Copy Loss Research Articles

Abstract P002: Timing onset of homologous recombination deficiency before cancer diagnosis in BRCA1/2 mutation carriers

Abstract Introduction: Identification of individuals at risk of developing cancer is key to prevention, particularly among carriers of germline BRCA1/2 mutations. Cancers that develop in this population display loss of the wildtype copy BRCA1/2 and a reproducible pattern of somatic passenger mutations manifesting homologous recombination deficiency (HRD) (Davies et al. 2017). It is not known how much time elapses between the bi-allelic loss of genes responsible for homologous recombination deficiency and the development of cancer. A better understanding of this process and estimating the time between cancer initiation to diagnosis in patients with germline BRCA1/2 mutations would inform early detection strategies in the high-risk group. Methods: Passenger mutations in the cancer genome capture the life history of each cancer. We analyzed patterns of somatic mutations in whole genomes of 27 cancers from patients with germline BRCA1/2 mutations who developed cancers of different tissues with whole genome duplications (WGD). We conducted a meta-analysis of data from the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Consortium (n=14) and the SCANB breast cancer biobank (n=13) (Staaf et al. 2019). Whole genome duplications in tumors allow for approximate timing of onset of HRD. The “molecular clock” mutations in characteristic CpG context accumulate with age and are used to time genomic events. Following bi-allelic BRCA1/2 loss, additional types of somatic mutations accumulate, notably the single point mutation signature SBS3 and HRD-associated indel and rearrangement signatures. Somatic mutations are assigned to the most likely signature which generated them. In tumors that carry it, whole genome duplication enables relative timing of other genomic events. Mutations that occur before and after the duplication are distinguished using allele frequencies (Gerstung et al. 2020). Results: Using this methodology, we observe that HRD-associated SBS3 accounts for as much as 40-60% of the duplicated mutations, suggesting that HRD developed prior to WGD. We use the “molecular clock” to time the intervals before and after WGD, and we estimate that WGD occurs six to thirteen years (interquartile range) prior to the most recent common ancestor of diagnosed tumor. Next, using the relative proportions of SBS3 and clock signatures deduced from post-WGD mutations, we infer how long before WGD SBS3 started to accumulate, providing an even closer bound for HRD development time point than the WGD timing alone. Conclusions: We establish a lower bound on the dormancy period of homologous recombination deficiency prior to cancer diagnosis which would inform early detection strategies in the high-risk group of BRCA1/2 carriers. Citation Format: Dominik Glodzik, Doga Gulhan, Peter J. Park. Timing onset of homologous recombination deficiency before cancer diagnosis in BRCA1/2 mutation carriers. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P002.

Expanding the clinical and molecular features of trichorhino- phalangeal syndrome with a novel variant.

Tricho-rhino-phalangeal syndrome (TRPS) is a rare, autosomal dominant disorder characterized by typical craniofacial features, ectodermal and skeletal findings. TRPS type 1 (TRPS1) is caused by pathogenic variations in the TRPS1 gene, which relates to the vast majority of cases. TRPS type 2 (TRPS2) is a contiguous gene deletion syndrome involving loss of functional copies of the TRPS1, RAD21, and EXT1. Herein, we reported the clinical and genetic spectrum of seven TRPS patients with a novel variant. We also reviewed the musculoskeletal and radiological findings in the literature. Seven Turkish patients (three female, four male) from five unrelated families aged between 7 to 48 years were evaluated. The clinical diagnosis was confirmed by either molecular karyotyping or TRPS1 sequencing analysis via next-generation sequencing. Both TRPS1 and TRPS2 patients had some common distinctive facial features and skeletal findings. All patients had a bulbous nose with hypoplastic alae nasi, brachydactyly, short metacarpals and phalanges in variable stages. Low bone mineral density (BMD) was identified in two TRPS2 family members presenting with bone fracture, and growth hormone deficiency was detected in two patients. Skeletal X-ray imaging revealed cone-shaped epiphysis of the phalanges in all, and multiple exostoses were present in three patients. Cerebral hamartoma, menometrorrhagia and long bone cysts were among the new/rare conditions. Three pathogenic variants in TRPS1 were identified in four patients from three families, including a frameshift (c.2445dup, p.Ser816GlufsTer28), one missense (c.2762G > A), and a novel splice site variant (c.2700+3A > G). We also reported a familial inheritance in TRPS2 which is known to be very rare. Our study contributes to the clinical and genetic spectrum of patients with TRPS while also providing a review by comparing with previous cohort studies.

In vivo modelling of cutaneous T-cell lymphoma: The role of SOCS1.

