Chromosome Arms 19q Research Articles

Amplification of MYC and Its Enhancer Correlates With Genetic Ancestry in Lung Squamous Cell Carcinoma.

In lung squamous cell carcinoma (LUSC), Black patients show significantly higher incidence and lower overall survival than White patients. Although socioeconomic factors likely contribute to this survival disparity, genomic factors have yet to be elucidated in LUSC. Using 416 LUSC tumor samples in the Cancer Genome Atlas (TCGA), we assessed genomic and transcriptomic profiles by ancestry. We replicated our analyses in pan-cancer data from TCGA, the American Association of Cancer Research (AACR) Genomics Evidence Neoplasia Information Exchange (GENIE), and Columbia University Medical Center. We found increased MYC amplification, LUSC-specific MYC enhancer amplification, and chromosome arm 8q (chr8q) gain to be significantly associated with genetic AFR (African) ancestry in LUSC in TCGA. Furthermore, expression of MYC target genes was significantly enriched in AFR samples. Local ancestry analysis identified correlation of chr8q gain with AFR ancestry at the MYC locus in TCGA. We also found a significant correlation between chr8q and AFR ancestry in multiple cancer types and pan-cancer in TCGA. Similarly, in a pan-cancer subset of AACR GENIE data, we found a significant correlation between chr8q gain and race. Together, our data suggest that ancestry may influence amplification of not only MYC but also its enhancer in LUSC. They also suggest a role for genetic ancestry in chr8q aneuploidy in cancer. These studies further define and expand patients who may benefit from future anti-MYC therapeutic approaches.

Integrative genomics identifies SHPRH as a tumor suppressor gene in lung adenocarcinoma that regulates DNA damage response.

Identification of driver mutations and development of targeted therapies has considerably improved outcomes for lung cancer patients. However, significant limitations remain with the lack of identified drivers in a large subset of patients. Here, we aimed to assess the genomic landscape of lung adenocarcinomas (LUADs) from individuals without a history of tobacco use to reveal new genetic drivers of lung cancer. Integrative genomic analyses combining whole-exome sequencing, copy number, and mutational information for 83 LUAD tumors was performed and validated using external datasets to identify genetic variants with a predicted functional consequence and assess association with clinical outcomes. LUAD cell lines with alteration of identified candidates were used to functionally characterize tumor suppressive potential using a conditional expression system both in vitro and in vivo. We identified 21 genes with evidence of positive selection, including 12 novel candidates that have yet to be characterized in LUAD. In particular, SNF2 Histone Linker PHD RING Helicase (SHPRH) was identified due to its frequency of biallelic disruption and location within the familial susceptibility locus on chromosome arm 6q. We found that low SHPRH mRNA expression is associated with poor survival outcomes in LUAD patients. Furthermore, we showed that re-expression of SHPRH in LUAD cell lines with inactivating alterations for SHPRH reduces their in vitro colony formation and tumor burden in vivo. Finally, we explored the biological pathways associated SHPRH inactivation and found an association with the tolerance of LUAD cells to DNA damage. These data suggest that SHPRH is a tumor suppressor gene in LUAD, whereby its expression is associated with more favorable patient outcomes, reduced tumor and mutational burden, and may serve as a predictor of response to DNA damage. Thus, further exploration into the role of SHPRH in LUAD development may make it a valuable biomarker for predicting LUAD risk and prognosis.

OTHR-26. MINIMALLY-INVASIVE MOLECULAR DIAGNOSIS OF DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMOR (DLGNT) USING CEREBROSPINAL FLUID CELL-FREE DNA SEQUENCING IN PEDIATRIC AND YOUNG ADULT PATIENTS

Abstract BACKGROUND Diffuse leptomeningeal glioneuronal tumor (DLGNT) presents with nodular leptomeningeal disease throughout the neuroaxis and is characterized by the presence of KIAA1549::BRAF fusion and chromosome arm 1p deletion. Concomitant gain of chromosome arm 1q is associated with poor prognosis. Diagnosis by meningeal biopsy is often non-diagnostic due to sampling error or yields insufficient material for molecular testing. We hypothesized that analysis of cell free DNA (cfDNA) from cerebrospinal fluid (CSF) could represent a superior diagnostic modality. METHODS We analyzed CSF samples from 9 suspected DLGNT patients, median age of 19.5 years with a 5:4 male:female ratio using MSK-IMPACT, a hybridization capture-based next-generation DNA sequencing panel, including matched germline. RESULTS Prior tissue biopsy was done in 7 patients; 6 were negative for the KIAA1549:BRAF fusion and 1 was positive. Of the 6 biopsies negative for the fusion, analysis of CSF cfDNA detected KIAA1549::BRAF fusion in 3/6 (50%) of patients, who were all subsequently started on MEK inhibitor therapy. The other 3 patients were negative for KIAA1549:BRAF fusion on both biopsy and CSF cfDNA. One patient received biopsy confirmation of KIAA1549::BRAF fusion after CSF cfDNA analysis detected the mutation. In addition, in one patient with prior tissue-confirmed KIAA1549::BRAF fusion, the fusion was not detected in CSF cfDNA during/after treatment with a MEK inhibitor, but additional somatic alterations and DNA copy number profiles were similar to those found in prior biopsies. Additional somatic alterations were identified in ATRX, BCOR, RARA, STAT5A, SPOP, PPM1D, MED12, KMT2C, RECQL andSETD2, including likely oncogenic secondary driver alterations that have not been previously reported in DLGNT. CONCLUSIONS Given the high molecular diagnostic yield and concordance with open biopsy, cfDNA CSF sequencing should be considered as a first line diagnostic maneuver for patients with suspected DLGNT, followed by surgical biopsy only if CSF testing is non-diagnostic.

