Unique Genomic Features Research Articles

Clinical, pathologic, and genomic hallmarks of KRAS-amplified non-small cell lung cancer.

9118 Background: KRAS-mutant non-small cell lung cancers (NSCLCs) have unique clinicopathologic and genomic features, and novel therapies are in development to target many KRAS-mutant tumor types. Whether KRAS amplification also defines a unique, potentially targetable, molecular subset of NSCLC is currently unknown. Methods: Clinicopathologic and genomic data were abstracted from multiple independent cohorts of pts whose cancers underwent tumor genomic profiling at the Dana-Farber Cancer Institute (Cohort 1), AACR Project GENIE v.13, The Cancer Genome Atlas (TCGA), and 212 other studies (Cohort 2). Patient (pt) outcomes were examined according to KRAS amplification status ( KRAS amplified vs KRAS non-amplified). Whole transcriptome sequencing and quantitative proteomics data from TCGA and the Cancer Cell Line Encyclopedia (CCLE) were used to correlate KRAS amplification with KRAS RNA levels and protein expression. Results: Among 15,341 pts with NSCLC, KRAS amplification was identified in 355 (2.3%) cases (median number of copies [range]: 11 [7-87]); the prevalence was similar in adenocarcinomas and squamous cell carcinomas. Compared to KRAS non-amplified cases, pts with KRAS-amplified NSCLC were more likely to be men (52.4% vs 45.6%, p = 0.01), have a history of smoking (82.1% vs 64.2%, p < 0.01), and higher median pack-years (35 vs 23.5, p < 0.01). However, median age was similar between the two groups (66 vs 66, p = 0.88). KRAS amplification was also associated with higher aneuploidy (median fraction of genome altered 30.4% vs 14.3%, p < 0.01), tumor mutational burden (p < 0.01 across different platforms), and increased median PD-L1 expression (20% vs 5%, p = 0.01). Of the 355 NSCLC samples with KRAS amplification, 152 (43%) had no concurrent oncogene driver mutations. In unbiased mutation enrichment analyses, KRAS-amplified tumors were enriched in concurrent mutations in KRAS, PALB2, POLE, and SLC34A2 (q < 0.1), while KRAS non-amplified tumors were enriched for oncogenic EGFR mutations (q < 0.1). Transcriptomic and proteomic profiling from the TCGA and CCLE cohorts demonstrated that KRAS amplification was associated with significantly increased KRAS mRNA (p < 0.01) and protein expression (p < 0.01), compared to KRAS non-amplified samples. In pts with available clinical outcomes data in the combined cohort (N = 9,335), the median overall survival (OS) from the date of diagnosis was significantly shorter in KRAS-amplified vs KRAS non-amplified cases (adjusted HR: 1.40, p < 0.01). KRAS amplification was confirmed to confer significantly worse survival outcomes in pts with both KRAS wild-type (N = 6,695, adjusted HR 1.37, p = 0.02) and KRAS-mutant (N = 2,640, adjusted HR 1.34, p = 0.02) NSCLC. These results were independently replicated in Cohort 1 and Cohort 2. Conclusions: KRAS amplification defines a novel molecular subset of NSCLC characterized by distinct clinicopathologic and genomic features and worse survival.

The clinical and genomic characteristics of KRAS G12D mutated cancers.

e15151 Background: KRAS mutations are relatively common in solid tumors. It is once considered to be “non-druggable” due to its high affinity to GTP/GDP and lack of other binding sites. Recently, irreversible inhibitors targeting specific KRAS mutations have been developed. We aimed to investigate the correlation of clinical and genomic factors with KRAS G12D mutation in pancreatic cancer (PC), colorectal cancer (CRC) and non-small-cell lung cancer (NSCLC). Methods: We retrospectively reviewed the clinical and genomic data of patients with PC (n = 300), CRC (n = 300), and NSCLC (n = 1800) who provided tumor tissues and matched peripheral blood samples (served as control to eliminate germline variants) for targeted sequencing of 450 cancer-related genes. Experiments were carried out in OrigiMed’s laboratory following the CAP/CLIA standards. Tumor mutation burden (TMB)-high was defined as TMB > 10 muts/Mb. Results: The prevalence of KRAS G12D mutation in PC, CRC and NSCLC was 38.3% (115/300), 15.0% (45/300) and 2.0% (36/1800), respectively. In PC and CRC, G12D was the major type of KRAS mutations, accounting for 44.9% (115/256) and 30.8% (45/146) of all the KRAS mutations, respectively. In contrast, G12C was the most common KRAS mutation in NSCLC (35.9% [79/220]), followed by G12D (16.4% [36/220]). Patients were divided into three groups according to the KRAS mutation status: the G12D (mutated in KRAS G12D), non-G12D (mutated in KRAS, but not G12D), and wild-type (WT; no KRAS mutation) groups. Clinical features including age, sex and disease stage were not associated with the KRAS grouping in PC and CRC. However, the KRAS grouping was significantly correlated with sex in NSCLC (Female vs Male: 1.9% vs 2.1% in G12D, 4.4% vs 13.9% in non-G12D, 93.7% vs 84.1% in WT, P < 0.001). In PC, KRAS G12D and BRAF mutations were mutually exclusive (P < 0.01), while in CRC, KRAS G12D mutation was found to be co-occurring with PIK3CA mutation (P < 0.05). For NSCLC, KRAS G12D mutation was mutually exclusive with EGFR mutation (P < 0.01) and co-occurred with STK11 mutation (P < 0.05). TMB was comparable between the KRAS grouping in PC and CRC. In contrast, TMB was significantly higher in the non-G12D group (median 11.7 [range 0.7-89.8] muts/Mb) compared to the G12D (median 6.9 [range 0-30.9] muts/Mb; P < 0.001) and WT (median 4.6 [range 0-215.2] muts/Mb; P < 0.001) groups in NSCLC. A higher proportion of TMB-high was also observed in the non-G12D group (58.5%) compared to the G12D (22.2%) and WT (22.6%) groups in NSCLC. Conclusions: Patients with KRAS G12D mutation possessed unique clinical and genomic characteristics. It was more prevalent in PC and CRC and was correlated with sex in NSCLC. KRAS G12D mutation co-occurred with STK11 mutation and showed a relatively low TMB value in NSCLC, suggesting that immunotherapy might be ineffective. While KRAS non-G12D mutated NSCLC patients may be favorable to ICI. Our results provide insights into the precision medicine of KRAS G12D-mutated cancers.