Mycosis fungoides (MF), the most common type of Cutaneous T cell Lymphoma (CTCL), is characterized by an inflamed skin intermixed with proliferating malignant mature skin-homing CD4+ T cells. Detailed genomic analyses of MF skin biopsies revealed several candidate genes possibly involved in genesis of these tumors and/or potential targets for therapy. These studies showed, in addition to common loss of cell cycle regulator CDKN2A, activation of several oncogenic pathways, most prominently and consistently involving JAK/STAT signaling. SOCS1, an endogenous inhibitor of the JAK/STAT signaling pathway, was identified as a recurrently deleted gene in MF, already occurring in the earliest stages of the disease. To explore the mechanisms of MF, we create in vivo mouse models of autochthonous CTCLs and these genetically engineered mouse models (GEMMS) can also serve as valid experimental models for targeted therapy. We describe the impact of allelic deletion of Socs1 in CD4 T cells of the skin. To achieve this, we crossed inducible Cre-transgenic mice in the CD4 lineage with transgenic mice carrying floxed genes of Socs1. We first determined optimal conditions for Socs1 ablation with limited effects on circulating CD4 T-cells in blood. Next, we started time-course experiments mimicking sustained inflammation, typical in CTCL. FACS analysis of the blood was done every week. Skin biopsies were analyzed by immunocytochemical staining at the end of the experiment. We found that the Socs1 knockout transgenic group had thicker epidermis of treated skin compared with the control group and had more CD3 and CD4 in the skin of the transgenic group compared to the control group. We also noted more activation of Stat3 by staining for P-Stat3 in Socs1 knockout compared to wt CD4+T cells in the skin. The results also indicated that single copy loss of Socs1 in combination with sustained inflammation is insufficient to start a phenotype resembling early stage mycosis fungoides within eight weeks in these mice. In sum, we developed and optimized an autochthonous murine model permitting selective knockout of Socs1 in skin infiltrating CD4 T-cells. This paves the way for more elaborate experiments to gain insight in the oncogenesis of CTCL.

Abstract B066: Elucidating the role of insulin receptor isoform expression in metastatic pancreatic ductal adenocarcinoma

Abstract Introduction: While type 2 diabetes mellitus (T2DM) has been shown to be both a risk factor and consequence of pancreatic ductal adenocarcinoma (PDAC), the bidirectional relationship between T2DM and PDAC remains to be fully understood. Due to spatial proximity of insulin-producing beta cells, PDAC tumor cells are presumably exposed to a highly concentrated insulin microenvironment, which may contribute to the dysregulation of the insulin receptor gene INSR. There are two isoforms of insulin receptor: IR-A and IR-B. IR-A activity has been associated with oncogenic function and is upregulated in several cancer types. This leads to questions regarding the role of INSR and more specifically, how the ratio of the two isoforms may potentiate tumor aggressiveness in PDAC. Results: Using data from whole-genome and RNA sequencing of metastatic PDAC (mPDAC) from both the PanGen (n=70; NCT01855477) and Personalized Oncogenomics (n=22; NCT02155621) trials, we identified somatic copy loss of INSR in 39/92 (42%) patient tumors, with the majority of such cases (35/39; 90%) showing heterozygous copy loss. There was no statistical association between INSR copy status and PDAC transcriptomic subtypes. INSR expression was lower in basal-like versus classical subtype tumors (p=1.1e-4), and INSR expression was further attenuated in basal-like tumors with heterozygous copy loss of INSR (p=0.0041). The ratio of IR-A:IR-B expression was heterogenous across samples, and heightened IR-A:IR-B ratio was significantly (p<0.05) associated with expression of genes linked to the PI3K-Akt signaling axis (CCDC88A, THEM4) and glucose metabolism (HK1, G6PC, PKLR) in basal-like tumors. Interestingly,HK1 and IGF1R were significantly upregulated, independent of IR-A:IR-B ratio, among basal-like (p 9.1e-9 and p=1.4e-5, respectively) compared to classical subtype tumors. Conclusion: These data indicate that heightened IR-A:IR-B ratio is associated with the expression of key pathways converging on PI3K signalling and glucose metabolism in mPDAC, while highlighting that several IR-A:IR-B-associated gene correlations are unique to basal-like tumors. Such findings warrant further investigation of relative INSR isoform expression in mPDAC and pre-clinical models to elucidate the role of the oncogenic isoform, IR-A, in driving tumor aggressiveness in basal-like subtype tumors. Citation Format: Lan V. Tao, James T. Topham, Joanna M. Karasinska, Erica S. Tsang, Andrew Metcalfe, Hassan Ali, Dawn Ashforth, Rachel Goodwin, Patricia A. Tang, Oliver F. Bathe, Janessa Laskin, Marco Marra, Daniel J. Renouf, David F. Schaeffer. Elucidating the role of insulin receptor isoform expression in metastatic pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B066.

Impact of CDKN2A/B, MTAP, and TERT Genetic Alterations on Survival in IDH Wild Type Glioblastomas

PurposePoor outcomes in IDH wild-type (IDHwt) glioblastomas indicate the need to determine which genetic alterations can indicate poor survival and guidance of patient specific treatment options. We sought to identify the genetic alterations in these patients that predict for survival when adjusting particularly for treatments and other genetic alterations.MethodsA cohort of 167 patients with pathologically confirmed IDHwt glioblastomas treated at our institution was retrospectively reviewed. Next generation sequencing was performed for each patient to determine tumor genetic alterations. Multivariable cox proportional hazards analysis for overall survival (OS) was performed to control for patient variables.ResultsCDKN2A, CDKN2B, and MTAP deletion predict for worse OS independently of other genetic alterations and patient characteristics (hazard ratio [HR] 2.192, p = 0.0017). Patients with CDKN2A copy loss (HR 2.963, p = 0.0037) or TERT mutated (HR 2.815, p = 0.0008) glioblastomas exhibited significant associations between radiation dose and OS, while CDKN2A and TERT wild type patients did not. CDKN2A deleted patients with NF1 mutations had worse OS (HR 1.990, p = 0.0540), while CDKN2A wild type patients had improved OS (HR 0.229, p = 0.0723). Patients with TERT mutated glioblastomas who were treated with radiation doses < 45 Gy (HR 3.019, p = 0.0010) but not those treated with ≥ 45 Gy exhibited worse OS compared to those without TERT mutations.ConclusionIn IDHwt glioblastomas, CDKN2A, CDKN2B, and MTAP predict for poor prognosis. TERT and CDKN2A mutations are associated with worse survival only when treated with lower radiation doses, thus potentially providing a genetic marker that can inform clinicians on proper dose-fractionation schemes.

