically modified (GM) or biotech seed was evaluated. The coefficient of variation, cv, among 500-seed sample test results taken from a lot with truly 1.0% was computed to be 44.5%. Increasing sample size to 1000 seeds reduced the cv among sample test results to 31.5%. The effects of sample size and accept/reject limits on the buyer's risk (bad lots accepted) and the seller's risk (good lots rejected) was also evaluated assuming a tolerance of 1.0% GM seed. Increasing sample size decreases both the buyer's and seller's risks at the same time. Using an accept/reject limit below the regulatory tolerance decreases the buyer's risk, but increases the seller's risk. Using an accept/reject limit above the regulatory tolerance decreases the seller's risk but increases the buyer's risk.

Introduction In recent years, immune checkpoint inhibitors (ICIs) have attracted widespread attention and made breakthroughs in progress towards the treatment of various cancers. However, ICI therapy is selective, and its effects on many patients are not ideal. It is therefore critical to identify prognostic biomarkers of response to ICI therapy. The PI3K/Akt pathway plays important roles in tumor formation and metastasis. However, there are no published reports clarifying the relationship between PI3K/Akt pathway mutations and prognosis for colon adenocarcinoma (COAD) patients receiving immunotherapy. Methods We collected data from a COAD cohort from The Cancer Genome Atlas (TCGA) database, including whole-exome sequencing (WES) data, RNA-seq data, and clinical data. We also collected data, including clinical prognosis and targeted sequencing data, from a cohort of COAD patients receiving immunotherapy. We collected 50 COAD patients (Local-COAD) from the Zhujiang Hospital of Southern Medical University and performed targeted sequencing. We analyzed the effects of PI3K/Akt pathway mutations on the patients' clinical prognosis, immunogenicity, and immune microenvironments. Gene set enrichment analysis (GSEA) was used to analyze the significantly upregulated and downregulated signaling pathways. We used these results to hypothesize potential mechanisms by which PI3K/Akt mutations could affect the prognosis of COAD patients. Results Univariate and multivariate Cox analyses and Kaplan-Meier (KM) survival curves showed that patients with PI3K-Akt mutations had better overall survival (OS) than those without PI3K-Akt mutations. Genes with significant mutation rates in the two cohorts were screened by panoramic view. CIBERSORT was used to analyze changes in 22 types of immune cells to identify immune activated cells. Similarly, patients in the PI3K/Akt-mutated type (PI3K/Akt-MT) group had significantly increased immunogenicity, including increases in tumor mutation burden (TMB), neoantigen load (NAL), and MANTIS score. Using GSEA, we identified upregulated pathways related to immune response. Conclusion PI3K/Akt pathway mutation status can be used as an independent predictor of response to ICI treatment in COAD patients. PI3K/Akt mutations are correlated with improved OS, higher immunogenicity, greater immune response scores, and increases in activated immune cells.

The primary focus of the paper is to establish both numerical and experimental methods to control the concentration of samples in a microreactor well. The concentration of the reacting samples is controlled by varying the initial sample size and electric field. Further, the paper numerically investigates the feasibility of mixing and reacting nanoliter samples with a wide variation in reaction rates in the microreactor driven by electro-osmotic pumping. Two discrete samples are measured and transported to the microreactor simultaneously by electro-osmotic pinching and switching. The transported samples are mixed in the microreactor and floated for 4.5 s for reaction to occur. It is seen that the normalized concentration of the product increases from 0.25 to 0.45 during that period. Also the effects of sample size and applied electric field on sample concentration during the switching process are studied. It is found that the normalized final sample concentration increases from 0.03 to 0.11 with an increase in sample size from 60 to 150 µm, at a constant electric field. Further, by increasing the electric field from 100 to 1000 V cm−1, at a constant sample size, there is a significant decrease in the final concentration of the sample from 0.14 to 0.04. Our studies also show that the normalized product concentration depends on the reaction rate and increases from 0.28 to 0.48 as the reaction rate increases from 10 L mol−1 s−1 to 105 L mol−1 s−1. However, the increase in the reaction rate beyond 105 L mol−1 s−1 does not influence the product concentration for the present design of the microreactor. Our microreactor with improved mixing can be used for assessing reactions of biological samples. The optimized sample size along with a controlled electric field for sample injection forms the basis for developing a prototype of a microreactor device for high throughput drug screening.