Comparative Genomic Landscape of Urothelial Carcinoma of the Bladder Among Patients of East and South Asian Genomic Ancestry.

Despite the low rate of urothelial carcinoma of the bladder (UCB) in patients of South Asian (SAS) and East Asian (EAS) descent, they make up a significant portion of the cases worldwide. Nevertheless, these patients are largely under-represented in clinical trials. We queried whether UCB arising in patients with SAS and EAS ancestry would have unique genomic features compared to the global cohort. Formalin-fixed, paraffin-embedded tissue was obtained for 8728 patients with advanced UCB. DNA was extracted and comprehensive genomic profiling was performed. Ancestry was classified using a proprietary calculation algorithm. Genomic alterations (GAs) were determined using a 324-gene hybrid-capture-based method which also calculates tumor mutational burden (TMB) and determines microsatellite status (MSI). Of the cohort, 7447 (85.3%) were EUR, 541 (6.2%) were AFR, 461 (5.3%) were of AMR, 74 (0.85%) were SAS, and 205 (2.3%) were EAS. When compared with EUR, TERT GAs were less frequent in SAS (58.1% vs. 73.6%; P = .06). When compared with non-SAS, SAS had less frequent GAs in FGFR3 (9.5% vs. 18.5%, P = .25). TERT promoter mutations were significantly less frequent in EAS compared to non-EAS (54.1% vs. 72.9%; P < .001). When compared with the non-EAS, PIK3CA alterations were significantly less common in EAS (12.7% vs. 22.1%, P = .005). The mean TMB was significantly lower in EAS vs. non-EAS (8.53 vs. 10.02; P = .05). The results from this comprehensive genomic analysis of UCB provide important insight into the possible differences in the genomic landscape in a population level. These hypothesis-generating findings require external validation and should support the inclusion of more diverse patient populations in clinical trials.

Identification, classification, and functional characterization of novel sponge-associated acidimicrobiial species

Sponges are known to harbour an exceptional diversity of uncultured microorganisms, including members of the phylum Actinobacteriota. While members of the actinobacteriotal class Actinomycetia have been studied intensively due to their potential for secondary metabolite production, the sister class of Acidimicrobiia is often more abundant in sponges. However, the taxonomy, functions, and ecological roles of sponge-associated Acidimicrobiia are largely unknown. Here, we reconstructed and characterized 22 metagenome-assembled genomes (MAGs) of Acidimicrobiia from three sponge species. These MAGs represented six novel species, belonging to five genera, four families, and two orders, which are all uncharacterized (except the order Acidimicrobiales) and for which we propose nomenclature. These six uncultured species have either only been found in sponges and/or corals and have varying degrees of specificity to their host species. Functional gene profiling indicated that these six species shared a similar potential to non-symbiotic Acidimicrobiia with respect to amino acid biosynthesis and utilization of sulfur compounds. However, sponge-associated Acidimicrobiia differed from their non-symbiotic counterparts by relying predominantly on organic rather than inorganic sources of energy, and their predicted capacity to synthesise bioactive compounds or their precursors implicated in host defence. Additionally, the species possess the genetic capacity to degrade aromatic compounds that are frequently found in sponges. The novel Acidimicrobiia may also potentially mediate host development by modulating Hedgehog signalling and by the production of serotonin, which can affect host body contractions and digestion. These results highlight unique genomic and metabolic features of six new acidimicrobiial species that potentially support a sponge-associated lifestyle.

Clinicopathologic, Genomic, and Immunophenotypic Landscape of ATM Mutations in Non-Small Cell Lung Cancer.