PSXII-1 CNV and Cnvr Detection from High-Density SNP Genotyping in Holstein Cattle

Abstract Copy number variation (CNV) represents an important source of genetic variation complementary to single nucleotide polymorphism (SNP) and provides valuable insights into the genetic architecture of complex traits in humans and livestock animals. However, many factors affect CNV detection, including sample size. Previous studies in Holstein cattle have only used hundreds of individuals. Therefore, in the present study, CNVs were identified from high-density genotype array data in 3,529 Holstein cows from 16 herds located in 7 U.S. states. CNVs were derived from 546,802 autosomal SNP markers with MAF &amp;gt; 0.05 and call rate &amp;gt; 0.90 using the intensity signals at each SNP position in a hidden Markov model implemented in PennCNV software. The log R ratio (LRR) values were corrected based on the guanine-cytosine content in the genomic regions located 500 kb upstream and downstream of each marker. After detection, CNVs presenting LRR ≤ 0.30, B allele frequency drift &amp;lt; 0.01, waviness factor ≤ 0.05, number of SNPs &amp;gt; 10 and length &amp;gt; 1 kb were merged into CNV regions (CNVR) using CNVRanger. A total of 1,881 non-redundant CNV events spanning the entire genome were identified in 1,771 cows, with a mean of 10 CNVs per individual and ranging from 1 to 27. The highest percentage of CNV calls was located on BTA7 (16%), while only 9 CNVs were identified on BTA9. The CNV calls were grouped into 685 CNVRs covering 3.07% of the bovine autosomal genome. The number of CNVRs with copy loss and gain were 356 and 310, and both types were observed in 19 regions. The chromosomal distribution of CNVR revealed that BTA7 harbors 38 CNVRs, while only 2 were detected on BTA28. These results will support further research on the contribution of CNVs and CNVRs to the genetic architecture of complex traits in Holstein cattle.

Direct Comparison of HPV16 Viral Genomic Integration, Copy Loss, and Structural Variants in Oropharyngeal and Uterine Cervical Cancers Reveal Distinct Relationships to E2 Disruption and Somatic Alteration.

Simple SummaryHPV16 causes approximately 60% of uterine cervical cancer and 95% of HPV-driven oropharynx cancers. Despite both being HPV-associated, these tumors are very different. We directly compare integration of the HPV16 genome into the human genome in these two diseases, finding that the viral gene E2 is frequently lost in cervical cancer, but usually maintained in oropharyngeal cancer. We also found that oropharyngeal cancers with integration have many more integration sites per tumor and these more frequently occur in genomic regions with a high density of genes.Squamous cell carcinoma of the oropharynx caused by HPV type 16 (HPV16+ OPSCC) is the most common HPV-associated malignancy in the USA and has many molecular differences from uterine cervical squamous cell carcinoma (UCSCC). Our understanding of HPV oncogenesis relied on studies of UCSCC revealing a consensus model reliant on HPV integration with a loss of E2. Here, we compare patterns of HPV integration in UCSCC and OPSCC by analysis of affinity capture sequencing of the HPV16 genome in 104 OPSCC and 44 UCSCC tumors. These cohorts were contemporaneously sequenced using an identical strategy. Integration was identified using discordant read pair clustering and assembly-based approaches. Viral integration sites, structural variants, and copy losses were examined. While large-scale deep losses of HPV16 genes were common in UCSCC and were associated with E2 loss, deep copy losses of the HPV16 genome were infrequent in HPV16+ OPSCC. Similarly, structural variants within HPV16 favored E2 loss in UCSCC but not OPSCC. HPV16 integration sites were non-random, with recurrent integration hot-spots identified. OPSCC tumors had many more integration sites per tumor when compared to UCSCC and had more integration sites in genomic regions with high gene density. These data show that viral integration and E2 disruption are distinct in UCSCC and OPSCC. Our findings also add to growing literature suggesting that HPV tumorigenesis in OPSCC does not follow the model developed based on UCSCC.