Methylenetetrahydrofolate reductase (MTHFR) is involved in DNA synthesis and repair. We here aimed to investigate two common polymorphisms, C677T and A1298C, with genotype and haplotype frequencies in colorectal cancer (CRC) cases among Jordanian. 131 CRC cases were studied for MTHFR C677T and A1298C polymorphisms, compared to 117 controls taken from the general population, employing the PCR-RFLP technique. We found the frequency of the three different genotypes of MTHFR C677T among Jordanians to be CC: 61.7%, CT: 35.2%, and TT 3.1% among CRC cases and 50.9%, 38.8% and 10.3% among controls. Carriers of the TT genotype were less likely to have CRC (OR=0.25; 95%CI: 0.076-0.811; p=0.021) as compared to those with the CC genotype. Genotype analysis of MTHFR A12987C revealed AA: 38.9%, AC: 45%, and CC 16% among CRC cases and 37.4%, 50.4% and 12.2% among controls. There was no significant association between genetic polymorphism at this site and CRC. Haplotype analysis of MTHFR polymorphism at the two loci showed differential distribution of the TA haplotype (677T-1298A) between cases and controls. The TA haplotype was associated with a decreased risk for colorectal cancer (OR=0.6; 95% CI: 0.4-0.9, p=0.03). The genetic polymorphism of MTHFR at 677 and the TA haplotype may modulate the risk for CRC development among the Jordanian population. Our findings may reflect an importance of genes involved in folate metabolism in cancer risk.

Bayesian methods allow borrowing of historical information through prior distributions. The concept of prior effective sample size (prior ESS) facilitates quantification and communication of such prior information by equating it to a sample size. Prior information can arise from historical observations; thus, the traditional approach identifies the ESS with such a historical sample size. However, this measure is independent of newly observed data, and thus would not capture an actual "loss of information" induced by the prior in case of prior-data conflict. We build on a recent work to relate prior impact to the number of (virtual) samples from the current data model and introduce the effective current sample size (ECSS) of a prior, tailored to the application in Bayesian clinical trial designs. Special emphasis is put on robust mixture, power, and commensurate priors. We apply the approach to an adaptive design in which the number of recruited patients is adjusted depending on the effective sample size at an interim analysis. We argue that the ECSS is the appropriate measure in this case, as the aim is to save current (as opposed to historical) patients from recruitment. Furthermore, the ECSS can help overcome lack of consensus in the ESS assessment of mixture priors and can, more broadly, provide further insights into the impact of priors. An R package accompanies the paper.

In several cancer models, it was shown that tumor mutation load correlates with response to immune checkpoint blockade therapy. However, whole exome sequencing of FFPE samples is not ideal for implementation on molecular pathology laboratories. In colorectal cancer (CRC), mutation load is correlated with microsatellite instability (MSI), which can therefore be used as surrogate marker for mutation load. In this study, we aimed at determining whether a target NGS-based mutation panel can be used for routine testing of mutation load in cancer, by assessing its performance in identifying MSI+ and MSI- CRC samples. The Oncomine Tumor Mutation Load Assay, which targets 409 genes corresponding to 1.7Mb of sequencing data, was used. It is a two-pool panel, optimized for FFPE samples, and only requires 20 ng of DNA in total. A cohort of 16 CRC samples was used. These included six cases MSI+, known to present a hypermutable phenotype caused by the loss of DNA mismatch repair activity, and 10 MSI- samples, which typically have a lower rate of somatic mutation. DNA was isolated from 10 uM slide cuts using the MagMAX FFPE DNA/RNA Ultra Kit. A multiplex of eight samples per 540 sequencing chip was sequenced using an Ion S5 XL system. In our data set, we observed an average of 67.2 mutations per Mb for the MSI+ samples and an average of 29.7 mutations per Mb for the MSI- samples (p=0.004). Using a cut-off of 40.0 mutations per Mb, we were able to classify correctly every sample as MSI+ or MSI-. Interestingly, we observed a tri-modal distribution of the samples, suggesting there might be a sub-group of MSI+ CRC cases that show a level of mutation load comparable to that of MSI- CRC cases. It remains to be seen whether these MSI+, low mutation load CRC cases are non-responders to treatment with immunotherapy. A validation cohort of CRC cases is currently being analyzed to validate our findings. In this study, we demonstrate it is viable to use a target and routine-amenable NGS-based sequencing gene panel to identify CRC samples with different levels of mutation load. It remains to be seen whether mutation load is a better predictor of response to immunotherapy than MSI status in CRC. This approach also needs to be validated in different tumor types. Citation Format: Jose Costa, Joana Reis, Margarida Fernandes, Rafaela Silva, Luis Cirnes, Ruchi Chaudhary, Fatima Carneiro, Jose C. Machado. Assessing tumor mutation load using an NGS-based, routine-friendly target gene panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1712.

