CDKN2A Mutations Research Articles

Mesotheliomas and Benign Mesothelial Tumors: Update on Pathologic and Imaging Findings

A diverse spectrum of benign entities and malignant neoplasms originate from the monotonous mesothelium that lines the serosal membranes of the pleural, pericardial, and peritoneal cavities. The mesothelium of myriad sites shows a common origin from the lateral plate mesoderm; primary mesothelial tumors thus demonstrate similar pathogenesis, imaging findings, and treatment options. Significant changes have been made in the 2021 World Health Organization (WHO) classification schemata of the pleural and pericardial tumors on the basis of recent advances in pathology and genetics. While malignant mesotheliomas are biologically aggressive malignancies that occur primarily in patients exposed to asbestos with attendant poor survival rates, well-differentiated papillary mesothelial tumors and adenomatoid tumors charter a benign clinical course with an excellent prognosis. Mesothelioma in situ is a newly characterized entity represented by recurrent unexplained pleural effusions without any identifiable mass at imaging or thoracoscopy. Immunohistochemical markers based on BAP1, MTAP, CDKN2A, and TRAF7 gene mutations help differentiate diffuse mesotheliomas from benign mesothelial proliferations and localized mesotheliomas. Cross-sectional imaging modalities, including US, CT, MRI, and fluorine 18-fluorodeoxyglucose (FDG) PET/CT, permit diagnosis and play a major role in staging and assessing surgical resectability. Imaging studies are invaluable in providing noninvasive and quantitative assessment of tumor response in patients with unresectable disease. Owing to significant overlap in patient characteristics and pathomorphology, accurate diagnosis based on advanced histopathology techniques and genetic abnormalities is imperative for optimal management and prognostication. While patients with nonepithelioid pleural mesotheliomas benefit from immunotherapy, novel targeted therapies for CDKN2A-, NF2-, and BAP1-altered mesotheliomas are under consideration. © RSNA, 2023 Quiz questions for this article are available through the Online Learning Center.

Prognosis-related molecular subtyping in head and neck squamous cell carcinoma patients based on glycolytic/cholesterogenic gene data

BackgroundHead and neck squamous cell carcinoma (HNSCC) remains an unmet medical challenge. Metabolic reprogramming is a hallmark of diverse cancers, including HNSCC.MethodsWe investigated the metabolic profile in HNSCC by using The Cancer Genome Atlas (TCGA) (n = 481) and Gene Expression Omnibus (GEO) (n = 97) databases. The metabolic stratification of HNSCC samples was identified by using unsupervised k-means clustering. We analyzed the correlations of the metabolic subtypes in HNSCC with featured genomic alterations and known HNSCC subtypes. We further validated the metabolism-related subtypes based on features of ENO1, PFKFB3, NSDHL and SQLE expression in HNSCC by Immunohistochemistry. In addition, genomic characteristics of tumor metabolism that varied among different cancer types were confirmed.ResultsBased on the median expression of coexpressed cholesterogenic and glycolytic genes, HNSCC subtypes were identified, including glycolytic, cholesterogenic, quiescent and mixed subtypes. The quiescent subtype was associated with the longest survival and was distributed in stage I and G1 HNSCC. Mutation analysis of HNSCC genes indicated that TP53 has the highest mutation frequency. The CDKN2A mutation frequency has the most significant differences amongst these four subtypes. There is good overlap between our metabolic subtypes and the HNSCC subtype.ConclusionThe four metabolic subtypes were successfully determined in HNSCC. Compared to the quiescent subtype, glycolytic, cholesterogenic and mixed subtypes had significantly worse outcome, which might offer guidelines for developing a novel treatment strategy for HNSCC.

Molecular landscape and clinical implication of CCNE1-amplified esophagogastric cancer.

440 Background: Cyclin E1 ( CCNE1) amplification ( CCNE1Amp) drives chromosomal instability (CIN) and is associated with immune cell exclusion, liver metastasis, therapeutic resistance, and poor survival in gastric adenocarcinoma (GA). However, the clinical relevance of CCNE1Amp in a larger real-world population is unknown. Therefore, we profiled the molecular landscape of CCNE1-amplified (amp) esophagogastric cancer (EGC) and explored treatment response outcomes. Methods: Tumors were analyzed by next-generation sequencing (NGS) of DNA (592 genes, NextSeq; WES, NovaSeq) and RNA (WTS, NovaSeq) (Caris Life Sciences, Phoenix, AZ). Deficiency in mismatch repair or microsatellite instability (dMMR/MSI-H) was tested by fragment analysis, IHC, and NGS. Tumor mutational burden (TMB) was measured by totaling somatic mutations per tumor (high>10 mt/MB). PD-L1 was tested by IHC (22C3, 1+). Real world overall survival (OS) was extracted from insurance claims and calculated from first of treatment to last contact using Kaplan-Meier survival curves for molecularly defined cohorts. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q<0.05). Results: CCNE1Amp (copy number, CN ≥6) was identified in 142/2276 (6.2%) esophageal adenocarcinoma (EA), 101/1449 (7.0%) esophagogastric junction carcinoma (EJC), 109/2607 (4.2%) GA, and 6/751 (0.8%) esophageal squamous cell carcinoma (ESCC) samples. Metastatic sites such as lymph node and liver showed higher frequency of CCNE1Amp compared to primary sites. TMB, dMMR/MSI-H, and PD-L1 expression were similar in CCNE1-amp vs non-amp tumors. CCNE1Amp was enriched in CIN type tumors (defined by increased TP53 and ERBB2 amp) and was associated with decreased mutation of CDKN2A and CDH1. In addition, CCNE1-amp tumors showed increased HER2 positivity/ ERBB2 amplification by IHC (14.3% vs 10.1%) or CN analysis (19.1% vs 10.3%). Differential gene expression analysis demonstrated increased liver X receptor/retinoid X receptor (LXR/RXR) pathway activation in tumors harboring CCNE1Amp. While there were no differences in OS between CCNE1-amp vs non-amp GA, CCNE1Amp was associated with worse OS after trastuzumab in the IHC HER2+ cohort (N=9 vs 28; HR (95% CI): 2.95 (1.18 – 7.34), p = 0.015). By contrast, there was a trend toward improved OS after immunotherapy in CCNE1-amp GA (N=9 vs 194; HR (95% CI): 0.34 (0.11 –1.07), p = 0.054). Conclusions: CCNE1Amp is associated with a distinct molecular profile in EGC and resistance to HER2-targeted therapy. While CCNE1-amp tumors are thought to be generally “immune-cold,” CCNE1-amp GA demonstrated potentially improved outcomes with immunotherapy. Further investigation of CCNE1Amp as a predictive biomarker is warranted, particularly as novel therapeutics selectively targeting CCNE1Amp are under clinical investigation.