EPEN-27. GAINING A CLUE FROM 1Q: A HIGH-RISK CHROMOSOMAL GAIN IN PEDIATRIC POSTERIOR FOSSA EPENDYMOMA

Abstract BACKGROUND Ependymomas are neuroepithelial tumours that can be classified into distinct molecular subgroups, each exhibiting a unique clinical outcome. Therapeutic efforts against the most aggressive and abundant subgroup of pediatric ependymoma (posterior fossa group A, PFA) have traditionally been hampered by a lack of known recurrent driver mutations. However, approximately 25% of PFA tumours exhibit gains of the chromosome arm 1q which is associated with an extremely poor prognosis despite aggressive treatment. By interrogating the multi-omic landscape of PFAs harbouring this copy number variation, we aimed to uncover genetic dependencies from which actionable therapeutics could be derived. METHODS Transcriptomic (bulk/single-cell RNA sequencing), proteomic (LC-MS/MS) and phosphoproteomic analyses were conducted on patient tissues and primary patient-derived cell lines to gain insight into the pathways that drive 1q gain pathogenesis. Chromatin immunoprecipitation sequencing of H3K27me3 and H3K27ac marked histones was carried out to profile the epigenetic landscape of these tumours, and whole genome CRISPR knockout screens were performed to reveal the contribution of essential genes unique to tumours harboring 1q gains. RESULTS Through the comparison of 1q gain and balanced PFA samples, our multi-omic analyses identified the up-regulation and essentiality of known glioma oncogenic drivers previously uncharacterized in PFA. The investigation of essential candidate genes showed a convergence on pathways related to ciliogenesis, with genetic vulnerabilities informing in vitro drug screening. Candidate genes were further validated by in vivo knockdown using established xenograft mouse models for pre-clinical testing. CONCLUSIONS This combined approach has allowed for the derivation of a functional cancer signature underlying 1q gains in PFA and presents novel therapeutic targets for this high-risk subgroup. PFA ependymoma is a deadly malignancy whose complex biology has so far eluded therapy, and new insights from this project will help to identify the first effective therapies for this vulnerable patient population.

Влияние хронического облучения на показатели цитогенетических маркеров старения у жителей прибрежных сел реки Теча

The understanding of the exposure effects on the human health could be improved by analyzing the influence of the chronic low dose rate exposure on the senescence of the immune system cells. It will also help to develop the measures aimed at the mitigation of the adverse effects. The objective of the study is to investigate the influence of the chronic low dose rate exposure on the senescence of the immune system cells using the cytogenetic markers. In the course of the research the authors evaluated the cellular senescence markers — genome instability and telomere depletion — in T-lymphocytes of the individuals exposed in the Southern Urals (exposure doses were 0.001 Gy — 4.7 Gy, the age of examined people was 40–89 years). The data analysis has demonstrated that the effect of chronic exposure on the T-cell senescence was indirect. Unstable chromosome aberrations occurred statistically significantly more frequently in exposed people aged 40–59 years (p = 0.012). Frequency of lymphocytes with micronuclei in exposed individuals differed in men and women (p = 0.001). Statistically significant decrease in the telomere length was revealed (for the chromosome arms 1q, 3p, 3q, 20p, 20q, 13q, 15p, 22q (p < 0.05); 19p, 21q (p < 0.01)).

GLI1 Coamplification in Well-Differentiated/Dedifferentiated Liposarcomas: Clinicopathologic and Molecular Analysis of 92 Cases