Abstract A38: Cd70 genetic perturbation limits the development of an effective CD8+ T-cell immune response to Bcl6-driven diffuse large B-cell lymphoma

Abstract Multiple immunomodulatory pathways shape the development of anti-tumor immune responses to lymphoid malignancies. We previously defined the recurrent genetic alterations in diffuse large B-cell lymphoma (DLBCL) and identified associated substructure and additional potential genetic bases for immune escape. CD70 was the most commonly perturbed immune response pathway component in our cohort of primary DLBCLs; alterations included inactivating mutations and copy loss. CD70 co-stimulation of CD27+ T cells induces antigen-dependent T-cell expansion and immune surveillance of normal and malignant B cells. Given the frequent co-association of CD70 alterations and BCL6 translocations in our DLBCL patient series, we assessed the consequences of Cd70 deficiency on Bcl6-driven lymphomagenesis in a murine model. We crossed previously generated Cd70 −/- and Bcl6 tg/+ mice to obtain Cd70 −/−; Bcl6 tg/+ animals. In our aging cohorts, Cd70− / −; Bcl6tg/+ mice developed significantly increased numbers of histopathologically confirmed DLBCLs at earlier timepoints, compared to Bcl6 tg/+ animals. Both the Cd70 −/−; Bcl6 tg/+ and Bcl6 tg/+ mice that were euthanized for symptoms exhibited massive splenomegaly and lymphomatous splenic infiltration. None of the wild-type (WT) and Cd70 −/- animals developed lymphoma. To characterize potential differences in anti-tumor responses in Cd70 −/−; Bcl6 tg/+ versus Bcl6 tg/+ mice, we harvested spleens from asymptomatic animals in each cohort at 6, 14 and 18 months (mo). Cd70 −/−; Bcl6 tg/+ mice exhibited significantly earlier onset splenomegaly than Bcl6 tg/+ animals (both in comparison with WT mice). We performed single cell RNA sequencing of splenic cell suspensions from each murine cohort at the above-mentioned predetermined timepoints (6, 14 and 18 mo) and describe genotype-related changes in splenic CD8+ T-cell infiltration in this abstract. Our study revealed an age-related decline in the percentages of naive CD8+ T cells in all genotypes, with more striking and earlier changes in Cd70 −/−; Bcl6 tg/+ animals. Cd70 −/−; Bcl6 tg/+ and Bcl6 tg/+ mice exhibited a selective and significant expansion of CD8+ cytotoxic T cells (CTLs), which expressed Gzmb and Prf1 and the exhaustion markers, Pdcd1, Lag3, Tigit, Tox and Tim3, and exhibited clonal expansion. At 6 mo, prior to splenic enlargement and the development of symptoms, CD8+ CTLs in Cd70 −/−; Bcl6 tg/+ animals expressed significantly higher levels of exhaustion markers than those in Bcl6 tg/+ mice. Consistent with this finding, there was a more limited expansion and a subsequent contraction of these splenic CD8+ CTLs in Cd70 −/−; Bcl6 tg/+ mice, in comparison to Bcl6 tg/+ animals. Taken together, these findings suggest that initial anti-tumor immune responses are less effective in Cd70 −/−; Bcl6 tg/+ mice than in Bcl6 tg/+ animals and highlight the likely importance of CD70/CD27 co-stimulation in CD8+ T-cell response to Bcl6-driven DLBCL. Citation Format: Elisa Mandato, Eleonora Calabretta, Gali Bai, Li Song, Yanbo Sun, Vignesh Shanmugam, Julia Paczkowska, Il-Kyu Choi, Robert Redd, Ming Tang, Lee N Lawton, Donna Neuberg, Scott Rodig, Franziska Michor, Baochun Zhang, Margaret A Shipp. Cd70 genetic perturbation limits the development of an effective CD8+ T-cell immune response to Bcl6-driven diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A38.

108P Copy loss enrichment at metastatic disease progression in hormone receptor-positive (HR+)/HER2-negative metastatic breast cancer patients treated with endocrine therapy and CDK4/6 inhibition

Blood copy number burden (bCNB), derived from a low-pass whole genome sequencing assay (PredicineCNBTM) is a comprehensive measure of copy number variation (CNV), including amplifications and deletions across the entire genome. We previously reported that increasing bCNB levels in blood samples collected from HR+/HER2-negative metastatic breast cancer patients at baseline and during treatment with endocrine therapy and CDK4/6 inhibition (ET + CDK4/6i) preceded radiographic detection of disease progression.

Genome-wide evaluation of copy gain and loss variations in three Afghan sheep breeds

Copy number variation (CNV) is one of the main sources of variation between different individuals that has recently attracted much researcher interest as a major source for heritable variation in complex traits. The aim of this study was to identify CNVs in Afghan indigenous sheep consisting of three Arab, Baluchi, and Gadik breeds using genomic arrays containing 53,862 single nucleotide polymorphism (SNP) markers. Data were analyzed using the Hidden Markov Model (HMM) of PennCNV software. In this study, out of 45 sheep studied, 97.8% (44 animals) have shown CNVs. In total, 411 CNVs were observed for autosomal chromosomes and the entire sequence length of around 144 Mb was identified across the genome. The average number of CNVs per each sheep was 9.13. The identified CNVs for Arab, Baluchi, and Gadik breeds were 306, 62, and 43, respectively. After merging overlapped regions, a total of 376 copy number variation regions (CNVR) were identified, which are 286, 50, and 40 for Arab, Baluchi, and Gadik breeds, respectively. Bioinformatics analysis was performed to identify the genes and QTLs reported in these regions and the biochemical pathways involved by these genes. The results showed that many of these CNVRs overlapped with the genes or QTLs that are associated with various pathways such as immune system development, growth, reproduction, and environmental adaptions. Furthermore, to determine a genome-wide pattern of selection signatures in Afghan sheep breeds, the unbiased estimates of FST was calculated and the results indicated that 37 of the 376 CNVRs (~ 10%) have been also under selection signature, most of those overlapped with the genes influencing production, reproduction and immune system. Finally, the statistical methods used in this study was applied in an external dataset including 96 individuals of the Iranian sheep breed. The results indicated that 20 of the 114 CNVRs (18%) identified in Iranian sheep breed were also identified in our study, most of those overlapped with the genes influencing production, reproduction and immune system. Overall, this is the first attempts to develop the genomic map of loss and gain variation in the genome of Afghan indigenous sheep breeds, and may be important to shed some light on the genomic regions associated with some economically important traits in these breeds.