ATM is the most commonly mutated DNA damage and repair gene in non-small cell lung cancer (NSCLC); however, limited characterization has been pursued. Clinicopathologic, genomic, and treatment data were collected for 5,172 patients with NSCLC tumors which underwent genomic profiling. ATM IHC was performed on 182 NSCLCs with ATM mutations. Multiplexed immunofluorescence was performed on a subset of 535 samples to examine tumor-infiltrating immune cell subsets. A total of 562 deleterious ATM mutations were identified in 9.7% of NSCLC samples. ATM-mutant (ATMMUT) NSCLC was significantly associated with female sex (P = 0.02), ever smoking status (P < 0.001), non-squamous histology (P = 0.004), and higher tumor mutational burden (DFCI, P < 0.0001; MSK, P < 0.0001) compared with ATM-wild-type (ATMWT) cases. Among 3,687 NSCLCs with comprehensive genomic profiling, co-occurring KRAS, STK11, and ARID2 oncogenic mutations were significantly enriched among ATMMUT NSCLCs (Q < 0.05), while TP53 and EGFR mutations were enriched in ATMWT NSCLCs. Among 182 ATMMUT samples with ATM IHC, tumors with nonsense, insertions/deletions, or splice site mutations were significantly more likely to display ATM loss by IHC (71.4% vs. 28.6%; P < 0.0001) compared with tumors with only predicted pathogenic missense mutations. Clinical outcomes to PD-(L)1 monotherapy (N = 1,522) and chemo-immunotherapy (N = 951) were similar between ATMMUT and ATMWT NSCLCs. Patients with concurrent ATM/TP53 mutations had significantly improved response rate and progression-free survival with PD-(L)1 monotherapy. Deleterious ATM mutations defined a subset of NSCLC with unique clinicopathologic, genomic, and immunophenotypic features. Our data may serve as resource to guide interpretation of specific ATM mutations in NSCLC.

Abstract 6499: Towards a combined immunohistochemistry-based subtyping of ovarian endometrioid carcinoma

Abstract Background: Ovarian endometrioid carcinoma (OEC) is an uncommon, understudied histotype that is distinguished from other histotypes by unique genomic features and high hormone receptor expression. There is growing interest in the biological relevance of subtyping using immunohistochemistry (IHC). Methods: Women with primary invasive OEC diagnosed from 1989 to 2010 were retrospectively identified from the population-based Alberta Cancer Registry, Canada. Pathology slide review and IHC staining for Napsin A, TP53, and WT1 were used to reduce histologic misclassification, resulting in N=166 participants (low-stage N=138, high-stage N=28), excluding those who received neo-adjuvant chemotherapy. The following markers were stained and scored on tissue microarrays: TP53 (normal/abnormal), PMS2/MSH6 (mismatch repair proficient [MMRp]/deficient [MMRd]), PGR (loss/retained), and CTNNB1 (nuclear/cytoplasmic). Using a hierarchical decision tree by stage group (low/high), we categorized tumors as TP53 abnormal, MMRd, PGR loss, nuclear CTNNB1, and no specific immunohistochemical profile (NSIP). Distributions and five-year disease specific survival (DSS) was estimated for each group; hazard ratios (HRs) adjusted for age were estimated using Cox regression with NSIP as referent group. Results: Among low-stage cases, five-year survival was as follows: 80% for TP53 abnormal (N=10, 7%), 87% for MMRd (N=15, 11%), 80% for PGR loss (N=10, 7%), 98% for nuclear CTNNB1 (N=63, 46%), and 95% for NSIP group (N=40, 29%). Overall, IHC group was associated with DSS among low stage cases (LRT p-value=0.006), and the group with nuclear CTNNB1 had a small but statistically significant DSS advantage (HR=0.089, 95% CI 0.011-0.742, p=0.025). TP53 and PGR loss suggested shorter DSS than the referent NSIP group, but not statistically significantly so (HR=1.26 and 3.08, respectively). Among high-stage cases, five-year DSS was as follows: 57% for TP53 abnormal (N=7, 25%), 75% for MMRd (N=8, 29%), 33% for nuclear CTNNB1 (N=3, 11%), and 17% for NSIP (N=10, 36%). There were no high-stage cases in the PGR-loss group. Nuclear CTNNB1 group had a shorter DSS among high-stage cases, but this was not statistically significantly so (HR=1.16, p=0.85). Conclusion: Pending replication in additional datasets, results suggest that OEC cases in nuclear CTNNB1 or NSIP IHC groups may have a favorable prognosis in which de-escalation of therapy can be considered. In addition, abnormal TP53 and loss of PGR may identify a high-risk subset of low-stage OEC. Citation Format: Brooke Jorgensen, Stacey Winham, Julie Cunningham, Sebastian Armasu, Chen Wang, Hunter Atkinson, Bryan McCauley, Linda Kelemen, Martin Koebel, Ellen Goode. Towards a combined immunohistochemistry-based subtyping of ovarian endometrioid carcinoma [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 6499.