491 Education level is associated with increased sun exposure, but also ultraviolet radiation protection and melanoma awareness in a high-risk population in Switzerland

The global incidence of melanoma has risen rapidly over the last decades in Caucasian populations with Switzerland reporting among the highest rates in Europe. Ultraviolet (UV) radiation is one of the main risk factors for melanoma. Our aim was to investigate the UV protection behavior and melanoma awareness in a high-risk cohort for melanoma. In this prospective monocentric study comparing 2D- and 3D-imaging with deep-learning based risk assessments and routine skin cancer screenings by dermatologists for early detection of melanoma, we assessed general melanoma awareness and UV protection habits in high-risk patients for melanoma (≥ 100 nevi, ≥ 5 dysplastic nevi, known CDKN2A mutation and/or positive family history) and melanoma patients by questionnaires.

Next-generation Sequencing as a Potential Diagnostic Adjunct in Distinguishing Between Desmoplastic Melanocytic Neoplasms.

Desmoplastic melanomas (DMs) are often challenging to diagnose and ancillary tests, such as immunohistochemistry, have limitations. One challenge is distinguishing DM from benign desmoplastic melanocytic neoplasms. In this study, we explored the utility of next-generation sequencing data in the diagnosis of DMs versus desmoplastic Spitz nevi (DSN) and desmoplastic nevi (DN). We sequenced 47 cases and retrieved 12 additional previously sequenced clinical cases from our dermatopathology database. The 59 total cases were comprised of 21 DMs, 25 DSN, and 13 DN. The DMs had the highest tumor mutation burden at 22 mutations/megabase (m/Mb) versus the DSN (6m/Mb) and DN (8m/Mb). Truncating mutations in NF1 resulting in a loss-of-function were exclusive to the DM cohort, identified in 8/21 (38%) cases. Importantly, missense mutations in NF1 were nonspecific and seen with similar frequency in the different cohorts. Other mutations exclusive to the DMs included truncating mutations in TP53 , CDKN2A , and ARID2 . Among the DSN, 17/25 (68%) had an HRAS mutation or receptor tyrosine kinase fusion consistent with other Spitz tumors. Two cases in the DN cohort had missense mutations in BRAF without additional progression mutations and 2 other cases had mutations in GNAQ , supporting a diagnosis of a sclerosing blue nevus. The remainder of the DN had nonspecific mutations in various signaling pathways with few progression mutations. Overall, our study provides preliminary data that next-generation sequencing data may have the potential to serve as an ancillary diagnostic tool to help differentiate malignant and benign desmoplastic melanocytic neoplasms.

Integrated multi-omics analysis identifies CD73 as a prognostic biomarker and immunotherapy response predictor in head and neck squamous cell carcinoma.

Advances in tumor immunotherapy have been developed for patients with advanced recurrent or metastatic (R/M) HNSCC. However, the response of most HNSCC patients to immune checkpoint inhibitors (ICI) remains unsatisfactory. CD73 is a promising target for tumor immunotherapy, but its role in HNSCC remains insufficient. In this study, we aim to explore the function of CD73 in HNSCC. Transcriptomic and clinical data of TCGA-HNSC were downloaded from UCSC Xena for analysis of CD73 mRNA expression and prognosis. Immunohistochemical assay were performed to validate the expression of CD73 in tumor tissues and its relationship with CD8+ T cells. GSEA analysis was performed with the "clusterProfiler" R package. Immune infiltration analysis was calculated with ESTIMATE, CIBERSORT and MCP-counter algorithms. Single-cell transcriptomic data was originated from GSE103322. Cell clustering, annotation and CD73 expression were from the TISCH database. Correlation data between CD73 and tumor signatures were obtained from the CancerSEA database. Somatic mutation data were obtained from TCGA-HNSC and analyzed by "maftools" R package. Immune efficacy prediction was performed using TIDE algorithm and validated with the IMvigor210 cohort. Compared with normal tissues, both mRNA and protein expressions of CD73 were elevated in tumor tissues (P = 9.7×10-10, P = 7.6×10-5, respectively). Kaplan-Meier analysis revealed that patients with high expression of CD73 had worse overall survival (log-rank P = 0.0094), and CD73 could be used as a diagnostic factor for HNSCC (AUC = 0.778). Both bulk RNA-seq and single-cell RNA-seq analysis showed that high CD73 expression can promote EMT and metastasis, samples with high CD73 expression had reduced CD8+ T cells. Furthermore, it was found that CD73-high group was more prone to have mutations in TP53, HRAS and CDKN2A, and were negatively correlated with TMB (P = 0.0055) and MSI (P = 0.00034). Mutational signature analysis found that CD73 was associated with APOBEC signature. Immunotherapy efficacy analysis showed that CD73-high group was less sensitive to immune efficacy. Our results demonstrate that CD73 has an inhibitory effect on the tumor microenvironment, and is more likely to be unresponsive to ICI therapy. Collectively, targeting CD73 may provide new insights for tumor targeted therapy and/or immunotherapy.