Molecular Characterization of Mesothelioma: Impact of Histologic Type and Site of Origin on Molecular Landscape.

Mesothelioma is an aggressive malignancy with heterogeneous outcomes that are partly driven by the differential efficacy of existing therapies across histologic types and sites of origin. Large-scale molecular analysis of mesothelioma and its subtypes has the potential to inform future therapeutic strategies. We analyzed 1,294 mesotheliomas {980 pleural (malignant pleural mesothelioma [MPM]) and 314 peritoneal (malignant peritoneal mesothelioma [MPeM])} using next-generation sequencing, determined programmed death ligand-1 (PD-L1) expression and histology in a subset of cases, and assessed MTAP/CDKN2A copy-number status by fluorescence in situ hybridization and T-cell infiltration in an independent cohort. The molecular landscape of MPM was characterized by inactivating alterations in CDKN2A (49%), BAP1 (44%), CDKN2B (42%), MTAP (34%), and NF2 (33%). Compared with epithelioid MPM, nonepithelioid (ie, biphasic and sarcomatoid) MPM had identical tumor mutational burden (median 1.25 mut/Mb, P = .63), more commonly expressed PD-L1 (74% v 51%, P = .02), and was more likely to harbor MTAP, CDKN2A, and CDKN2B copy loss (P < .05). Fluorescence in situ hybridization confirmed that homozygous MTAP loss was enriched in nonepithelioid MPM. Relative to MPM, MPeM had comparable tumor mutational burden and PD-L1 expression. The molecular profile of MPeM was similar to MPM, with the distinction that PBRM1 alterations occurred at higher frequency (16% v 7%, P < .01). ALK rearrangements were only observed in MPeM. Regardless of histology and location, the molecular landscape of mesothelioma primarily consists of inactivating alterations in tumor suppressor genes, with enrichment of certain alterations in distinct subsets (eg, MTAP loss in nonepithelioid tumors). Given the limited efficacy of current therapies for this disease, novel approaches targeting recurring alterations should be explored.

Loss of COP9 signalosome genes at 2q37 is associated with IMiD resistance in multiple myeloma

AbstractThe acquisition of a multidrug refractory state is a major cause of mortality in myeloma. Myeloma drugs that target the cereblon (CRBN) protein include widely used immunomodulatory drugs (IMiDs), and newer CRBN E3 ligase modulator drugs (CELMoDs), in clinical trials. CRBN genetic disruption causes resistance and poor outcomes with IMiDs. Here, we investigate alternative genomic associations of IMiD resistance, using large whole-genome sequencing patient datasets (n = 522 cases) at newly diagnosed, lenalidomide (LEN)-refractory and lenalidomide-then-pomalidomide (LEN-then-POM)-refractory timepoints. Selecting gene targets reproducibly identified by published CRISPR/shRNA IMiD resistance screens, we found little evidence of genetic disruption by mutation associated with IMiD resistance. However, we identified a chromosome region, 2q37, containing COP9 signalosome members COPS7B and COPS8, copy loss of which significantly enriches between newly diagnosed (incidence 5.5%), LEN-refractory (10.0%), and LEN-then-POM-refractory states (16.4%), and may adversely affect outcomes when clonal fraction is high. In a separate dataset (50 patients) with sequential samples taken throughout treatment, we identified acquisition of 2q37 loss in 16% cases with IMiD exposure, but none in cases without IMiD exposure. The COP9 signalosome is essential for maintenance of the CUL4-DDB1-CRBN E3 ubiquitin ligase. This region may represent a novel marker of IMiD resistance with clinical utility.

Pediatric-Type Indolent B-Cell Lymphomas With Overlapping Clinical, Pathologic, and Genetic Features.

Pediatric-type follicular lymphoma (PTFL) and pediatric nodal marginal zone lymphoma (PNMZL) are rare pediatric-type indolent B-cell lymphomas (PedIBCL) that differ clinicopathologically from their adult counterparts. Accurate diagnosis is important to avoid overtreatment but is often challenging. The mutational landscape of PTFL is known and may aid diagnosis, but the genetic features of PNMZL are not well understood. We analyzed 21 cases of PedIBCL according to their clinicopathologic findings and classified them into PTFL (n=11), PNMZL (n=2), and “mixed type” tumors (n=8) showing ambiguous histology. We also analyzed 2 cases of adult B-cell lymphomas showing features of PedIBCL. Targeted sequencing of 121 lymphoma-related genes was performed. The median age of PedIBCL patients was 16 years (range: 3 to 47), and all but 1 PTFL patient were male. All patients presented with limited-stage disease, and only 1 relapsed. There were no significant differences in clinical features among the 3 PedIBCL groups. The most frequently mutated genes were MAP2K1, TNFRSF14, KMT2C, IRF8, and NOTCH2. The genetic features of all groups were similar to the established mutational landscape of PTFL. The 2 adult B-cell lymphomas cases also had MAP2K1, TNFRSF14, and IRF8 mutations, but the clinical features were not typical of PedIBCL. In summary, this study demonstrated that PTFL and PNMZL are similar diseases with overlapping clinical, pathologic, and genetic features; mixed type tumors can also occur. Atypical adult cases with similar histologic features were also observed. Therefore, the disease spectrum of PedIBCL may be much broader than is currently believed.