Abstract ED4-4: Lobular/pleomorphic Breast Cancer

Abstract Invasive lobular carcinoma (ILC) accounts for up to 15% of all invasive breast cancers. The general marker to identify lobular cancer is the loss or low expression of E-cadherin which is the result of genetic alterations of the E-cadherin gene which accounts for about 95%. The majority of classical ILCs are of low grade and express both hormone receptors strongly and rarely any HER2. Those not expressing the hormone-receptors are categorised as apocrine breast cancers which is a small group. Recently it has been shown that ILC is a breast cancer type which has unique genomic features. Desmedt and colleagues genomically profiled 430 ILCs, to characterize the genomic alterations present in these tumors compared to invasive ductal carcinoma (now cold unfortunately NST non-specific subtype). The most frequent altered gene was CDH1 which results in a mRNA or protein loss of the cell adhesion molecule E-cadherin. Amongst the genes found to be altered at a high frequency are ESR1 and PIK3CA/PTEN. Currently those have little or no clinical impact in early breast cancer there might be consequences for metastatic breast cancer. The molecular alterations result in a less cohesive tissue connection and influence imaging as well as surgical procedures and in the end the prognosis in those patients. The size and extend of ILC is often underestimated by imaging and clinical examination. MRI thought to be the optimal imaging turned out to have no influence on positive margins and is no longer considered standard for all ILCs. Several data suggest that ILCs have an improved outcome the first 5 years after primary diagnosis but thereafter the survival curves in general lie below those for non lobular breast cancer. ILC has a distinct metastatic pattern and often metastasise into the GI tract and the ovaries and less frequently to the lung. Breast conserving surgery as well as mastectomy can be used when indicated based on tumor size and patients preference. Because ILC more often presents at higher stage, mastectomy is more frequently performed. But even after neoadjuvant chemotherapy we could demonstrate, despite response even resulting in pCR, mastectomy was more frequently conducted in ILC patients compared to non-ILC. In this analysis 71% of the non-ILC did receive BCS compared to only 59% in ILC (p Systemic therapy follows the general recommendations based on stage and histopathological profile. Neoadjuvant chemotherapy results in significantly lower rates of pathological responses. All analyses showed response rate in single digit numbers (far below 10%) but the less classical ILCs with either grade 3 or hormone-receptor negativity have a higher chance of achieving a pCR. Those ILCs with grade 3 and hormone-receptor negativity (apocrine cancer) have a pCR rate comparable to invasive ductal carcinoma (IDC). It is not surprising that lobular breast cancer does not respond as well as IDC to neoadjuvant chemotherapy because their biological features are, as described above, more luminal A like (high hormone receptor positivity, low grade, low proliferation). Because many patients with ILC present with extended diseases, they might still benefit from neoadjuvant chemotherapy even if they do not achieve a pathologic complete response, downstaging is an option and would potentially lead to less mastectomies. Because of the luminal A like type of the majority of ILCs endocrine therapy seems to be the treatment of choice. Some data even suggested a better outcome for ILC breast cancer when treated with an aromatase inhibitor compared to tamoxifen. But other data are controversial in this regard and did not confirm the data from BIG 1-98. In conclusion the molecular profile of ILC seems to be distinct but does not result in different treatment paradigms. We should not underestimate the necessity of optimal treatment, including chemotherapy for invasive lobular breast cancer patients. Citation Format: Sibylle Loibl. Lobular/pleomorphic Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr ED4-4.

A novel higher polyhydroxybutyrate producer Halomonas halmophila 18H with unique cell factory attributes

For cost-competitive biosynthesis of polyhydroxybutyrate (PHB), the screening of efficient producers and characterization of their genomic potential is fundamental. In this study, 94 newly isolated halophilic strains from Turkish salterns were screened for their polyhydroxyalkanoates (PHAs) biosynthesis capabilities through fermentation. Halomonas halmophila 18H was found to be the highest PHB producer, yielding 63.72 % of its biomass as PHB. The PHB produced by this strain was physically and chemically characterized using various techniques. Its genome was also sequenced and found to be large (6,713,657 bp) and have a GC content of 59.9 %. Halomonas halmophila 18H was also found to have several copies of PHB biosynthesis genes, as well as 20 % more protein-coding genes and 1075 singletons compared to other high PHB producers. These unique genomic features make it a promising cell factory for the simultaneous production of PHAs and other biotechnologically important secondary metabolites.

Can circulating cell free DNA be a promising marker in ovarian cancer? – a genome-scale profiling study in a single institution

BackgroundCell-free DNA (cfDNA) is emerging as a potential biomarker for the detection of ovarian cancer (OC). Recently, we reported a method based upon cfDNA whole-genome sequencing data including the nucleosome distribution (nucleosome footprinting NF), terminal signature sequence (motif), DNA fragmentation (fragment), and copy number variation (CNV).In the present study, we explored whether multiomics early screening technology in cfDNA can be applied for early screening of ovarian cancer.MethodsFifty-nine patients with OC and 100 healthy controls were included in this prospective study. Cell-free DNA was extracted from plasma and analyzed by low-pass whole-genome sequencing. Genomic features were obtained for all samples of the cohort, including copy number variation (CNV), 5’-end motifs, fragmentation profiles, and nucleosome footprinting (NF). An integrated scoring system termed the OC score was developed based on the performance of these four features.ResultsAll four features showed diagnostic potential for OC. Based on the unique genome features of cfDNA, the OC score has high accuracy in distinguishing OC patients from healthy controls (AUC 97.7%; sensitivity 94.7%; specificity 98.0%) as a new comprehensive diagnostic method for OC. The OC score showed a gradual trend from healthy controls to OC patients with different stages, especially for early OC monitoring of concern, which achieved a satisfactory sensitivity (85.7%) at a high specificity.ConclusionsThis is the first study evaluating the potential of cell-free DNA for the diagnosis of primary OC using multidimensional early screening technology. We present a promising method to increase the accuracy of prediction in patients with OC.

