Somatic TP53 Mutations Research Articles

Abstract 3683: Prognostic value and biological correlates of LINE1 hypomethylation in oropharyngeal cancer

Abstract Background. We have recently demonstrated that LINE-1 hypomethylation is a negative prognostic factor in oropharyngeal squamous cell carcinoma (OPSCC). HPV-positive patients with LINE-1 methylation <55% showed an almost 5-fold higher risk of death than hypermethylated once, with an overall survival as poor as HPV-negative patients. Somatic TP53 mutations are a common feature in HPV-negative OPSCC, whereas their presence has been reported in only a small subset of HPV-related OPSCC. At present, little is known about the correlation between LINE-1 methylation and p53 expression status and/or TP53 mutation in OPSCC. Methods. Since 2019, an ongoing prospective study has been enrolling patients with OPSCC in nine cancer centers in Northern Italy. HPV16 E6 DNA was quantified using real-time quantitative PCR (qPCR). LINE-1 methylation was evaluated by methylation-specific qPCR. p53 expression was detected by immunohistochemical staining. TP53 exon mutations were analyzed by Next Generation Sequencing. Results. Up to date, 134 OPSCC patients have been enrolled, including cancer of the tonsil (n=74; 55.2%), base of tongue (n=47; 35.1%), and other subsites (n=13; 9.7%). HPV16 DNA was found in 54.3% of cases: 65.7% in base of tongue, 57.1% in tonsil, and 16.7% in other subsites (p<0.001). LINE-1 methylation was higher in HPV16-positive patients (median: 55.8%, interquartile rage [IQR]: 42.5-68.5) than HPV16-negative ones (median: 40.7; IQR: 23.9-59.1; p=0.003). Nevertheless, the median LINE-1 methylation was lower (50.7%) among HPV-16 OPSCC patients who relapsed than those who did not (56.0%), thus confirming the findings previously reported. However, data from the present investigation are presently not mature to purse the study aims (median follow-up: 12.3 months). The correlation between LINE-1 hypomethylation and p53 expression has also been evaluated in OPSCC patients. p53 expression was categorized into three groups (0%, 1-49%, and ≥50%) based on the overall intensity of nuclear staining of the tumor cells. p53 was considered null, overexpressed, or wild type when its nuclear staining was 0 %, ≥50%, or ranged between 1% and 49%, respectively. Of note, the highest level of LINE-1 methylation was observed in OPSCC tissues with nuclear p53 staining between 1% and 49% (57.8%), whereas OPSCC patients with p53 expression ≥50% showed a decline of LINE-1 methylation levels (40.6; p=0.026). Interestingly, LINE-1 methylation was lower in OPSCC patients harboring TP53 mutations in both HPV16-negative (24.8%) and HPV16-positive (24.0%) OPSCC patients, even if statistical significance was reached only in the HPV-negative group (p=0.015). Conclusion. Preliminary results confirm the potential of LINE-1 methylation to identify HPV16-positive patients with poor prognosis. In addition, this study suggest that TP53 mutations might influence LINE-1 methylation, irrespective of HPV status. Citation Format: Mariateresa Casarotto, Roberto Guerrieri, Paolo Boscolo-Rizzo, Monica Schiappacassi, Sara D'Andrea, Valentina Lupato, Susanna Chiocca, Marta Tagliabue, Rita De Berardinis, Anna Menegaldo, Alice Piccinato, Paola Stritoni, Mohssen Ansarin, Doriano Politi, Giuseppe Fanetti, Giorgio Giurato, Jerry Polesel, Elisabetta Fratta. Prognostic value and biological correlates of LINE1 hypomethylation in oropharyngeal cancer [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 3683.

Abstract 4781: Evaluation of a TP53 biomarker for lung cancer risk in nasal brushings and peripheral blood

Abstract Background and Purpose: Early detection of lung cancer through annual screening with low-dose computed tomography (LDCT) scans enables potentially curative surgical or stereotactic body radiation therapy (SBRT) treatment and thereby significantly reduces mortality. However, LDCT screening among those currently eligible based primarily on age and smoking criteria yields a large number of false-positive findings and many lung cancers occur among individuals who currently are ineligible. A biomarker that identifies lung cancer risk independent from that caused by age and cigarette smoking might increase specificity and increase the number of people eligible for LDCT screening. Recently we demonstrated that a biomarker measuring TP53 somatic mutations in bronchial brush biopsy specimens from grossly normal airway epithelium was significantly associated with cancer status independent of age and smoking status. Additionally, the TP53 biomarker was synergistic with traditional risk score models (e.g. PLCOM2012 risk score). The goal of this pilot study was to test the TP53 biomarker in nasal brush biopsy specimens, a potential surrogate tissue obtained using less invasive collection methods. Methods: Nasal brushings from 10 lung cancer and 11 control subjects were collected at the University of Toledo and Vanderbilt University Medical Center under approved institutional protocols. Genomic DNA (gDNA) was extracted with the Qiagen AllPrep DNA/RNA kit using a modified protocol. Amplifiable copies of gDNA were measured using quantitative PCR. For each test a sample comprising 25,000 amplifiable copies of gDNA was combined with 50,000 copies of a TP53 internal standard and a complexity control, then PCR amplified, purified and barcoded. Samples were pooled to create two NGS libraries, each with approximately equal numbers of cases and controls, and sequenced on an Illumina MiSeq (Two V3 flow cells, 2 x 250 paired-end). Bioinformatic analysis was conducted using Qiagen CLC Workbench. Variants were called using the Basic Variant Detection Tool. Poisson Exact Test was utilized to determine the significance of endogenous variants relative to internal standard variants at the same base position and a Bonferroni correction was applied to minimize false discovery. Comparison of variant numbers between cases and controls was conducted using an f-test followed by a paired t-test. Results and Conclusions: Consistent with previous results in airway epithelium, in this pilot study the TP53 mutation biomarker measured in nasal brushing was associated with lung cancer (p=0.0085). Measurement of the TP53 biomarker in nasal brush specimens in a larger case-control study currently is underway along with a pilot study in gDNA from peripheral blood. Citation Format: Erin L. Crawford, Daniel J. Craig, Patrick T. Gorman, Mohamed Omballi, Rami Ahmad, Eric L. Grogan, Stephen A. Deppen, James C. Willey. Evaluation of a TP53 biomarker for lung cancer risk in nasal brushings and peripheral blood [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 4781.