P-547 Confined placental mosaicism for inherited segmental aneuploidies in miscarriages traces early karyotype self-correction events

Abstract Study question Does tissue-specific compartmentalization of segmental aneuploidies in first-trimester miscarriages reflect their postzygotic de novo origin or correction of inherited copy number variants (CNV)? Summary answer Most of mosaic CNVs (84%) in spontaneous abortions with normal karyotype arose de novo, but 16% resulted from postzygotic tissue-specific correction of maternally inherited variants. What is known already Chromosomal mosaicism is a hallmark of early human embryogenesis affecting significantly developmental potential of blastocysts, it’s implantation and delivery rates. Most of the currently available data are related to mosaic aneuploidies for a whole chromosome in IVF cycles, whereas little is known about origin and fate of mosaic segmental CNVs due to inherent limitations of NGS and aCGH for detection of copy number changes less for 5-10 Mb in size after single-cell whole genome amplification. Study design, size, duration Chorionic villi (CV) and extraembryonic mesoderm (EM) of miscarriages as well as maternal and paternal peripheral blood DNA samples were evaluated by aCGH+SNP arrays to detect the prevalence and tissue-specific distribution of segmental aneuploidies. As far as CV and EM are derivatives of trophectoderm and inner cell mass lineages, comparative analysis of both tissues allows to investigate the origin of mosaic CNVs by postzygotic de novo appearance or correction of the inherited variants. Participants/materials, setting, methods Eleven trios (mother, father, CV and EM samples from spontaneous abortion with normal karyotype by G-banding) were included in the study. Mean gestational age was 7.9 weeks. Mean maternal and paternal ages were 30.8 and 30.5 years, respectively. aCGH+SNP analysis was performed by SurePrint G3 Human CGH + SNP 4 × 180K microarrays (Agilent Technologies) with 13 kb median aCGH probes spacing. Main results and the role of chance Altogether 116 CNVs were detected in CV and EM samples from 11 abortions. Fifty four (46.6%) variants were present in both tissues, while 29 (25%) were confined by CV and 33 (28.4%) by EM. Most of tissue-specific segmental aneuploidies in CV (90%) and EM (79%) arose de novo, whereas others were inherited from parents. It is important that 5 maternally inherited microduplications in some clinically significant regions like 1p21.1 (103 kb in size), 2p21 (3.56 kb), 3p22.3 (128 kb), 7q36.3 (42.7 kb), and Xq28 (182 kb) were confined by EM, whereas two at 4p16.3 (22.3 kb) and 19q13.43 (2.2 kb) by CV. No cases of tissue-specific maternally inherited microdeletions or paternal CNVs were observed in our study. One can suggest that confined placental mosaicism for inherited maternal microduplications arisen by trisomy rescue mechanism with loss of additional maternal chromosome copy in CV or EM after its divergence around the time of blastocyst implantation. This hypothesis is confirmed by comparative SNP-haplotype analysis of DNA samples from both CV and EM tissues as well as parental samples in the subchromosomal regions of microduplications and adjusted loci. Limitations, reasons for caution Control group of induced (social) abortions of the same developmental terms are required to estimate the prevalence and distribution of mosaic segmental aneuploidies in embryonic and extraembrionic tissues during normal human embryo development. Wider implications of the findings Tracing of tissue-specific mosaic segmental aneuploidies in first-trimester miscarriages allows to estimate the level of karyotype instability and self-correction during human embryo development. This study was supported by the Russian Science Foundation, № 21-65-00017, https://rscf.ru/project/21-65-00017/ Trial registration number not applicable

Impact of STK11 copy loss on clinical outcomes to PD-(L)1 blockade in non–small cell lung cancer.