GLI1(12q13.3) amplification is identified in a subset of mesenchymal neoplasms with a distinct nested round cell/epithelioid phenotype. MDM2 and CDK4 genes are situated along the oncogenic 12q13-15 segment, amplification of which defines well-differentiated liposarcoma (WDLPS)/dedifferentiated liposarcoma (DDLPS). The 12q amplicon can occasionally include GLI1, a gene in close proximity to CDK4. We hereby describe the first cohort of GLI1/MDM2/CDK4 coamplified WD/DDLPS. The departmental database was queried retrospectively for all cases of WD/DDLPS having undergone next-generation (MSK-IMPACT) sequencing with confirmed MDM2, CDK4, and GLI1 coamplification. Clinicopathologic data was obtained from a review of the medical chart and available histologic material. Four hundred eighty-six WD/DDLPS cases underwent DNA sequencing, 92 (19%) of which harbored amplification of the GLI1 locus in addition to that of MDM2 and CDK4. These included primary tumors (n = 60), local recurrences (n = 29), and metastases (n = 3). Primary tumors were most frequently retroperitoneal (47/60, 78%), mediastinal (4/60, 7%), and paratesticular (3/60, 5%). Average age was 63 years, with a male:female ratio of 3:2. The cohort was comprised of DDLPS (86/92 [93%], 6 of which were WDLPS with early dedifferentiation) and WDLPS without any longitudinal evidence of dedifferentiation (6/92, 7%). One-fifth (13/86, 17%) of DDLPS cases showed no evidence of a well-differentiated component in any of the primary, recurrent, or metastatic specimens. Dedifferentiated areas mostly showed high-grade undifferentiated pleomorphic sarcoma-like (26/86,30%) and high-grade myxofibrosarcoma-like (13/86,16%) morphologies. A disproportionately increased incidence of meningothelial whorls with/without osseous metaplasia was observed as the predominant pattern in 16/86 (19%) cases, and GLI1-altered morphology as described was identified in a total of 10/86 (12%) tumors. JUN (1p32.1), also implicated in the pathogenesis of WD/DDLPS, was coamplified with all 3 of MDM2, CDK4, and GLI1 in 7/91 (8%) cases. Additional loci along chromosomal arms 1p and 6q, including TNFAIP3, LATS1, and ESR1, were also amplified in a subset of cases. In this large-scale cohort of GLI1 coamplified WD/DDLPS, we elucidate uniquely recurrent features including meningothelial whorl-like and GLI-altered morphology in dedifferentiated areas. Assessment of tumor location (retroperitoneal or mediastinal), identification of a well-differentiated liposarcoma component, and coamplification of other spatially discrete genomic segments (1p and 6q) might aid in distinction from tumors with true driver GLI1 alterations.

Abstract 409: Deciphering chromosomal instability in consensus molecular subtypes (CMS) in CRC: Insights from an integrative multi-omics approach

Abstract CRC is a heterogeneous disease with different molecular subtypes, which can have a profound impact on treatment response and patient outcomes. CMS classification based on gene expression profiles has been shown to differentiate patients into clinically relevant sub-groups. However, deep multi-omics characterization of the CMS classes based on high quality patient data is lacking. Through the integration of multi-omics data, we aim to enhance our understanding of CRC’s molecular heterogeneity and thereby facilitate the development of personalized therapeutic strategies. We performed a multi-omics analysis of CRC patients to identify potential new features of CMS groups. 890 fresh-frozen, surgically resected CRC tumor and adjacent normal samples were used to analyze whole genome and RNA sequencing data. DNA and RNA was prepared using Qiagen AllPrep Universal and KAPA Hyper Prep kit (DNA), as well as TruSeq Stranded Total mRNA kits and sequenced on a NovaSeq6000 system. We identified CMS subtypes based on RNA-Seq data and performed all statistical analyses using R. Our analysis revealed distinct molecular characteristics across the four CMS subtypes, including unique driver mutations, signaling cascades, and immune cell infiltration profiles. The poor prognosis associated with CMS4 patients was corroborated by our clinical data. Notably, we observed high global chromosomal instability (CIN) profiles for both CMS2 and CMS4 and identified recurrent global aneuploidy patterns within these subtypes that link alterations of known cancer genes to these large-scale structural events. We found higher levels of CIN in CMS2 compared to CMS4, which appears to be predominantly driven by numerical, rather than structural CIN. Specifically, in CMS2, SMAD2/4 is commonly impacted by deletion on chromosome arm 18q, while PLCG1 is more influenced by amplifications on 20q respectively, suggesting potential values as CRC CMS biomarkers. In conclusion, our integrative multi-omics analysis offers a comprehensive understanding of CRC molecular subtypes, which can inform personalized treatment strategies. Our findings underscore the importance of considering not only specific driver mutations but also large-scale structural rearrangements in the context of CMS subtypes. Citation Format: Julia Bischof, Jonathan Woodsmith, David Church. Deciphering chromosomal instability in consensus molecular subtypes (CMS) in CRC: Insights from an integrative multi-omics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 409.

Abstract C049: Genomic correlates with ancestry in lung squamous cell carcinoma

Abstract Outcome data from lung squamous cell carcinoma (SCC) show significantly lower overall and progression-free survival in Black patients. However, whether any genomic factors correlate with race and ancestry in this lung SCC, and whether this may contribute to disparities, has not been elucidated. We analyzed clinically annotated genomic data from 501 lung SCC tumor samples in the Cancer Genome Atlas (TCGA), with available ancestry admixture data. Here, we analyzed clinical data, gene expression profiles, mutations, and copy number changes to identify significantly differential events between tumors from patients of European (EUR) or African (AFR) ancestry. In tumors from AFR patients, we identified more frequent gain of chromosome arm 8q and/or the chr8q24 locus, where the oncogene MYC and its enhancers are located. 8q24 gain is associated both with global AFR ancestry and local AFR ancestry. Furthermore, expression of MYC targets is significantly increased in AFR samples relative to EUR samples, all of which suggest a role for the MYC pathway in differential cancer progression. MYC activity correlates with decreased immune infiltration and gene signaling, and patients with AFR ancestry are more likely to have decreased immune infiltrate and pathway activity. Our findings are consistent with observations in prostate cancer; this is the first study to find MYC enhancer amplification correlating with African ancestry in lung SCC. We are currently testing additional cohorts, including AACR GENIE, to validate these findings. Future studies will elucidate patient-specific treatment modalities. Citation Format: Sejal Jain, Alexandria Yao, Fatemeh Momen-Heravi, Diane Allen-Gipson, Xiaoyang Zhang, Brian S. Henick, Jian Carrot-Zhang, Alison M. Taylor. Genomic correlates with ancestry in lung squamous cell carcinoma [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C049.