Idasanutlin and Navitoclax Induce Synergistic Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

We report here investigation of the combination of idasanutlin and navitoclax in the context of T-cell acute lymphoblastic leukemia (T-ALL), including detailed mechanistic characterization and in vivo patient derived xenograft results. T-ALL is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age, ranging from 70-75% for pediatric patients to 21.7% for patients aged 60-79. An unmet clinical need remains for the development of better therapies for T-ALL. Notch pathway activation mutations are present in the majority of T-ALL and are thought to induce transformation, in part, through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, and critical to our approach, greater than 95% of primary T-ALL cases and 75% of relapsed cases maintain wild type p53. Based on these genetic data, we hypothesized that treatment with MDM2 inhibitors, by bypassing the loss of ARF, would increase p53 expression and lead to T-ALL cell apoptosis. To test this hypothesis, we initially treated T-ALL patient derived xenograft (PDX) lines in vitro with single-agent idasanutlin (a second generation MDM2 inhibitor) and observed upregulation of pro-apoptotic p53 target genes Bax and Puma1. However, only modest T-ALL cell apoptosis was observed in response to MDM2 inhibition alone. Based on these observations, particularly the induction of pro-apoptotic p53 targets, we hypothesized that inhibition of pro-survival BH3 domain proteins would synergize with MDM2 inhibition to kill T-ALL cells. Of note, a prior study showed that most T-ALL (~62%) respond to treatment with navitoclax (ABT-263), an inhibitor of BCL2, BCLXL, and BCLW, although most responses were transient and dose-limiting thrombocytopenia was observed. Combination therapy with idasanutlin could permit on-target inhibition with tolerable dosing. In vitro treatment of a panel of 5 different PDX lines derived from pediatric and adult T-ALL with dual idasanutlin and navitoclax led to robust cell death in all lines. In addition, treatment of a T-ALL PDX line with a matrix of dose combinations showed strong evidence of synergic activity. Synergy was assessed by ZIP (zero interaction potency) score, a statistical method developed by B Yadav et al. (Comput. Struct. Biotechnol. J. 2015) that compares the change in potency of individual dose response curves in the presence of a second agent. Generally, a ZIP score greater than 5 is considered significant; Idasanutlin and navitoclax combination therapy had a global ZIP score=16-18, with the most synergic dose area ZIP score=40-52. Combined treatment resulted in marked apoptosis, with strong induction of pro-apoptotic p53 targets, including PUMA and BAX. Finally, we assessed the in vivo response of four different T-ALL xenografts to dual treatment. In each case, human T-ALL cells were injected into immunodeficient NSG mice and randomized into treatment groups. Monotherapy with idasanutlin or navitoclax resulted in a modest but significant decrease in T-ALL burden in all cases, with the exception of an ETP T-ALL, which was resistant to idasanutlin monotherapy. A marked response to combination treatment with idasanutlin and navitoclax was seen in all four T-ALL xenografts. Specifically, the combination was found to be synergistic in each case based on a modified Bliss Independence test, analyzing average daily change in tumor burden for synergy interaction. Furthermore, overall survival was significantly increased in the combination treatment group. These data suggest that the rational combination of idasanutlin and navitoclax is highly active, with induction of an apoptotic cell death in T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract A001: Clinical-genomic analysis of KRAS wild-type pancreatic cancer confirms alternative targetable drivers and provides insight for age and risk related clinical stratification