9059 Background: STK11 is among the most commonly altered genes in non-squamous lung cancers. While STK11 mutation is associated with diminished efficacy of immune checkpoint inhibition (ICI), particularly in KRAS mutated tumors, it is not known whether STK11 copy deletion influences outcomes to ICI. Methods: Patients with advanced non-squamous non-small cell lung cancer (NSCLC) treated with ICI whose tumors underwent genomic profiling were included. Clinical outcomes to ICI were analyzed according to KRAS mutation and STK11 deletions. STK11 copy number variations (CNVs) were determined using an internal informatic pipeline. Kaplan-Meier methodology was used to estimate event-time distributions. Results: Of 559 patients with non-squamous NSCLCs (Nsq-NSCLC), 40.4% (N = 226) had a KRAS mutation ( KRASmut), 18.4% (N = 103) had an oncogenic STK11 mutation ( STK11mut), and 22.5% had either single (N = 123), or bi-allelic (N = 3) deletion ( STK11del). Given that 32.5% of STK11del cases had a concurrent oncogenic STK11 mutation in our cohort, to isolate the impact of STK11del on ICI outcomes we excluded samples with STK11mut from this analysis. In all comers with NSCLC, STK11del had no impact on objective response rate (22.2% versus 23.8%, P = 0.8), progression-free (PFS, HR 0.90, P = 0.30), and overall survival (OS, HR 0.96, P = 0.79) to ICI. When we examined the impact of STK11del on clinical outcomes to PD-(L)1 blockade among KRASmut cases we found that STK11del was associated with a numerically lower ORR (13.3% versus 30.0%, P = 0.12), and a significantly shorter PFS (HR 0.57, P = 0.018) compared to cases without STK11del. No difference in OS were observed between these groups (HR 0.77, P = 0.39). Among KRASwt NSCLCs, STK11del cases had a similar ORR (22.6% versus 22.9%, P = 0.99), PFS (HR 0.92, P = 0.63), and OS (HR 1.18, P = 0.32) to PD-(L)1 inhibition compared to cases without STK11del. Among KRASmut but not KRASwt NSCLCs, cases with STK11del had significantly lower PD-L1 expression compared to those without STK11 deletions (27.5% versus 70%, P = 0.01). Conclusions: STK11 deletion is associated with low response rate and short progression-free survival among KRAS mutant NSCLCs. Future analyses will incorporate additional cases to increase sample size and immunopathologic findings to assess impact of mono and bi-allelic deletion on protein expression.

Genomic correlates of acquired resistance to PD-(L)1 blockade in patients with advanced non-small cell lung cancer (NSCLC).

9021 Background: Despite improvements in survival with immune checkpoint inhibition (ICI), the majority of patients develop acquired resistance to ICI after an initial benefit. However, the mechanisms underlying acquired resistance to ICI in NSCLC are largely unknown. Methods: Patients with advanced NSCLC treated with ICI at the Dana-Farber Cancer Institute (DFCI), and whose tumors underwent genomic profiling before and after ICI, with no intervening therapies, were included. Mutations, tumor mutational burden (TMB), copy number variations (CNVs), and PD-L1 tumor proportion score (TPS) were compared between pre- and post-ICI samples. Acquired resistance was defined as the development of disease progression after an initial objective response, or stable disease ≥3 months with PD-(L)1 blockade. Results: Among 1763 patients with advanced NSCLC who received ICI, 45 had matched pre- and post-ICI tissue samples available for genomic profiling. Putative mechanisms of resistance were identified in 55% of cases (N = 25). Five patients (20%) acquired an STK11 mutation, one patient (4%) acquired a KEAP1 mutation, and another patient (4%) developed concurrent KEAP1 and SMARCA4 mutations. A patient (4%) with KRAS G12C-mutant NSCLC developed concurrent STK11 and KEAP1 mutations at resistance. In 3 cases (12%) with pre-existing STK11 or KEAP1 mutations prior to ICI administration, we identified acquired copy losses of STK11 and KEAP1, respectively, resulting in bi-allelic inactivation of these genes. Acquired beta-2-microglobulin ( B2M) mutations were detected in 3 patients (12%), one of whom developed concurrent B2M copy loss, indicating bi-allelic inactivation. Eight additional patients (32%) developed B2M gene deletions. Other acquired alterations that have been implicated in ICI resistance included CDKN2A/B loss (N = 10, 40%), including 5 with bi-allelic deletion, acquired PTEN deletions (N = 5, 20%), and MDM2 amplification (N = 2, 8%). When we examined alterations in immune checkpoint genes, we identified acquired CD274 (PD-L1) and PDCD1LG2 (PD-L2) loss in 8% of cases (N = 2), and bi-allelic deletion in one case (4%). Intervening ICI did not affect TMB (median TMB: 8.7 [pre-ICI] vs 9.1 [post-ICI] mut/Mb, P = 0.6), PD-L1 expression (median PD-L1 TPS: 3% [pre-ICI] vs 5.0% [post-ICI] mut/Mb, P = 0.5), or aneuploidy levels (as fraction of genome altered [FGA]) (median FGA: 18.4% [pre-ICI] vs 21.1% [post-ICI], P = 0.2), indicating that acquired gene level CNVs were not a reflection of increased cancer aneuploidy. In a control cohort of 30 patients with pre- and post-chemotherapy matched samples which underwent genomic profiling, no acquired mutations in STK11, KEAP1, SMARCA4, or B2M were detected. Conclusions: Mechanisms of acquired resistance to PD-(L)1 blockade are heterogenous, and new therapeutic strategies are required to delay and overcome ICI resistance in patients with NSCLC.

Improved genome assembly of Chinese sucker (Myxocyprinus asiaticus) provides insights into the identification and characterization of pharyngeal teeth related maker genes in Cyprinoidei

The Chinese sucker, Myxocyprinus asiaticus (M. asiaticus, Catostomidae, Cypriniformes), is the only living species of Catostomidae in Asia. There are more than 75 species of this family in North America. The fossil record of this group dates back to the early Eocene. As the Chinese sucker is located at the base of the Cyprinoidei phylogeny, this species is also important in clarifying the evolutionary relationships within Cyprinoidei. Here, we assembled a high-quality genome of the Chinese sucker, contig N50 (40.26 ​Mb), which is nearly ten times longer than the previous version (4.19 ​Mb). Phylogenetic analysis identified that Chinese sucker together with Cyprinidae groups are paraphyletic with respect to Cobitoidea. The specific whole genome duplication event of the Chinese sucker was estimated to have occurred ∼25.9 million years ago. Analysis of population historical changes indicated a trend of reduction for the Chinese sucker and T. tibetana. Since Dlx genes play a key role in Cypriniformes pharyngeal teeth development, we conducted a genome-wide identification of Dlx genes, and found that these genes were doubled in whole genome duplication events, followed by the loss of specific copies. Transcriptome results showed that the expression levels of these paralogous genes were similar. This genomic resource provides useful information for the protection of Chinese sucker and functional study of Dlx genes.