Multiplex Detection of Copy Number Alterations in Multiple Myeloma Including Emerging Therapeutic Targets By Digitalmlpa

Recent advances in immunotherapy have highlighted that in multiple myeloma (MM), besides risk-determining copy number alterations (CNAs) there is a need for investigation of CNAs in CAR-T cell therapy-related targets (e.g. BCMA, GPRC5D, FcRH5). DigitalMLPA technology has been successfully used for characterization of recurrent CNAs on chromosome arms 1p, 1q, 13q and 17p in newly diagnosed and relapsed MM cases, as well as in cancer cell lines of MM origin. Adding probes aimed at CAR-T cell therapy-related target genes complements the already included predictive target probes ( IKZF1/3, IRF4, MYC, RPL5, SLAMF7 and BRAF V600E point mutation) in SALSA® digitalMLPA™ Probemix D006 Multiple Myeloma. In addition, due to a large overlap of affected genomic regions between hematologic neoplasms, we aimed to demonstrate usefulness of this multiplex assay not only on cell lines of MM origin, but also of myeloid and lymphoid lineages. Methods. D006 Multiple Myeloma digitalMLPA probemix contains (i) probes targeting chromosomal arms with recurrent CNAs, including 1p (33 probes), 1q (29 probes), 13q (23 probes), 17p (20 probes); (ii) probes for promising predictive targets such as BCMA, CRBN, GPRC5D, FcRH5, IKZF1/3, IRF4, MYC, RPL5, SLAMF7 - 2-4 probes per gene, and BRAF V600E mutation-specific probe; (iii) 278 probes for subtelomeric, pericentromeric and middle regions of chromosomal arms for all chromosomes; and (iv) control probes for quality control and troubleshooting. DigitalMLPA reactions were performed with 20-40 ng DNA from 29 cancer cell lines of lymphoid and myeloid lineage, 16 of which were of MM origin. Commercial blood-derived genomic DNA samples from healthy individuals were used as references for data normalization using data analysis software Coffalyser digitalMLPA™. Results. Losses of 1p were among the most frequent CNAs in MM cell lines (n=13) and were mainly local (1p21.1-p22.1), including homozygous deletion of CDKN2C in MM cell lines co-occurring with TENT5C subclonal loss in 2 of those. 13q loss (encompassing predominantly the whole arm) was detected in 13 MM cell lines. 1q and 17p losses were present in 12 MM cell lines. One MM cell line showed loss of BCMA (LOPRA-1) and one of CRBN (EJM), 6 had loss of GPRC5D, 9 had MYC gain. None of the MM cell lines carried the BRAF V600E mutation. In the EOL-1 myeloid lineage cell line, trisomy of chromosomes 4, 6, 8 and 19 were correctly identified by increased ratios for all probes covering these chromosomes: 24, 27, 20 and 11 probes respectively; and in the HG-3 lymphoid lineage cell line 11q25 gain and 13q loss was detected. Examples of CNAs detected by D006 Multiple Myeloma probemix are described in Table 1. Copy number ratios of predictive target genes in MM cell lines are shown in Table 2. Ratios between 0.7-1.3 (green cells) indicate normal copy number, ratios below 0.7-1.3 range indicate copy number loss (red cells) and higher ratios indicate gains (blue cells). CNAs detected by digitalMLPA were highly concordant with those reported in public databases. Conclusions. digitalMLPA technology is well suited for multiplex CNA detection of routinely analyzed genomic regions in MM and provides a unique opportunity to research molecular genetic markers of emerging significance in MM in the same reaction. In addition, this MM assay also gives the possibility to analyze gross CNAs in other hematopoietic neoplasms with overlapping affected regions. Input DNA requirements that are a fraction of current WGS, simple protocol and possibility of combining of digitalMLPA libraries with other NGS libraries make D006 Multiple Myeloma digitalMLPA probemix a reliable, cost-effective and robust method to detect well-established and emerging CNAs.

Acute megakaryoblastic leukaemia shows high frequency of chromosome 1q aberrations and dismal outcome.