Abstract Approximately 8-10% of pancreatic cancers do not harbor mutations in KRAS. Understanding the unique molecular and clinical features of this subset of pancreatic cancer (PC) is important to guide patient stratification for clinical trials of molecularly targeted agents. To this end we investigated a cohort of 795 PC patients from Dana-Farber Cancer Institute who had undergone somatic genomic characterization with OncoPanel, a targeted next-generation sequencing panel with coverage of more than 400 cancer-associated genes. A total of 9.2% (73/795) of cases in our cohort were KRAS WT. The KRAS WT cohort was statistically enriched for MSI-H PC and acinar cell carcinomas (p = 0.0035, p < 0.0001 respectively). Actionable alterations in alternative MAPK drivers were identified in 44% (32/73) of KRAS WT cases. BRAF alterations accounted for 56% (18/32) of detected alternative MAPK drivers, the majority of which (72%) were Class II which exhibit dimer-dependent constitutive activity. Receptor Tyrosine Kinase (RTK) fusion events in BRAF, NTRK1, NRG1, NTRK3, ROS1, and FGFR2 accounted for 25% (8/32) of detected alternative MAPK drivers in KRAS WT tumors. BRAF in-frame deletions showed increased sensitivity to dual pan-RAF and MEK inhibition in organoid models and one patient with a ROS1 fusion received prolonged clinical benefit from targeted therapy. In addition to alternative MAPK drivers, mutations in GNAS (p = 0.0014) and ARID2 (p = 0.045) were significantly enriched in KRAS WT PC, whereas TP53 mutations were significantly less frequent in KRAS WT cases. Interestingly, although not statistically significant, rates of mutation in the other canonical tumor suppressor genes (CDKN2A, SMAD4) were also lower in KRAS WT PC. Clinically, KRAS Mutant (MUT) PC were associated with a decreased overall survival (OS) compared to the KRAS WT cohort [median OS 17.5 vs 24.0 months, HR 1.38, p = 0.036], however this relationship was no longer significant after accounting for other clinical factors. For patients with KRAS WT PC, those with SMAD4 alterations had a significantly decreased OS (HR 6.24, p < 0.001), whereas presence of TP53 or CDKN2A mutations had no significant impact. Lastly, we found that KRAS WT PC was associated with a younger age of onset. Interestingly, we noted that KRAS WT PC patients with a younger age of onset had tumors with few oncogenic alterations whereas no such association was seen in KRAS MUT patients. Validation of this finding in a separate dataset is required and is currently ongoing. In summary, our clinical and genomic characterization of KRAS WT PC identifies a high prevalence of alternative MAPK drivers that are amenable to targeted therapies. Our cohort also recapitulates the previously reported clinical characteristics of KRAS WT PC and identifies the presence of SMAD4 alterations as significantly associated with decreased overall survival in KRAS WT PC. Additional analysis from multiple sources will be critical to risk stratify these patients further and to validate age-related findings. Citation Format: Harshabad Singh, Rachel B. Keller, Kevin S. Kapner, Julien Dilly, Srivatsan Raghavan, Chen Yuan, Eizabeth Cohen, Michael Tolstorukov, Emma Hill, Elizabeth Andrews, Lauren K. Brais, Annacarolina Da Silva, Kimberly Perez, Douglas A. Rubinson, Benjamin L. Schlechter, Michael H. Rosenthal, Jason L. Hornick, Valentina Nardi, Yvonne Li, Hersh Gupta, Andrew Cherniack, Mathew L. Meyerson, James M. Cleary, Jonathan A. Nowak, Brian M. Wolpin, Andrew A. Aguirre. Clinical-genomic analysis of KRAS wild-type pancreatic cancer confirms alternative targetable drivers and provides insight for age and risk related clinical stratification [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 A001.

Genetic and Prognostic Analysis in Blastoid and Pleomorphic Mantle Cell Lymphoma: A Multicenter Analysis in China

Blastoid and pleomorphic mantle cell lymphoma (B/PMCL), which are characterized by more highly aggressive features and an unfavorable outcome, are still a diagnostic and therapeutic challenge in MCL. In this study, we aimed to integrated analysis of clinical, genomic characteristics and treatment options of patients with blastoid/pleomorphic mantle cell lymphoma in China from multicenter. Second generation sequencing was performed in evaluable biopsy specimens. Seventy-four patients with blastoid (n=43) and pleomorphic (n=31) were included in the analysis. Median age of the cohort was 60.0 years with a male-to-female ratio of 2.89:1. 3-year progression free survival (PFS) and overall survival (OS) rates were 37.6% and 46.0%, respectively. The most common gene mutations in blastoid/pleomorphic MCL were TP53, ATM, NOTCH1, NOTCH2, NSD2, SMARCA4, CREBBP, KMT2D, FAT1, TRAF2. Progression of disease within 12 months (POD12) can distinguish the worst prognosis in blastoid and pleomorphic variants. Which carried a distinct mutation profile (TP53, SMARCA4, NSD2, NOTCH2, KMT2D, PTPRD, CREBBP and CDKN2A mutations) compared with non-POD12 patients. Gene with POD≤12 was enriched in BCR signaling while gene with POD>12 were enriched in TRAF3-dependent IRF activation pathway. First-line high dose cytarabine exposure obtained survival benefits in these population, and BTKi-combined treatment in front-line had somewhat improvement in survival with no significant difference in statistic. In conclusion, B/P MCL have inferior outcomes and distinct genomic profile, patients with POD12 display a distinct mutation profile and worst prognosis, new therapeutic drugs and clinical trials need to be further explored in B/P MCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular Characterization of Adult Acute Lymphoblastic Leukemia Identifies a Subgroup with Myeloid Mutations and Pre-Existing Clonal Hematopoiesis