Molecular analysis of adenocarcinomas of the rete testis demonstrates frequent alterations in genes involved in cell cycle regulation.

Adenocarcinomas of the rete testis (ACRT) are rare and aggressive testicular neoplasms that present predominantly in older men and have a tendency for early systemic spread. Their morphology spans a wide spectrum, including tumors with glandular, solid, papillary, micropapillary, glomeruloid, cribriform, and sarcomatoid growth patterns, or a combination thereof. The genomic alterations associated with these tumors have not been studied previously. We assessed eight ACRT published in prior clinicopathologic series using a solid tumor DNA sequencing panel. Pathogenic variants were identified in 6/8 cases. More specifically, four cases demonstrated inactivation of genes involved in cell cycle regulation, including CDKN2A, BAP1, TP53, and RB1. CDKN2A was the only recurrently affected gene, with pathogenic variants detected in 3/8 cases. One of these three cases had molecular evidence of concurrent homozygous (i.e. biallelic) NF2 inactivation by a frameshift variant and loss of the wildtype copy of the gene. One case had an internal tandem duplication in AKT, which has been previously described in juvenile granulosa cell tumor and sclerosing pneumocytoma and results in downstream activation of PI3K signaling. The remaining case with positive molecular findings harbored two concurrent truncating SETD2 variants. Multiple arm-level and chromosome-level copy number events were present in 3/8 cases, all of which harbored variants in genes involved in cell cycle regulation. In summary, ACRT are rare tumors with frequent inactivation of genes that play a major role cell cycle regulation, and a subset harbors variants that are potentially amenable to targeted therapy.

Molecular assessment of testicular adult granulosa cell tumor demonstrates significant differences when compared to ovarian counterparts

Testicular adult granulosa cell tumor (AGCT) is a rare type of sex-cord stromal tumor that affects patients of a wide age range and has the potential for late metastasis. In the testis, the diagnosis of AGCTs often requires the exclusion of other more common types of sex-cord stromal tumors. Immunohistochemistry is of limited utility, being used mostly to confirm sex-cord lineage and to exclude other entities when morphology is not typical. Unlike ovarian AGCTs, which are molecularly homogeneous and harbor a specific activating FOXL2 mutation (c.7558C > T p.C134W) in >90% of cases, the molecular characteristics of testicular AGCTs remain largely unknown. In the current study, we analyzed 13 testicular AGCTs diagnosed at multiple institutions using massively parallel DNA sequencing to evaluate single nucleotide variants, copy number alterations, and structural variants. In all, 10/13 cases were sequenced successfully. Notably, the FOXL2 c.7558C > T (p.C134W) mutation was identified in only a single case (1/10, 10%). The remaining cases were molecularly heterogeneous, with largely nonrecurrent genetic variants. Putative driver events in individual cases included a well-characterized gain-of-function NRAS mutation, as well as inactivation of ATM and TP53, among others. The only highly recurrent finding was single copy loss of 22q (7/10 cases, 70%). Comparatively, the frequencies of FOXL2 c.7558C > T (p.C134W) and 22q loss in 12 metastatic ovarian AGCTs identified in our database were 92% (11/12) and 42% (5/12), respectively. The results of the present study suggest that testicular AGCTs are different from their ovarian counterparts in that they appear to be molecularly heterogeneous and only rarely harbor FOXL2 mutations.

A Validation Framework for Somatic Copy Number Detection in Targeted Sequencing Panels

Somatic copy number alterations (SCNAs) in tumors are clinically significant diagnostic, prognostic, and predictive biomarkers. SCNA detection from targeted next-generation sequencing panels is increasingly common in clinical practice; however, detailed descriptions of optimization and validation of SCNA pipelines for small targeted panels are limited. This study describes the validation and implementation of a tumor-only SCNA pipeline using CNVkit, augmented with custom modules and optimized for clinical implementation by testing reference materials and clinical tumor samples with different classes of copy number variation (CNV; amplification, single copy loss, and biallelic loss). Using wet-bench and in silico methods, various parameters impacting CNV calling, including assay-intrinsic variables (establishment of normal reference and sequencing coverage), sample-intrinsic variables (tumor purity and sample quality), and CNV algorithm-intrinsic variables (bin size), were optimized. The pipeline was trained and tested on an optimization cohort and validated using an independent cohort with a sensitivity and specificity of 100% and 93%, respectively. Using custom modules, intragenic CNVs with breakpoints within tumor suppressor genes were uncovered. Using the validated pipeline, re-analysis of 28 pediatric solid tumors that had been previously profiled for mutations identified SCNAs in 86% (24/28) samples, with 46% (13/28) samples harboring findings of potential clinical relevance. Our report highlights the importance of rigorous establishment of performance characteristics of SCNA pipelines and presents a detailed validation framework for optimal SCNA detection in targeted sequencing panels.