Acute megakaryoblastic leukaemia (AMKL) is associated with poor prognosis. Limited information is available on its cytogenetics, molecular genetics and clinical outcome. We performed genetic analyses, evaluated prognostic factors and the value of allogeneic haematopoietic stem cell transplantation (allo-HSCT) in a homogenous adult AMKL patient cohort. We retrospectively analysed 38 adult patients with AMKL (median age: 58 years, range: 21-80). Most received intensive treatment in AML Cooperative Group (AMLCG) trials between 2001 and 2016. Cytogenetic data showed an accumulation of adverse risk markers according to ELN 2017 and an unexpected high frequency of structural aberrations on chromosome arm 1q (33%). Most frequently, mutations occurred in TET2 (23%), TP53 (23%), JAK2 (19%), PTPN11 (19%) and RUNX1 (15%). Complete remission rate in 33 patients receiving intensive chemotherapy was 33% and median overall survival (OS) was 33 weeks (95% CI: 21-45). Patients undergoing allo-HSCT (n = 14) had a superior median OS (68 weeks; 95% CI: 11-126) and relapse-free survival (RFS) of 27 weeks (95% CI: 4-50), although cumulative incidence of relapse after allo-HSCT was high (62%). The prognosis of AMKL is determined by adverse genetic risk factors and therapy resistance. So far allo-HSCT is the only potentially curative treatment option in this dismal AML subgroup.

METB-07. MINIMALLY-INVASIVE DIAGNOSIS OF DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMOR (DLGNT) USING CEREBROSPINAL FLUID CELL-FREE DNA SEQUENCING IN PEDIATRIC AND YOUNG ADULT PATIENTS

Abstract Diffuse leptomeningeal glioneuronal tumor (DLGNT) presents with nodular leptomeningeal disease throughout the neuroaxis. Histology shows variable presence of a low density intraparenchymal lesion containing OLIG2-expressing neoplastic cells. Molecularly, DLGNT is characterized by the presence of KIAA1549::BRAF fusion and chromosome arm 1p deletion. Concomitant gain of chromosome arm 1q is associated with poor prognosis. Diagnosis by meningeal biopsy is often non-diagnostic and we hypothesized analysis of cell free DNA (cfDNA) from cerebrospinal fluid (CSF) could represent a superior diagnostic modality. We analyzed CSF samples from 7 patients, median age of 18 years, with DLGNT including matched germline DNA. MSK-IMPACT, a hybridization capture-based next-generation DNA sequencing panel, was utilized to analyze the DNA. Prior tissue biopsy was done in 5/7 patients. Of these, biopsy was non-diagnostic in 3 patients and positive for the KIAA1549:BRAF fusion in 2. Analysis of CSF cfDNA detected KIAA1549::BRAF fusion in 5/7 (71%) of patients, including all patients with prior non-diagnostic biopsy or tissue quantity insufficient for molecular testing. Analysis of CSF cfDNA copy number profiles revealed 1p-/1q+ in 6 patients and 1p- only in the remaining 1 patient, fully concordant with available prior tissue testing in all instances. In 2 patients with prior tissue-confirmed KIAA1549::BRAF fusion, the fusion was not detected in CSF cfDNA during/after treatment with a MEK inhibitor, but additional somatic alterations and DNA copy number profiles were similar to the prior biopsies. Additional somatic alterations were identified in ATRX, BCOR, RARA, STAT5A, SPOP, PPM1D, MED12, KMT2C, RECQL and SETD2, including likely oncogenic secondary driver alterations that have not been previously reported in DLGNT. Given the high molecular diagnostic yield and concordance with open biopsy, cfDNA CSF sequencing should be considered as a first line diagnostic maneuver for patients with MRI findings suspicious for DLGNT, followed by open biopsy only if CSF is non-diagnostic.

Abstract 1414: The role of chromosome arm 3p and human papillomavirus on proliferation, differentiation, and metastasis in squamous cancers

Abstract Squamous Cell Carcinoma (SCC) is an aggressive form of cancer that develops in the mucosal epithelium of the digestive and respiratory tracts as well as on the skin. Two major risk factors for development of SCC are carcinogen exposures, such as alcohol and tobacco, and Human Papillomavirus (HPV) infection. Viral- and carcinogen-driven SCCs have differential responses to radiation, chemotherapy, and immunotherapy, with HPV+ SCCs responding better to these treatments than HPV- carcinomas. Analysis of aneuploidy patterns of genomic samples from The Cancer Genome Atlas (TCGA) have shown that squamous cancers cluster together and are characterized by gain of chromosome arm 3q gain and loss of chromosome arm 3p. However, while HPV+ SCCs show similar rates of 3q gain, they have significantly lower rates of 3p loss, suggesting that 3p deletion and HPV infection might have overlapping roles in cancer initiation and/or progression. In addition, the degree to which these two paths of tumor development contribute to differences in disease progression and outcome has been largely unexplored. We have identified a strong correlation between 3p deletion and high epithelial-mesenchymal transition (EMT) gene signature across all cancer types. Interestingly, interferon signaling has been found to be upregulated in non-squamous cancers upon 3p deletion but downregulated in squamous cancers with 3p deletion. SCCs positive for HPV infection show decreased expression of EMT genes and increased expression of genes in interferon signaling and cell cycle checkpoint pathways. Together, the results of these TCGA analyses suggest mechanisms by which chromosome 3p deletion and HPV infection may affect cancer progression and are currently being validated in vitro in a panel of squamous cancer cell lines. Additionally, we will use a targeted CRISPR-Cas9 system developed in our lab to delete chromosome 3p, as well as transfect HPV oncoproteins, in normal keratinocytes to create isogenic models. With these models, we will study the role of individual aneuploidy events in tumorigenesis, tumor development, and metastasis in the context of HPV positive and negative SCC. Citation Format: Samyukta Mallick, Alison M. Taylor. The role of chromosome arm 3p and human papillomavirus on proliferation, differentiation, and metastasis in squamous cancers [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 1414.