The Somatic Mutational Landscape of Mismatch Repair Deficient Prostate Cancer.

Prostate cancers with mismatch repair deficiency (MMR-d) have aggressive clinical and histological features, and they are potentially responsive to immunotherapy. However, its rarity prevents the analysis of the underlying biology. Here, we collected the genomic data of 2664 primary prostate tumors and 1409 metastatic prostate tumors from the GENIE and TCGA databases. A total of 69 (2.59%) primary and 60 (4.26%) metastatic MMR-d tumors were identified among these tumors. Single nucleotide variant (SNV) frequencies of 34 candidate genes (including KMT2D (46.4%), ZFHX3 (33.3%), JAK1 (31.9%), and RNF43 (27.5%)) and 16 candidate genes (including KMT2D (33.3%) and JAK1 (28.3%)) were higher in MMR-d primary tumors and MMR-d metastatic tumors, respectively. The tumor mutation burden (TMB) was higher in primary MMR-d tumors. Homozygous deletions of EPCAM and EPAS1 were enriched in MMR-d primary tumors, while EPCAM deletions were enriched in metastatic MMR-d tumors. For genomic rearrangement events, TMPRSS2-ETS fusions were less frequent in primary MMR-d tumors. Our study indicates MMR-d prostate cancers have unique genomic features. These may play an important role in providing therapeutic targets for the treatment of this subset of prostate cancer patients.

Molecular characterization of plasmacytoid urothelial carcinoma and the impact on treatment implications

Bladder cancer researchers and clinicians have increasingly viewed tumor biology through the lens of genomic and molecular alterations, drastically improving our knowledge of the underlying disease biology. This understanding has led to significant advances in treatment options that allow implementation of a personalized approach to cancer treatment. Large-scale genomic studies initially focused on the most common forms of bladder cancer. However, as genomic and molecular technologies become more widespread and are applied to less common variant histologies, we are gaining additional insight into the unique molecular and genomic characteristics driving the biology of variant histologies of bladder cancer. In this review, we summarize the current state of knowledge of molecular alterations underlying the distinct tumor biology of plasmacytoid urothelial carcinoma and how these alterations may impact treatment options.

Abstract A026: Identifying unique genomic and clinical characteristics of radiation-associated rectal cancer

Abstract Introduction: Prostate cancer patients treated with radiation therapy (RT) are at risk of developing radiation-associated rectal cancer (RARC). Previous literature reports worse overall survival in these patients compared to those with non-radiation associated primary rectal cancer (PRC), yet the genomic and clinical characteristics of RARC are not well understood. We compared molecular profiles of RARC and PRC patients to better understand the mechanism of RARC tumorigenesis and its interplay with clinical outcomes. Methods: We performed a retrospective analysis of 22 patients who had stage I-IV rectal cancer (within 12 cm of the anal verge) and had received previous RT for prostate cancer. Patients were excluded if they had a latency period between cancers of &amp;lt;5 years or additional metachronous cancers. Tumors were profiled using MSK-IMPACT, a targeted DNA-sequencing panel that identifies somatic mutations, copy number changes and fusions in a panel of 341-505 genes using a combination of solid tissue and matched blood specimens. The cohort included patients who had received external beam radiation therapy (EBRT, n=8), brachytherapy (BT, n=7), and combined EBRT-BT (n=7). All the RARC patients were microsatellite stable (MSS). To compare molecular features while accounting for clinical differences, we performed propensity score matching (1:1 ratio) using a background set of 616 patients with MSS PRC who had also been sequenced with MSK-IMPACT. Patients were matched for gender, age at diagnosis, distance from anal verge, tumor size, tumor location (anterior, lateral or posterior) and stage. Results: When compared to the general background set of 616 MSS PRC cases, RARC patients tended to be older at diagnosis (median 74 vs. 54 years, P=&amp;lt;0.0001). RARC tumors were more likely to have size &amp;lt;4cm at diagnosis (70% vs. 36%, P=0.01), to originate in the distal rectum, within 0-4 cm from the anal verge (55% vs. 25%, P=&amp;lt;0.001) and also to develop along the anterior rectal wall (41% vs 22%, P=&amp;lt;0.001). We then compared the genomic features of RARC to the clinically matched PRC set. RARC exhibited lower tumor mutational burden than PRC (median 4.4 vs. 6.0 mut/Mb, P=0.011), but no difference was observed in terms of chromosomal instability measured as the fraction of genome altered by copy number changes [median 0.11 vs. 0.17, P=0.49]. RARC patients also exhibited significantly lower frequency of APC mutations (41% vs. 82%, P=0.012) compared to PRC patients. Conclusion: We have identified clinical and molecular differences between RARC and PRC, including distal and anterior locations as well as lower TMB and lower frequency of APC mutations in RARC patients. The results provide clinically relevant insights into the unique attributes of RARC and may ultimately help to improve prognostication and treatment for this unique population. Citation Format: Dana M. R. Omer, Anisha Luthra, Hannah M. Thompson, Jonathan B. Yuval, Paul B. Romesser, Philip B. Paty, Julio Garcia-Aguilar, Francisco Sanchez-Vega. Identifying unique genomic and clinical characteristics of radiation-associated rectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A026.