Acute lymphoblastic leukemia (ALL) represents 20% of adult leukemias. Genomic risk stratification of ALL relies on the data generated from large cohorts of pediatric and young adult patients, where older patients with ALL have been underrepresented. It is imperative to understand the unique leukemogenic pathways in this group in order to develop novel therapeutic approaches. Clonal hematopoiesis (CH) is a precursor lesion for development of myeloid leukemias and mature lymphoid neoplasms (Niroula, Nat Med 2021), but its impact on the biology and outcomes of ALL is unknown. Based on our observations on high incidence of myeloid mutations in older adults with ALL, we hypothesized that CH constitutes a fertile genomic background for lymphoblastic leukemogenesis, and ALL with myeloid mutations is a unique entity with distinct clinical and molecular characteristics. We studied 400 adult patients with ALL treated at the University of Chicago, Moffitt Cancer Center, and City of Hope between 2014 and 2022 (Fig 1A). Genetic profiling of pre-treatment bone marrow samples was performed with a comprehensive panel covering 147 myeloid and lymphoid genes. Overall, 33% of ALL cases had at least one myeloid CH-associated mutation with the following frequencies: TP53 (18%), DNMT3A (5%), TET2 (4%), ASXL1 (3%), RUNX1 (3%), IDH2 (2%), BCORL1 (2%), U2AF1 (1%), CUX1 (1%), BCOR (1%), CBL (1%), and SF3B1 (1%). We further investigated the association between mutations and clinical variables by calculating the odds-ratios (OR) with adjustments for multiple testing by using the Benjamini-Hochberg method. Myeloid CH mutations in TP53, DNMT3A, TET2, and ASXL1 genes were more common in older adults with ALL. Mutations in TP53 and DNMT3A were enriched in therapy-related B-ALL group (OR 2.6, 95% CI= 1.3-5.5, q= .007 and OR 4.7, CI= 1.2-18.1, q= .01, respectively), and were more commonly associated with hypodiploid (OR 7.1, 95% CI= 1.5-32.7, q=.004 and OR 5.2, 95% CI= 0.6-48, q=.1, respectively) and hyperdiploid (OR 5.9, 95% CI= 2.7-13.1, q=<0.001 and OR 3.9, 95% CI= 1.1-16.5, q=.04, respectively) cytogenetics. We stratified the ALL cohort into three groups: TP53-mutated ALL, non-TP53 CH-associated ALL, and ALL without myeloid CH mutations. Patients in both groups of CH-associated B-ALL had worse overall survival (OS) than patients without myeloid CH mutations (median OS, 23 vs 25 vs 48 months, respectively, p=.001). In a multivariable analysis of OS for B-ALL, adjusting for age and therapy-related ALL, both TP53 (HR 1.95, 95% CI=1.2-3.17, p=.006) and non-TP53 myeloid CH (HR 1.95, 95% CI=1.14-3.35, p=.01) were associated with worse OS. Based on the data indicating high allelic frequencies of myeloid CH mutations in ALL, we hypothesized that these mutations are ancestral events arising in HSCs. To gain insights into the clonal architecture, we performed single cell DNA+protein sequencing using the Mission Bio, lnc platform. Data from 4,180 cells obtained from a CH-associated B-ALL patient are shown in Fig 1B. Cells were clustered by immunophenotype, and mutations were overlayed into 8 clusters. IDH2 R140Q mutation was shared between ALL and myeloid compartments. PTPN11 mutation was more prevalent in B-lymphoblasts. We observed a similar pattern in 7 additional patients, indicating that both lymphoblasts and non-malignant myeloid cells share CH mutations in ALL patients. Next, we investigated whether CH can be detected as a precursor lesion prior to ALL. In a patient with therapy-related B-ALL, a TET2 CH clone was detectable 5 years prior to ALL, and additional SF3B1 and CDKN2A mutations were detectable at the time of B-ALL diagnosis. We confirmed the presence of these mutations in B-lymphoblasts by single cell sequencing. This patient had persistent TET2/SF3B1 mutated CH despite achieving remission from B-ALL. We observed a similar pattern in four additional patients, for whom the CH mutations were detectable several years prior to their ALL diagnosis and were present in the dominant ALL clone. Collectively, these data support a model in which ALL can arise from pre-existing clones harboring myeloid CH mutations in a significant fraction of older adults. ALL with myeloid mutations is a distinct clinicopathologic entity with inferior survival outcomes. Future studies may decipher mechanisms driving lymphoid bias of CH-mediated leukemogenesis that can be targeted to prevent and treat ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Rapid Generation of Murine CLL/Richter Syndrome Models By Multiplexed CRISPR/Cas9 Editing of Common CLL Driver Genes