Purpose: The association between Human Papillomavirus (HPV) infection and the development of cervical cancer and anogenital cancer has been established. Moreover, molecular studies have detected HPV DNA in tumor tissues of head and neck cancer, esophageal cancer, and some skin cancers. Viral genes expression and multiplication occur exclusively in the nuclei of the infected cells and are tightly linked to the state of differentiation of the cells. Integration of viral DNA into host genome is essential for carcinogenesis since it promotes disruption of HPV E2 gene, leading to abnormal expression of E6 and E7 oncoproteins. Both E6 and E7 can form specific complexes with tumor suppressor gene p53 and pRB respectively. However, the role of HPV infection in colorectal cancer (CRC) and its mechanisms of carcinogenesis have not been properly elucidated. Our objectives were to evaluate the presence of HPV infection in patients with CRC; to probe the viral integrations status of HPV 16 in CRC and its adjacent normal colonic mucosa, and to determine the mRNA expression levels of the viral oncogenes HPV-16 E6 and HPV-16 E7. Experimental Designs: To determine the association of HPV and CRC, we conducted an age-and-gender matched case-control study using tumor and tumor-adjacent colorectal tissues from patients with CRC (cases) and without CRC (controls). Presence of L1, E2, and E6 genes was analyzed to determine whether a full-length HPV genome existed in tumor and normal tissue samples. Head-to-tail junctions of HPV-16 genomes were analyzed to determine the integration the HPV-16 genome in the HPV-16 (+) cases. Moreover, we determined the mRNA expression levels of E6 and E7 in the HPV-16 (+) cases. Hypothesis testing was performed using Pearson Chi-square analysis of proportions using SPSS 17. Results: A total of 45 (ß-actin+) CRC patients (mean age 61 ±11 years, 24 males) and 47 controls (mean age was 60 ± 9 years, 18 males) were evaluated. Tumors were located in proximal colon (n=7) and distal colon (n=38). HPV DNA was identified in 19 of 45 (42.2%) patients with CRC and in 2 of 47 (4.3%) controls (OR = 16.4; 95% CI 3.5-76.3, p < 0.001). HPV-16 was identified in 12 of 19 (63.2%) HPV (+) CRC patients and in none (0%) HPV(+) controls. The twelve HPV-16 CRC cases showed integration of HPV-16. Three of 12 (25%) HPV16 (+) cases expressed E6 and E7 mRNA. Among the 12 CRC cases where both tumor and adjacent tissue was available, 2 of 9 (16.7%) exhibited HPV-16 DNA in both tumor and tumor-adjacent tissues.Conclusion: These results suggest that colorectal HPV infection is common among patients with CRC. The presence of HPV in tumors located proximal to the rectum suggests that this infection might not result from direct spread from anogenital sites. Moreover, our finding of highly frequent integration of viral DNA and the active expression of E6 and E7 viral oncogenes in the host strongly suggests that HPV oncoproteins may play a role in the pathogenesis of colorectal carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 552. doi:1538-7445.AM2012-552

To explore the relationship between sample size in the groups and statistical power of ANOVA and Kruskal-Wallis H test with an imbalanced design. The sample sizes of the two tests were estimated by SAS program with given parameter settings, and Monte Carlo simulation was used to examine the changes in power when the total sample size varied or remained fixed. In ANOVA, when the total sample size was fixed, increasing the sample size in the group with a larger mean square error improved the statistical power, but an excessively large difference in the sample sizes between groups led to reduced power. When the total sample size was not fixed, a larger mean square error in the group with increased sample size was associated with a greater increase of the statistical power. In Kruskal-wallis H test, when the total sample size was fixed, increasing the sample size in groups with large mean square errors increased the statistical power irrespective of the sample size difference between the groups; when total sample size was not fixed, a larger mean square error in the group with increased sample size resulted in an increased statistical power, and the increment was similar to that for a fixed total sample size. The relationship between statistical power and sample size in groups is affected by the mean square error, and increasing the sample size in a group with a large mean square error increases the statistical power. In Kruskal-Wallis H test, increasing the sample size in a group with a large mean square error is more cost- effective than increasing the total sample size to improve the statistical power.