ONT-Based Alternative Assemblies Impact on the Annotations of Unique versus Repetitive Features in the Genome of a Romanian Strain of Drosophila melanogaster

To date, different strategies of whole-genome sequencing (WGS) have been developed in order to understand the genome structure and functions. However, the analysis of genomic sequences obtained from natural populations is challenging and the biological interpretation of sequencing data remains the main issue. The MinION device developed by Oxford Nanopore Technologies (ONT) is able to generate long reads with minimal costs and time requirements. These valuable assets qualify it as a suitable method for performing WGS, especially in small laboratories. The long reads resulted using this sequencing approach can cover large structural variants and repetitive sequences commonly present in the genomes of eukaryotes. Using MinION, we performed two WGS assessments of a Romanian local strain of Drosophila melanogaster, referred to as Horezu_LaPeri (Horezu). In total, 1,317,857 reads with a size of 8.9 gigabytes (Gb) were generated. Canu and Flye de novo assembly tools were employed to obtain four distinct assemblies with both unfiltered and filtered reads, achieving maximum reference genome coverages of 94.8% (Canu) and 91.4% (Flye). In order to test the quality of these assemblies, we performed a two-step evaluation. Firstly, we considered the BUSCO scores and inquired for a supplemental set of genes using BLAST. Subsequently, we appraised the total content of natural transposons (NTs) relative to the reference genome (ISO1 strain) and mapped the mdg1 retroelement as a resolution assayer. Our results reveal that filtered data provide only slightly enhanced results when considering genes identification, but the use of unfiltered data had a consistent positive impact on the global evaluation of the NTs content. Our comparative studies also revealed differences between Flye and Canu assemblies regarding the annotation of unique versus repetitive genomic features. In our hands, Flye proved to be moderately better for gene identification, while Canu clearly outperformed Flye for NTs analysis. Data concerning the NTs content were compared to those obtained with ONT for the D. melanogaster ISO1 strain, revealing that our strategy conducted to better results. Additionally, the parameters of our ONT reads and assemblies are similar to those reported for ONT experiments performed on various model organisms, revealing that our assembly data are appropriate for a proficient annotation of the Horezu genome.

A haplotype-resolved genome assembly of the Nile rat facilitates exploration of the genetic basis of diabetes

BackgroundThe Nile rat (Avicanthis niloticus) is an important animal model because of its robust diurnal rhythm, a cone-rich retina, and a propensity to develop diet-induced diabetes without chemical or genetic modifications. A closer similarity to humans in these aspects, compared to the widely used Mus musculus and Rattus norvegicus models, holds the promise of better translation of research findings to the clinic.ResultsWe report a 2.5 Gb, chromosome-level reference genome assembly with fully resolved parental haplotypes, generated with the Vertebrate Genomes Project (VGP). The assembly is highly contiguous, with contig N50 of 11.1 Mb, scaffold N50 of 83 Mb, and 95.2% of the sequence assigned to chromosomes. We used a novel workflow to identify 3613 segmental duplications and quantify duplicated genes. Comparative analyses revealed unique genomic features of the Nile rat, including some that affect genes associated with type 2 diabetes and metabolic dysfunctions. We discuss 14 genes that are heterozygous in the Nile rat or highly diverged from the house mouse.ConclusionsOur findings reflect the exceptional level of genomic resolution present in this assembly, which will greatly expand the potential of the Nile rat as a model organism.

Discovery and Evolution of Six Positive-Sense RNA Viruses Co-infecting the Hypovirulent Strain SCH733 of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a well-known phytopathogenic fungus with a wide host range. Identifying novel mycoviruses in phytopathogenic fungi is necessary to develop novel strategies for plant health protection and contribute to understanding the origin of viruses. Six new mycoviruses with positive single-stranded RNA genomes co-infecting the hypovirulent strain SCH733 of S. sclerotiorum were identified using a metatranscriptomic approach, and their complete genome sequences were molecularly determined. These mycoviruses belong to the following five families: Narnaviridae, Mitoviridae, Deltaflexviridae, Botourmiaviridae, and Ambiguiviridae. Three of these mycoviruses belong to existing International Committee on Taxonomy of Viruses (ICTV)-recognized species. Two of these newly identified mycoviruses have unique genomic features that are significantly different from those of all known mycoviruses. Phylogenetic analysis revealed that these six mycoviruses included close as well as distant relatives of known mycoviruses, thereby providing new insight into virus evolution and classification. Mycovirus horizontal transmission and elimination experiments revealed that Sclerotinia sclerotiorum narnavirus 5 is associated with hypovirulence of S. sclerotiorum, although we have not shown that it is independently responsible for the hypovirulence phenotype. This study broadens the diversity of known mycoviruses infecting S. sclerotiorum and provides a clue toward limiting hypovirulence in S. sclerotiorum.