Background: Recent large-scale next generation sequencing studies have revealed a highly heterogeneous genetic landscape in CLL. However, the majority of the genetic lesions are relatively infrequent and their functional role and clinical relevance are still unknown. Moreover, in vivo models to study how these lesions cooperate between themselves and affect the response to novel therapeutic agents are still lacking. To further understand the relative contribution of individual genetic lesions to CLL development, progression and response to treatment, we performed multiplex CRISPR/Cas9-editing of normal and leukemic murine B cells by targeting a large set of CLL driver genes. Materials and Methods: Multiplexed CRISPR/Cas9 editing was done on normal B1 and B2 B cells isolated from the peritoneal cavity of young (<4 months) del(13q14)MDR-/- mice or on leukemic B1 B cells isolated from the spleen of old (>12 months) Eμ-TCL1 mice using a recently established procedure (Chakraborty et al, Blood 2021). The effects of the introduced genetic lesions on leukemia cell growth and treatment resistance were investigated by analyzing the mutant allele frequency (MAF) of the injected cells and cells recovered from the spleens of untreated or venetoclax-treated mice. Results: To investigate whether multiplex CRISPR/Cas9 editing can be used to transform normal murine B cells derived from del(13q14)MDR-/- mice, we simultaneously targeted 12 putative CLL driver genes representative of cellular pathways that are commonly affected in CLL, including DNA damage response (TP53, ATM and POT1), chromatin modifiers (CHD2, SETD2), cell cycle (RB1), inflammation (SAMHD1) and the NF-kB (BIRC3, NFKBIE, TRAIL-R), NOTCH (FBXW7) and MYC pathways (MGA) (n=4 experiments). The transfected B1 and B2 B cells were transplanted in the peritoneal cavity of B cell-deficient NSG mice and analyzed at different timepoints within a range of 1 to 5 months. Both cellular subsets were detected in the peritoneal cavity of NSG mice at the initial timepoint, but B1 B cells preferentially expanded at the later timepoints, giving rise to CD19+/CD5+ leukemias 3-5 months after transplantation. An increase in MAF was observed for all targeted genes, suggesting that all of the introduced genetic lesions provide a growth advantage to the leukemic cells. To investigate the impact of selected genetic lesions on CLL progression and Richter transformation (RT), we simultaneously introduced activating mutations in NOTCH1 (targeting of the PEST domain) or inactivating mutations in MGA, ATM, TP53, CDKN2A, CDKN2B, BIRC3 and NFKBIE in CLL cells derived from Eμ-TCL1 transgenic mice (n=3 independent experiments). A rise in MAF was observed for all 8 genes following in vivo propagation of the edited cells, suggesting that all of these mutations are positively selected during leukemia progression. However, selection of NOTCH1, TP53, ATM, BIRC3 and NFKBIE mutations occurred more rapidly, reaching the maximum MAF already after the 1st passage in vivo, whereas the MAF of genetic lesions specifically associated with Richter transformation, i.e. CDKN2A, CDKN2B and MGA, rouse more slowly and increased during serial passages (Figure 1). Interestingly, a substantial increase in MAF was observed for CDKN2A and CDKN2B when cells from the 1st passage were placed in culture (from 44% to 93%, p=0.017, and from 64% to 94%, p=0.043, after 6 weeks in culture for CDKN2A and CDKN2B, respectively), consistent with our recent finding that biallelic disruption of these genes allows for spontaneous proliferation of Eμ-TCL1 leukemia cells and may represent the transforming factor in a subset of cases with Richter Transformation. In contrast, the MAF of the other five driver genes showed no or only minor changes during culture. The MAF of CDKN2A and CDKN2B also increased to a greater extent in mice treated with venetoclax compared to untreated mice, indicating that cells with such genetic lesions are more resistant to venetoclax treatment. Conclusions: This study shows that multiplex CRISPR/Cas9 editing can be used to rapidly generate murine leukemias with the genetic make-up of human CLL or Richter Syndrome. These models can represent an important preclinical tool to study how individual genetic lesions cooperate during CLL development and progression and how they affect the response to treatment with novel therapeutic agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Homologous Recombination Deficiency and Tumor Mutational Burden as Clinical Biomarkers for Melanoma

Abstract Introduction/Objective Homologous recombination deficiency (HRD) cancers have high levels of genetic instability, potentially being targets for platinum agents or poly-ADP ribose polymerase inhibitors. Tumor mutational burden (TMB), which is used to guide immunotherapy, is thought to be associated with HRD. This study aimed to evaluate the frequency of HRD and its correlation with other genomic parameters in melanoma. Methods/Case Report A total of 14 cases with the diagnosis of melanoma were found in the genomic database between March 1, 2021 to July 30, 2021. One case had an EWSR1-ATF1 translocation and was excluded. Copy number variations and absence of heterozygosity detection were analyzed by the NxClinical software (BioDiscovery, El Segundo, CA). HRD was scored by three DNA-based measures of genomic instability (loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale transitions (LST)). HRD-LOH is the number of LOH regions &amp;gt; 15 Mb and do not cross the centromere. HRD-TAI is the number of regions &amp;gt; 10 Mb with allelic imbalance that extend to one of the subtelomeres and do not cross the centromere. HRD-LST is the number of break points between regions &amp;gt; 10 Mb. A high HRD score and LOH percentage were defined as ≥ 42 and ≥ 16%, respectively. Results (if a Case Study enter NA) Of the 13 cases, 10 had a high TMB and 3 had a low TMB. A high HRD score was found in 3 cases; high LOH percentage was found in 4 cases. PDL-1 was expressed in 8 of the 11 cases tested. A high HRD score correlated with high LOH percentage by Pearson’s chi-squared test (p &amp;lt; 0.01). No significant association was found between TMB and HRD score or LOH percentage. Common alterations among cases with high TMB included mutations in NRAS (3/13; 23.1%), CDKN2A (2/13; 15.4%), BRAF (2/13; 15.4%), BRCA2 (2/13; 15.4%), RAF1 (2/13; 15.4%), RET (1/13; 7.7%), IDH1 (1/13; 7.7%), PALB2 (1/13; 7.7%), BRCA1 (1/13; 7.7%), ERBB2 (1/13; 7.7%), NF1 (1/13; 7.7%), MLH1 (1/13; 7.7%), ATM (1/13; 7.7%) and MET (1/13; 7.7%). One case had amplifications in CCND1, FGF 3/4/19 and MET. Among cases with low TMB, alterations included MYC amplification (1/3; 33.3%) and BRAF mutations (2/3; 66.6%). Conclusion In our study, HRD score is not associated with TMB status. There is a strong correlation between HRD score and LOH percentage, suggesting that LOH is a major component in the genomic scarring seen in HRD. HRD may be an independent biomarker of immunogenicity to guide immunotherapy.