Identification of recurrent 3q13.31 chromosomal rearrangement indicates LSAMP as a tumor suppressor gene in neuroblastoma.

Neuroblastoma (NB) is a childhood malignancy of the sympathetic nervous system. NB is mainly driven by copy number alterations, such as MYCN amplification, large deletions of chromosome arm 11q and gain of chromosome arm 17q, which are all markers of high‑risk disease. Genes targeted by recurrent, smaller, focal alterations include CDKN2A/B, TERT, PTPRD and ATRX. Our previous study on relapsed NB detected recurrent structural alterations centered at limbic system‑associated membrane protein (LSAMP; HUGO Gene Nomenclature Committee: 6705; chromosomal location 3q13.31), which is a gene frequently reported to be deleted or downregulated in other types of cancer. Notably, in cancer, LSAMP has been shown to have tumor‑suppressing functions. The present study performed an expanded investigation using whole genome sequencing of tumors from 35 patients, mainly with high‑risk NB. Focal duplications or deletions targeting LSAMP were detected in six cases (17%), whereas single nucleotide polymorphism‑microarray analysis of 16 NB cell lines detected segmental alterations at 3q13.31 in seven out of the 16 NB cell lines (44%). Furthermore, low expression of LSAMP in NB tumors was significantly associated with poor overall and event‑free survival. Invitro, knockdown of LSAMP in NB cell lines increased cell proliferation, whereas overexpression decreased proliferation and viability. These findings supported a tumor suppressor role for LSAMP in NB. However, the higher incidence of LSAMP aberrations in cell lines and in relapsed NB tumors suggested that these alterations were a late event predominantly in advanced NB with a poor prognosis, indicating a role of LSAMP in tumor progression rather than in tumor initiation. In conclusion, the present study demonstrated recurrent genomic aberrations of chromosomal region 3q13.31 that targeted the LSAMP gene, which encodes a membrane protein involved in cell adhesion, central nervous system development and neurite outgrowth. The frequent aberrations affecting LSAMP, together with functional evidence, suggested an anti‑proliferative role of LSAMP in NB.

Abstract A009: Survival disparities in lung squamous cell carcinoma: Clinical and genomic correlates with genetic ancestry

Abstract Many studies show racial and ethnic survival disparities in lung squamous cell carcinoma (LSCC), and self-identified African-Americans have significantly lower overall survival and progression-free survival than self-identified Caucasians. However, survival models based on genetic ancestry, rather than self-identified race, are needed to assess whether genetic ancestry influences this survival disparity or whether the cause of the disparity is largely socioeconomic. Using clinically annotated genomic data from 501 LSCC tumor samples in the Cancer Genome Atlas (TCGA), we compared clinical data, gene expression profiles, mutations, and copy number changes of tumors across ancestral groups to identify significantly differential events. We then applied the Cox Proportional Hazards model to predict overall survival as a function of genetic ancestry alongside other genetic, molecular, and clinical variables. We found a significant difference in metastatic stage between tumors from patients of African (AFR) and European (EUR) ancestry, which was significant in predicting survival. We identified increased copy number of chromosome arm 8q and locus chr8q24, as well as increased expression of MYC targets in AFR samples relative to EUR samples, all of which suggest a role for the MYC pathway in differential cancer progression. In addition, we found enrichment of amplifications at chr22q11 and chr16p11 in AFR samples compared to EUR samples. We identified top differentially mutated genes between groups, of which mutant SPRR3, PDGFA, FRS3, and C21orf62 correlate with survival. Notably, when survival was modeled as a function of both genetic ancestry and SPRR3 mutation, genetic ancestry was no longer significant as a predictor of survival. The PDGFRA receptor is also significantly differentially expressed between groups, suggesting a possible role of the PDGF axis in differential tumor growth. These results suggest potential genetic and clinical factors implicated in racial survival disparities. Further studies and more diverse sampling of cancer patient populations would allow validation of these differences and future elucidation of patient-specific treatment modalities. Citation Format: Sejal Jain, Alexandria Yao, Diane Allen-Gipson, Fatemeh Momen-Heravi, Alison M. Taylor. Survival disparities in lung squamous cell carcinoma: Clinical and genomic correlates with genetic ancestry [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A009.

Bioinformatics analysis of miRNAs in the neuroblastoma 11q-deleted region reveals a role of miR-548l in both 11q-deleted and MYCN amplified tumour cells

Neuroblastoma is a childhood tumour that is responsible for approximately 15% of all childhood cancer deaths. Neuroblastoma tumours with amplification of the oncogene MYCN are aggressive, however, another aggressive subgroup without MYCN amplification also exists; rather, they have a deleted region at chromosome arm 11q. Twenty-six miRNAs are located within the breakpoint region of chromosome 11q and have been checked for a possible involvement in development of neuroblastoma due to the genomic alteration. Target genes of these miRNAs are involved in pathways associated with cancer, including proliferation, apoptosis and DNA repair. We could show that miR-548l found within the 11q region is downregulated in neuroblastoma cell lines with 11q deletion or MYCN amplification. In addition, we showed that the restoration of miR-548l level in a neuroblastoma cell line led to a decreased proliferation of these cells as well as a decrease in the percentage of cells in the S phase. We also found that miR-548l overexpression suppressed cell viability and promoted apoptosis, while miR-548l knockdown promoted cell viability and inhibited apoptosis in neuroblastoma cells. Our results indicate that 11q-deleted neuroblastoma and MYCN amplified neuroblastoma coalesce by downregulating miR-548l.