Mesenchymal gene expression subtyping analysis for early-stage human papillomavirus-negative head and neck squamous cell carcinoma reveals prognostic and predictive applications.

Patients with oral cavity squamous cell carcinoma (OCSCC) are predominantly human papillomavirus (HPV)(−), and treatment typically involves surgical resection ± neck dissection, followed by radiation ± chemotherapy. We previously described four mRNA expression patterns (classical, atypical, basal, and mesenchymal), each with unique genomic features and prognosis. Here, we examine the clinical utility of gene expression subtyping in head and neck squamous cell carcinoma (HNSCC) and introduce potentially predictive applications in HPV(−) OCSCC. A retrospective genomic database analysis was performed including 562 HNSCC patients from MD Anderson (MDA-GSE41116) and The Cancer Genome Atlas (TCGA). Samples were assigned molecular subtypes (classical, atypical, basal, and mesenchymal) using an 88-gene classifier. HPV status was determined by gene expression. The clinical endpoint was overall survival censured at 36 months. The Kaplan–Meier plots and log-rank tests were used to investigate associations between clinical variables and survival. Of the 418 TCGA training patients who met analysis criteria, nearly 20% presented as stage I/II. Among node(−) OCSCC patients, the mesenchymal subtype is associated with worse survival (hazard ratio (HR) = 2.4, p = 0.021), offering a potentially actionable biomarker in otherwise early-stage, low-risk disease. This was confirmed in the MDA validation cohort. Node(−) non-mesenchymal OCSCC patients had far better survival compared to node(−) mesenchymal, and all node(+) patients had similarly poor survival. These findings suggest that the mesenchymal subtype is associated with poor survival in surgically resected, early-stage, node(−) OCSCC otherwise expected to have favorable outcomes. These findings highlight the potential value of gene expression subtyping as a pathology adjunct for prognostication and treatment decision-making in OCSCC patients.

1778P Comparative genomic alterations (GA) landscape in urothelial carcinoma of the bladder (UCB) in patients of South Asian ancestry (SAS)

UCB has a variable prevalence worldwide. We queried whether UCB arising in pts with SAS ancestry would feature unique genomic features when compared with patients (pts) of other, predominantly European, descent.

High infectivity and unique genomic sequence characteristics of Cryptosporidium parvum in China

Zoonotic Cryptosporidium parvum infections are mainly caused by IIa and IId subtypes. As most biological characterizations have been performed on IIa subtypes, the biological and genetic characteristics of IId subtypes in China are not clear. We evaluated the infection and genetic characteristics of IId isolates in interferon-γ-knockout mice using qPCR to quantify oocyst shedding, histological examination to monitor pathological changes and comparative genomic analyses to identify infectivity and virulence-associated differences. Compared with the reference IIa isolate, mice infected with the IId isolates had significantly higher and longer oocyst shedding and lower body weight gain. In addition, the four IId isolates examined differed significantly in infectivity (as indicated by the median infective dose), oocyst shedding duration, and pathogenicity. Comparative genomic analysis indicated that the IId isolates had three more subtelomeric genes than the reference IIa isolate and 5385-5548 nucleotide substitutions, with the hypervariable genes mostly in two blocks on chromosome 1. In contrast, the four IId isolates differed from each other by 77-1,452 nucleotides, with virulence-associated sequence differences mainly in nine genes within a 28-kb block on chromosome 6. These data indicate the newly emerged C. parvum IId subtypes in China have high animal infectivity and unique genomic characteristics.

Abstract 2142: Prognostic and predictive applications from mesenchymal gene expression subtype analysis for early-stage, HPV(-) head and neck squamous cell carcinoma