Molecular profile and its clinical impact of IDH1 mutated versus IDH1 wild type intrahepatic cholangiocarcinoma

IDH1-mutated cholangiocarcinomas (CCAs) are an interesting group of neoplasia with particular behavior and therapeutic implications. The aim of the present work is to highlight the differences characterizing IDH1m and IDH1wt CCAs in terms of genomic landscape. 284 patients with iCCA treated for resectable, locally advanced or metastatic disease were selected and studied with the FOUNDATION Cdx technology. A comparative genomic analysis and survival analyses for the most relevant altered genes were performed between IDH1m and IDH1wt patients. Overall, 125 patients were IDH1m and 122 IDH1wt. IDH1m patients showed higher mutation rates compared to IDH1wt in CDKN2B and lower mutation rates in several genes including TP53, FGFR2, BRCA2, ATM, MAP3K1, NOTCH2, ZNF703, CCND1, NBN, NF1, MAP3KI3, and RAD21. At the survival analysis, IDH1m and IDH1wt patients showed no statistically differences in terms of survival outcomes, but a trend in favor of IDH1wt patients was observed. Differences in prognostic values of the most common altered genes were reported. In surgical setting, in IDH1m group the presence of CDKN2A and CDKN2B mutations negatively impact DFS, whereas the presence of CDKN2A, CDKN2B, and PBRM1 mutations negatively impact OS. In advanced setting, in the IDH1m group, the presence of KRAS/NRAS and TP53 mutations negatively impact PFS, whereas the presence of TP53 and PIK3CA mutations negatively impact OS; in the IDH1wt group, only the presence of MTAP mutation negatively impact PFS, whereas the presence of TP53 mutation negatively impact OS. We highlighted several molecular differences with distinct prognostic implications between IDH1m and IDH1wt patients.

Indications for Digital Monitoring of Patients With Multiple Nevi: Recommendations from the International Dermoscopy Society.

In patients with multiple nevi, sequential imaging using total body skin photography (TBSP) coupled with digital dermoscopy (DD) documentation reduces unnecessary excisions and improves the early detection of melanoma. Correct patient selection is essential for optimizing the efficacy of this diagnostic approach. The purpose of the study was to identify, via expert consensus, the best indications for TBSP and DD follow-up. This study was performed on behalf of the International Dermoscopy Society (IDS). We attained consensus by using an e-Delphi methodology. The panel of participants included international experts in dermoscopy. In each Delphi round, experts were asked to select from a list of indications for TBSP and DD. Expert consensus was attained after 3 rounds of Delphi. Participants considered a total nevus count of 60 or more nevi or the presence of a CDKN2A mutation sufficient to refer the patient for digital monitoring. Patients with more than 40 nevi were only considered an indication in case of personal history of melanoma or red hair and/or a MC1R mutation or history of organ transplantation. Our recommendations support clinicians in choosing appropriate follow-up regimens for patients with multiple nevi and in applying the time-consuming procedure of sequential imaging more efficiently. Further studies and real-life data are needed to confirm the usefulness of this list of indications in clinical practice.

RGWAS: A Novel Genome-Wide Association Study to Link Genomics to Radiomics for Pancreatic Cancer

<h3>Purpose/Objective(s)</h3> Pancreatic cancer is a critical global health problem with the highest mortality rate among all major cancers. The 5-year survival rate remained at ∼10% despite decades of clinical and research efforts. Clearly, new and synergistic approaches are needed to battle this ferocious disease. In this study, a novel radiomics-based genome-wide association study (RGWAS) was conducted in a unique patient population, linking genomics to radiomics in pancreatic cancer. <h3>Materials/Methods</h3> A unique pancreatic cancer rapid autopsy dataset was included for the study, where comprehensive tissue samples were collected from tumors and normal organs within hours of patient death. On 28 patients, whole exome sequencing (WES) was conducted to identify significant somatic mutations with VarScan. Diagnostic dynamic contrast CT of abdomen was used to manually segment the primary tumor and metastatic lesions, from which ∼1000 radiomic features were extracted. Using LASSO, radiomic features most significantly associated with clinical endpoints such as overall survival and with interesting genetic variants (i.e., somatic SNVs) were identified. <h3>Results</h3> Based on WES, somatic mutations of KRAS, TP53, and CDKN2A occur at high rates in both primary and metastatic tumors. Interestingly, some highly occurring somatic mutations in the primary tumor, such as BRCA1 and MYC, were not top ranked in metastases. Instead, somatic mutations of IS2, the insulin receptor substrate 2, occur at a high rate in metastases. RGWAS found several significant peaks (P-value<10⁻⁵) for certain radiomic features, uncovering interesting correlations between genetic mutations and imaging phenotypes. Some of these radiomic features were consistently associated with genetic variants between primary and metastatic tumors. <h3>Conclusion</h3> A unique patient dataset with rapid autopsy tumor and normal tissue samples was analyzed using a novel integrative RGWAS approach. RGWAS linked relevant radiomic features to interesting oncogenes and clinical phenotypes. These findings provide promising genetic and radiomic feature candidates for follow-up work, and shed light on the mechanism of pancreatic cancer progression and metastasis.

Targeted-sequence of normal urothelium and tumor of patients with non-muscle invasive bladder cancer

During tumorigenesis, certain tissues are colonized by mutant clones with oncogenic driver mutations as precancer lesions. These mutations can facilitate clonal expansion and may contribute to malignant transformation. The molecular features of low-grade non-muscle invasive bladder cancer (NMIBC) and high-grade bladder cancer are so distinct that they are thought to follow different evolutionary tumorigenesis pathways. Although NMIBC accounts for most bladder tumors, the somatic mutation patterns in “precancer” urothelium of patients with NMIBC remain unclear. Here, we analyzed specimens of normal urothelium and bladder tumors from patients with low-grade and high-grade NMIBC and investigated the genomic evolution of the cancer. Somatic mutations were analyzed using 50 oncogene-targeted sequences and droplet digital polymerase chain reaction for TERT promoter mutations. Somatic mutations in TERT promoter, FGFR3, and CDKN2A were characteristically identified in the normal urothelium of patients with NMIBC. These mutations, consistently identified in both tumor and normal specimens, likely affect clonal expansion during the malignant transformation of NMIBC. Though larger samples and comprehensive study are warranted to confirm our results, the difference in mutational landscape of the precancerous urothelium of patients with bladder cancer could offer deeper understandings of genomic evolution in bladder tumorigenesis.