Gene expression profiling has shown that diffuse large B-cell lymphoma (DLBCL) clusters into three major subtypes based on similarity in expression patterns to their cell of origin (COO): germinal center B-cell-like (GCB-DLBCL), activated B-cell-like DLBCL (ABC-DBLCL) and primary mediastinal B-cell lymphoma (PMBCL). These subtypes of DLBCLs are associated with distinctly different overall survival rates after standard immunochemotherapy. However, clinical and prognostic heterogeneity remains within COO subsets and strategies are needed to further stratify patients to identify and target high-risk subsets. A comprehensive genomic analysis of COO on a clinically defined set of DLBCL cases has not been performed and the aim of this study was to use whole-exome sequencing (WES) data from 58 paired tumor-normal DLBCL samples to assess association of known DLBCL genomic alterations with cell COO as well as for identification of novel and relevant genetic biomarkers.To investigate genomic alterations associated with DLBCL subsets, we analyzed WES and genome wide copy number data from 58 paired tumor-normal DLBCL tumors. Gene expression profiling or Hans classification was performed to determine DLBCL COO subtype; 31 patients were classified as GCB and 27 as non-GCB. The WES data were used to 1) assess the association of known DLBCL genomic alterations with COO, and 2) identify novel alternations associated with COO. Statistical analysis was performed and the data were ranked by significance (p≤ 0.05) within each DLBCL subtype. In total, 45 genomic abnormalities were analyzed for their association with either GCB, non-GCB or both. Mutations in CREBBP, EZH2, MEF2B, FOXO1 and REL have also been reported as GCB driver mutations and we observed GCB patients with these mutations, but the mutation clustering was not wholly associated with GCB. MYC double-hits were exclusively found in the GCB-subtype group. For the non-GCB cases we found that mutations in MAP2K3 and MYD88 were significantly associated with this subtype(p< 0.05). In addition to mutational patterns, we identified several copy number alterations (CNA) across both groups. Chromosomal losses in GCB patients were found at chromosomes 10q11.21-10q24.23, 4q12-4q35.2, 3q12.1-3q29, 4p12-4p16.3, 10p11.21-10p15.3, and 14q11.2-14q24.3 whereas gains were localized to 7q11.1-7q36.3, 7p11.2-7p22.3, and 1q21-1q32.1 (p < 0.05). No CNA was observed to directly associate with non-GCB patients, however, a loss at 9p21 and gains at 9q24.1 and 18q21.33 trended with the non-GCB subtype, supporting previous reports. Loss at 10q23.31 or a gain in 2p13-2p12 have been reported as being specific for GCB and our data confirmed the association of 10q23.31 with GCB while a gain at 2p13-2p12 (REL)was found in both subtypes. To further understand genomic differences between DLBCL subtypes, we evaluated the relative percentage of each genomic feature. 18/45 (40%) were only observed in GCB patients whereas 2/45 (5%) were specific to the non-GCB subtype. The majority (25/45, 55%) overlapped between the two subtypes.Throughout our analysis we noted that 7 non-GCB cases lacked any of the driver mutations analyzed in the study. While all cases carried mutations, they consisted of low frequency mutations that were not specifically associated with COO. 2/7 cases had a gain at 9p24.1 that included CD274 and JAK2. Because 9p24.1 gains have not been fully defined in DLBCL, we reviewed all cases and identified 4 (7%) with a 9p24.1 gain, 3 of which were non-GCB and 1 GCB. One non-GCB case was EBV+ and none of the cases showed evidence of PMBCL. Of the 9p24.1 cases, three had RNAseq data available and we found that PDL1 and JAK2 expression was elevated (12 fold p< 0.01 and 7 fold p< 0.01, respectively) when compared to the 9p24.1 normal cases (n=32). While outcome was not the focus of the study, we did note that 6/7 cases that lacked driver mutations achieved event free survival at 24 months (EFS24).Taken together, this analysis has further characterized the genetic profile of each COO subtype and has identified novel GCB CNAs which require independent replication. Additionally, we identify a subgroup of non-GCB DLBCL patients that do not harbor known driver mutations and require further genomic study to better resolve the biology of these tumors. Together, these data provide insight on the genetic heterogeneity of DLBCL and identify genetic variants that may inform subtype specific therapy. DisclosuresNowakowski:Morphosys: Research Funding; Celgene: Research Funding; Bayer: Consultancy, Research Funding. Rimsza:NCI/NIH: Patents & Royalties: L.M. Rimsza is a co-inventor on a provisional patent, owned by the NCI of the NIH, using Nanostring technology for determining cell of origin in DLBCL..

The genetic etiology of intellectual disability remains elusive in almost half of all affected individuals. Within the Solve-RD consortium, systematic re-analysis of whole exome sequencing (WES) data from unresolved cases with (syndromic) intellectual disability (n = 1,472 probands) was performed. This re-analysis included variant calling of mitochondrial DNA (mtDNA) variants, although mtDNA is not specifically targeted in WES. We identified a functionally relevant mtDNA variant in MT-TL1 (NC_012920.1:m.3291T > C; NC_012920.1:n.62T > C), at a heteroplasmy level of 22% in whole blood, in a 23-year-old male with severe intellectual disability, epilepsy, episodic headaches with emesis, spastic tetraparesis, brain abnormalities, and feeding difficulties. Targeted validation in blood and urine supported pathogenicity, with heteroplasmy levels of 23% and 58% in index, and 4% and 17% in mother, respectively. Interestingly, not all phenotypic features observed in the index have been previously linked to this MT-TL1 variant, suggesting either broadening of the m.3291T > C-associated phenotype, or presence of a co-occurring disorder. Hence, our case highlights the importance of underappreciated mtDNA variants identifiable from WES data, especially for cases with atypical mitochondrial phenotypes and their relatives in the maternal line.