Abstract Introduction: Although often presented as one disease for genomic studies, head and neck squamous cell carcinoma (HNSCC) is treated according to clinical factors such as anatomic subsite, and tumor stage. Oral cavity squamous cell carcinoma (OCSCC) comprises 1/3 of all HNSCC and is predominantly HPV(-). Depending upon clinical stage, OCSCC treatment involves surgical resection +/- neck dissection, followed by radiation +/- chemotherapy. Our group and others previously described four mRNA expression patterns (classical, atypical, basal, and mesenchymal), each with unique genomic features and prognosis. Here, we further examine the clinical utility of gene expression subtyping in HNSCC and introduce the potential for predictive applications in HPV(-) HNSCC according to clinically relevant subgroups including oral cavity cancer. Experimental Procedures: Multiple independent HNSCC datasets were obtained from public repositories, totaling 562 patients, including from MD Andersen (MDA) (GSE41117) and the Cancer Genome Atlas (TCGA) sourced from the Genome Data Commons. Cases were included for further analysis if they had N stage and overall survival values. Samples were assigned molecular subtypes (basal, mesenchymal, atypical or classical) using a reduced gene set version of the classifier reported earlier. HPV status was determined by HPV gene expression. The clinical endpoint was overall survival at censured at 36 months. Kaplan-Meier (KM) plots and logrank tests were used to investigate associations between clinical variables and survival. Cox models were used to adjust for potential confounders. All statistics were performed using the survival package in R. Results: Of the 418 training patients from the TCGA dataset that met analysis criteria, nearly 20% presented with stage I and II tumors. In the clinically relevant subgroup of node(-) OCSCC patients, mesenchymal subtype was associated with worse survival (HR 2.4, p=0.021), offering a novel and potentially actionable biomarker in otherwise early-stage and low risk disease. Associations in the MDA validation cohort confirmed the association. Node(-) non-mesenchymal OCSCC patients had far better survival (no deaths observed) compared to node(-) mesenchymal and all node(+) patients which had similarly poor survival. Differential responses as a function of radiation will be presented. Summary and Conclusions: This study confirms that the mesenchymal subtype is associated with worse outcomes across all cases of HNSCC. However, we demonstrate that mesenchymal subtype is associated with poor survival, even in the clinically relevant setting of early-stage, node(-) OCSCC treated with surgical resection. These findings highlight the potential value of gene expression subtyping as an adjunct to pathology for deciding which treatment option is best suited for patients with HNSCC. Citation Format: D. Neil Neil Hayes, MD, MS, MPH, Greg Mayhew, Josh Uronis, Jose Zevallos. Prognostic and predictive applications from mesenchymal gene expression subtype analysis for early-stage, HPV(-) head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2142.

Abstract 2143: Clinicopathologic, genomic and immunophenotypic landscape of ATM mutations in non-small cell lung cancer

Abstract Introduction: Defective DNA damage repair machinery is a hallmark of cancer, resulting in increased mutation rates and genomic instability. In non-small cell lung cancer (NSCLC), ATM is mutated in ~10% of cases, representing the most commonly mutated DNA damage and repair gene. However, the clinicopathologic, genomic, and immunophenotypic correlates of ATM mutations in NSCLC are unknown. The impact of ATM mutation on clinical outcomes to PD-(L)1 blockade is also unclear. Methods: Clinicopathologic and genomic data were collected from 3592 patients (pts) with NSCLC who had consented to correlative studies at the Dana-Farber Cancer Institute (DFCI), and whose tumors underwent genomic profiling (OncoPanel). Multiplexed immunofluorescence (mIF) for CD8, PD1, PD-L1, FOXP3, and CK AE1/AE3 was performed on a subset of 416 NSCLC samples to examine tumor-infiltrating immune cells. ATM immunohistochemistry (IHC) was also performed on 184 ATM mutated NSCLCs with available tissue. ATM mutated (ATMMUT) tumors were defined as harboring loss-of-function mutations (nonsense, frameshift, splice site, known deleterious missense mutations). Missense mutation of unknown significance were excluded, unless deemed to affect protein function in silico. Tumors lacking ATM mutations or harboring benign ATM alterations were defined as ATM wild type (ATMWT). Results: A total of 399 deleterious ATM mutations were identified in 10.2% (365/3592) of samples; 138 (34.6%) mutations were truncating (nonsense, frameshift, and splice site mutations); the remaining 261 (65.4%) were missense mutations. Truncating mutations were significantly more likely to result in ATM loss by IHC compared to missense mutations (71.4% vs 28.9%, P&amp;lt;0.01) When we examined the genomic profiles of tumors with versus without deleterious ATM mutations, we found that ATMMUT NSCLCs were significantly enriched with KRAS, STK11, RBM10, and KDM5C co-mutations (P&amp;lt;0.01), while co-mutations in EGFR, CDKN2A and TP53 were nearly mutually exclusive (P&amp;lt;0.01). Among ATMMUT NSCLCs, those with ATM loss by IHC were significantly enriched with KRAS and STK11 co-mutations, while those with retained ATM expression were enriched with TP53 co-mutations (P&amp;lt;0.01). Pts with ATMMUT NSCLCs had similar outcomes to PD-(L)1 inhibition +/- chemotherapy, compared to ATMWT cases, and similar immune cell subsets infiltration (P&amp;gt;0.05). Pts with deleterious mutations in ATM and TP53 (ATMMUT/TP53MUT) had increased response rates to chemo-immunotherapy compared to those with ATMMUT/TP53WT, ATMWT/TP53MUT, or ATMWT/TP53WT genotypes (70% vs 56.2% vs 35.7% vs 27.4%, respectively, P=0.01), as well as increased tumor-stroma interface CD8+ T cells (P&amp;lt;0.01) and higher PD-L1 expression by mIF on tumor (P&amp;lt;0.01) and immune cells (P&amp;lt;0.01). Conclusion: Deleterious ATM mutations defined a subset of NSCLC with unique clinicopathologic, genomic, and immunophenotypic features. Citation Format: Biagio Ricciuti, Joao Victor Alessi, Xinan Wang, Arrien A. Bertram, Victor R. Vaz, Mizuki Nishino, James Lindsay, Kristen D. Felt, Bijaya Sharma, Lynette M. Sholl, Rodig Scott, Mark M. Awad, Michael L. Cheng. Clinicopathologic, genomic and immunophenotypic landscape of ATM mutations in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2143.