Melanoma-specific survival before and after inclusion in a familialmelanoma dermatologic surveillance program in CDKN2A mutation carriers and non-carriers.

Inherited mutations in the CDKN2A gene are among the strongest known risk factors for cutaneous melanoma. Further, previous studies have reported inferior melanoma-specific survival in CDKN2A mutation carriers. Here, the melanoma-specific survival was studied, depending on CDKN2A carrier status and if the melanomas had been diagnosed before or after families were included in a surveillance program. Melanoma-prone families participating in this study were identified through a nationwide preventive program starting in 1987. Information on melanoma tumours and deaths was obtained through the Swedish Cancer Registry and Cause of Death Registry. Kaplan-Meier and Cox proportional hazards regression models were used to assess melanoma-specific survival in four defined cohorts, CDKN2A mutation (MUT) carriers with first invasive melanoma before or after inclusion [MUT-pre (n=53) and MUT-post (n=43)] and likewise in CDKN2A wild type (WT) cases [WT-pre (n=255) and WT-post (n=122)]. The MUT-pre and MUT-post cases were diagnosed with their first invasive melanoma at a significantly younger ages (38 and 42 years, respectively) than the WT-pre and WT-post cases (48 and 57 years, respectively). The melanomas in the MUT-pre had significantly higher T stage compared with MUT-post (p=0.006), whereas no such difference was seen comparing WT-pre with WT-post (p=0.849). MUT-pre had compared with WT-pre, significantly worse melanoma-specific survival, unadjusted (HR 2.33, 95% CI 1.33-4.08, p=0.003) adjusted (HR 2.70, 95% CI 1.46-5.00, p=0.001). However, the MUT-post cases had compared with the WT-post cases, no significant survival differences. This study is the first to address the impact on survival from introducing a dermatologic surveillance program to familial melanoma cases with or without CDKN2A mutations. The CDKN2A-mut carriers appeared to have a clear benefit with less advanced melanomas diagnosed and better melanoma-specific survival after inclusion. Among the CDKN2A-wt cases, the effect of the inclusion on the studied outcomes was less evident.

Prognostic Factors of Low-Grade Gliomas in Adults.

Adult low-grade gliomas are a rare and aggressive pathology of the central nervous system. Some of their characteristics contribute to the patient’s life expectancy and to their management. This study aimed to characterize and identify the main prognostic factors of low-grade gliomas. The six-year retrospective study statistically analyzed the demographic, imaging, and morphogenetic characteristics of the patient group through appropriate parameters. Immunohistochemical tests were performed: IDH1, Ki-67, p53, and Nestin, as well as FISH tests on the CDKN2A gene and 1p/19q codeletion. The pathology was prevalent in females, with patients having an average age of 56.31 years. The average tumor volume was 41.61 cm3, producing a midline shift with an average of 7.5 mm. Its displacement had a negative impact on survival. The presence of a residual tumor resulted in decreased survival and is an independent risk factor for mortality. Positivity for p53 identified a low survival rate. CDKN2A mutations were an independent risk factor for mortality. We identified that a negative prognosis is influenced by the association of epilepsy with headache, tumor volume, and immunoreactivity to IDH1 and p53. Independent factors associated with mortality were midline shift, presence of tumor residue, and CDKN2A gene deletions and amplifications.

Comprehensive analyses of genomic features and mutational signatures in adenosquamous carcinoma of the lung.

Adenosquamous carcinoma (ASC) of the lung is a relatively rare tumor with strong aggressiveness and poor prognosis. The analysis of mutational signatures is becoming routine in cancer genomics and has implications for pathogenesis, classification, and prognosis. However, the distribution of mutational signatures in ASC patients has not been evaluated. In this study, we sought to reveal the landscape of genomic mutations and mutational signatures in ASC. Next-generation sequencing (NGS) technology was used to retrieve genomic information for 124 ASC patients. TP53 and EGFR were the most prevalent somatic mutations observed, and were present in 66.9% and 54.8% of patients, respectively. CDKN2A (21%), TERT (21%), and LRP1B (18.5%) mutations were also observed. An analysis of gene fusion/rearrangement characteristics revealed a total of 64 gene fusions. The highest frequency of variants was determined for ALK fusions, with six ALK-EML4 classical and two intergenic ALK fusions, followed by three CD74-ROS1 fusions and one ROS1-SYN3 fusion. EGFR 19del (45.6%), and EGFR L858R (38.2%) and its amplification (29.4%) were the top three EGFR mutations. We extracted mutational signatures from NGS data and then performed a statistical analysis in order to search for genomic and clinical features that could be linked to mutation signatures. Amongst signatures cataloged at COSMIC, the most prevalent, high-frequency base changes were for C > T; and the five most frequent signatures, from highest to lowest, were 2, 3, 1, 30, and 13. Signatures 1 and 6 were determined to be associated with age and tumor stage, respectively, and Signatures 22 and 30 were significantly related to smoking. We additionally evaluated the correlation between tumor mutational burden (TMB) and genomic variations. We found that mutations ARID2, BRCA1, and KEAP1 were associated with high TMB. The homologous recombination repair (HRR) pathway-related gene mutation displayed a slightly higher TMB than those without mutations. Our study is the first to report comprehensive genomic features and mutational signatures in Chinese ASC patients. Results obtained from our study will help the scientific community better understand signature-related mutational processes in ASC.