Monosomy 7 and Del(7q) Cause Selective Sensitivity to Inhibitors of Nicotinamide Phosphoribosyltransferase in Acute Myeloid Leukemia

Monosomy 7 and deletion of chromosome arm 7q (-7/-7q) occur in up to 10% of patients with acute myeloid leukemia (AML). In AML, -7/-7q are associated with resistance to chemotherapy and adverse prognosis. As such, treatment outcomes for this subgroup of patients remain particularly dismal, and targeted therapies are urgently needed. To identify targeted therapies for AML with -7/-7q we analyzed ex vivo drug sensitivities in 158 AML samples. The presence of -7/-7q (n=18) was determined using karyotyping and exome-sequencing of bone marrow (BM) mononuclear cells and a matched skin biopsy control. Mononuclear cells from fresh bone marrow aspirates of AML patients were incubated with a library of up to 525 oncology drugs for 72h, with each drug pre-plated in 5 concentrations. Cell viability was measured using the CellTiter Glo (CTG) assay, and the response of each sample was summarized as a drug sensitivity score (DSS), which is a modified integration of the dose-response inhibition curve, directly proportional to the sensitivity of a sample. We found that AML samples with -7/-7q were highly sensitive to the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor daporinad (p-value ~ 4.2e-06) (Figure A). NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway. The NAMPT gene is located at 7q22.3. Monosomy 7 and del(7q) lead to the deletion of one copy (i.e., haploinsufficiency) of NAMPT. Notably, a rare AML with del(7q) in which the deletion did not include the NAMPT gene (and which was thus diploid for NAMPT) was not sensitized to NAMPT inhibition. Taken together, this data demonstrates a significant association between the NAMPT haploinsufficiency in AML with -7/-7q and increased sensitivity to NAMPT inhibition. To further support our findings, we analyzed the sensitivity of AML cells without or with -7/-7q to four NAMPT inhibitors (daporinad, GMX1778, KPT-9274, and LSN3154567) using multiparametric flow cytometry. Viably frozen BM mononuclear cells from AML patients with (n=11) and without (n=51) -7/-7q were thawed and incubated with drugs in 8 concentrations for 72 hours. After incubation, cell viability was analyzed using the iQue Screener Plus flow cytometer with a panel consisting of CD45, CD3, CD34, CD117, CD14, and CD38 antibodies, to identify myeloid and lymphoid cell populations, as well as Annexin V and 7-AAD to discriminate live from dead cells. We found that CD34+ myeloblasts from AML with -7/-7q were selectively killed by all four NAMPT inhibitors, validating our initial results (p-value ~ 1.4e-02). An in-depth analysis of cellular subpopulations revealed that while CD34+CD38+ cells (representing more differentiated myeloblasts) were significantly more sensitive to NAMPT inhibition in -7/-7q samples compared to wild-type samples (p-value ~ 4.5e-04), the CD34+CD38- compartment (anticipated to contain the immature leukemic stem and progenitor cells) was not sensitized in this group (Figure B). Interestingly, the analysis of ex vivo drug sensitivities also revealed that while the majority of AML with -7/-7q are resistant to the FDA-approved BCL2 inhibitor venetoclax, they are highly sensitive to daporinad (Figure A). This finding proposes NAMPT inhibitors as a suitable alternative to venetoclax for -7/-7q patients. Additionally, since venetoclax has previously shown significant efficacy against the highly BCL2-dependent undifferentiated (CD34+CD38-) myeloblasts, its combination with NAMPT inhibitors represents a promising strategy for achieving total leukemic cell death in these patients. In conclusion, we have found that AML with NAMPT haploinsufficiency caused by -7/-7q shows selective sensitivity to NAMPT inhibition. This suggests that NAMPT inhibitors have the potential to be a potent targeted therapy for AML with -7/-7q, a disease with a particularly adverse prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Loss of genes in chromosome arms 5q and 16q in breast cancer.

Losses of genetic material from chromosomes 5q and 16q commonly occur in sub-sets of breast cancer. Their significance from a pathophysiologic point of view is not well-defined. This study uses publicly available genomic data from extensive breast cancer datasets to define the landscape of losses in chromosomal arms 5q and 16q in the two sub-types of breast cancer they most commonly occur, basal-like and luminal A cancers, respectively. It is shown that dozens of genes from these chromosomal arms are putatively hemi-deleted in few samples each. No individual gene from either 5q or 16q shows an incidence of deep deletion above 10% in the cohorts with basal-like and luminal A cancers or in the whole cohorts. A few tumor suppressor genes are deleted in a small number of samples, less than 5% in each cohort. Losses of 5q or 16q confer no survival advantage in either the basal-like or the luminal A cohorts from TCGA that harbor them. Results suggest that there are no individual genes in chromosomes 5q and 16q whose loss can be implicated in a dominant pathophysiologic sequence of events in breast cancer or its sub-sets.

Overexpression of the miR-17-92 cluster in colorectal adenoma organoids causes a carcinoma-like gene expression signature

Gain of chromosome arm 13q is one of the most prevalent DNA copy number alterations associated with colorectal adenoma-to-carcinoma progression. The oncogenic miR-17-92 cluster, located at 13q, was found to be overexpressed in colorectal cancer and in adenomas harboring 13q gain. However, to what extent overexpression of this group of microRNAs actually drives progression to cancer remains to be resolved. Therefore, we aimed to clarify the role of miR-17-92 cluster in the progression from colorectal adenoma to carcinoma.The miR-17-92 cluster was overexpressed in human colorectal adenoma organoids without 13q gain and downstream effects on mRNA expression were investigated, along with functional consequences in vitro and in vivo.Comparison of mRNA sequencing results of organoids overexpressing miR-17-92 and cultures transduced with control vector revealed a miR-17-92 expression signature. This signature appeared to be enriched in an independent series of colorectal cancers and adenomas with 13q gain, confirming that miR-17-92 expression is associated with malignant progression. However, tumor-associated characteristics, such as increased proliferation rate, were not observed in miR-17-92 overexpressing adenoma organoids in vitro. In addition, subcutaneous injection of these organoids in immunodeficient mice was insufficient to cause tumor outgrowth.In conclusion, this study showed that miR-17-92 expression contributes to 13q gain-associated adenoma-to-carcinoma progression, however, this is insufficient to cause malignancy.

Copy-number intratumor heterogeneity increases the risk of relapse in chemotherapy-naive stage II colon cancer.

Optimal selection of high-risk patients with stage II colon cancer is crucial to ensure clinical benefit of adjuvant chemotherapy. Here, we investigated the prognostic value of genomic intratumor heterogeneity and aneuploidy for disease recurrence. We combined targeted sequencing, SNP arrays, fluorescence in situ hybridization, and immunohistochemistry on a retrospective cohort of 84 untreated stage II colon cancer patients. We assessed the clonality of copy-number alterations (CNAs) and mutations, CD8+ lymphocyte infiltration, and their association with time to recurrence. Prognostic factors were included in machine learning analysis to evaluate their ability to predict individual relapse risk. Tumors from recurrent patients displayed a greater proportion of CNAs compared with non-recurrent (mean 31.3% versus 23%, respectively; p = 0.014). Furthermore, patients with elevated tumor CNA load exhibited a higher risk of recurrence compared with those with low levels [p = 0.038; hazard ratio (HR) 2.46], which was confirmed in an independent cohort (p = 0.004; HR 3.82). Candidate chromosome-specific aberrations frequently observed in recurrent cases included gain of the chromosome arm 13q (p = 0.02; HR 2.67) and loss of heterozygosity at 17q22-q24.3 (p = 0.05; HR 2.69). CNA load positively correlated with intratumor heterogeneity (R = 0.52; p < 0.0001). Consistently, incremental subclonal CNAs were associated with an elevated risk of relapse (p = 0.028; HR 2.20), which we did not observe for subclonal single-nucleotide variants and small insertions and deletions. The clinico-genomic model rated an area under the curve of 0.83, achieving a 10% incremental gain compared with clinicopathological markers (p = 0.047). In conclusion, tumor aneuploidy and copy-number intratumor heterogeneity were predictive of poor outcome and improved discriminative performance in early-stage colon cancer. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Preimplantation genetic testing for aneuploidy (PGT-A)-a single-center experience.

The aim of this study was to determine the prevalence and nature of human embryonic aneuploidy based on the preimplantation genetic testing for aneuploidy (PGT-A), the distribution of aneuploidy across the individual chromosomes, and their relationship to maternal age. This is a retrospective cohort study conducted at a single center. The study includes subjects who opted for PGT-A in their in vitro fertilization (IVF) cycle from 2016 to 2020. PGT-A was performed on 1501 embryos from 488 patients in 535 cycles. PGT-A was performed using NGS-based technique on Ion Torrent PGM (Life Technologies). Analysis was performed to determine the (i) frequency of the aneuploidy, (ii) the chromosome most commonly affected, (iii) relationship between maternal age and the rate of aneuploidy, and (iv) incidence of segmental aneuploidy. The overall frequency of aneuploidy was observed to be 46.8%. The incidence of aneuploidy rate was ~ 28% at maternal age < 30years which steadily increased to ~ 67% in women above 40years. High frequency of aneuploidy was observed in chromosomes 16, 22, 21, and 15. Segmental abnormalities, involving loss or gain of chromosomal fragments, were observed at a frequency of 5.3%, and highest incidence of segmental gain was observed on the q-arm of chromosome 9. The study provides important information regarding the frequency of the aneuploidy in IVF cohort and the most frequent chromosomal abnormality. The study further emphasizes the relationship between maternal age and aneuploidy. This study has important implications which help clinicians and genetic counselors in providing information in patient counseling.