Targeted DNA sequencing of high-grade serous ovarian carcinoma reveals association of TP53 mutations with platinum resistance when combined with gene expression.

High-grade serous ovarian carcinoma (HGSC) is the most common subtype of ovarian cancer and is among the most fatal gynecological malignancies worldwide, due to late diagnosis at advanced stages and frequent therapy resistance. In 47 HGSC patients, we assessed somatic and germline genetic variability of a custom panel of 144 known or suspected HGSC-related genes by high-coverage targeted DNA sequencing to identify the genetic determinants associated with resistance to platinum-based therapy. In the germline, the most mutated genes were DNAH14 (17%), RAD51B (17%), CFTR (13%), BRCA1 (11%), and RAD51 (11%). Somatically, the most mutated gene was TP53 (98%), followed by CSMD1/2/3 (19/19/36%), and CFTR (23%). Results were compared with those from whole exome sequencing of a similar set of 35 HGSC patients. Somatic variants in TP53 were also validated using GENIE data of 1287 HGSC samples. Our approach showed increased prevalence of high impact somatic and germline mutations, especially those affecting splice sites of TP53, compared to validation datasets. Furthermore, nonsense TP53 somatic mutations were negatively associated with patient survival. Elevated TP53 transcript levels were associated with platinum resistance and presence of TP53 missense mutations, while decreased TP53 levels were found in tumors carrying mutations with predicted high impact, which was confirmed in The Cancer Genome Atlas data (n = 260). Targeted DNA sequencing of TP53 combined with transcript quantification may contribute to the concept of precision oncology of HGSC. Future studies should explore targeting the p53 pathway based on specific mutation types and co-analyze the expression and mutational profiles of other key cancer genes.

Abstract B101: Characterizing somatic TP53 mutations in non-cancerous and high-risk fallopian tubes using ultra-deep sequencing

Abstract High-grade serous carcinoma (HGSC), the most common and pervasive subtype of ovarian cancer originates in the fallopian tube epithelium from precursor lesions carrying somatic TP53 mutations. These lesions may evolve into carcinomas, eventually spreading to the ovary and peritoneum1,2. Recent research in non-cancerous tissues and gynecologic liquid biopsies of individuals with and without HGSC have demonstrated that somatic TP53 mutations accumulate and clonally expand with age3–9. However, TP53 somatic evolution has not been characterized in the normal fallopian tube epithelium. In addition, it is not known whether TP53 mutations are more abundant in individuals with germline BRCA1/2 mutations (BRCA carriers), who are at a high risk of HGSC10. The goals of this study were to characterize age-related TP53 somatic mutations in non-cancer fallopian tube epithelium across the human lifespan and to determine whether the TP53 mutational landscape is altered in BRCA carriers. Our cohort consisted of 1) autopsy cases, spanning the ages of 0-91 years, from individuals without ovarian cancer (N=46) and 2) prophylactic surgery cases from BRCA carriers and non-carriers (N=26, ongoing analyses). Fallopian tube tissues were collected, frozen, and macrodissected using a 1mm biopsy punch, enriching for the fallopian tube epithelium. DNA was extracted from each sample, the sequenced for TP53 using ultra-deep duplex sequencing (mean depth: ~12,000x). In the normal fallopian tube epithelium collected across human lifespan, we found that the number of unique TP53 mutations and the size of pathogenic clones increased with age, showcasing somatic evolution and clonal expansion of TP53 in non-cancerous fallopian tube epithelium with age. We also found that the number of TP53 mutations in hotspot codons increased with age, suggesting that cancer-like mutations are positively selected as individuals age. We compared the normal fallopian tube TP53 mutations with HGSC TP53 mutations and demonstrated that the types of mutations (e.g. indels, splice, nonsense, missense, and silent mutations) in the aging fallopian tube closely mirrored the mutations types observed in HGSC. Data analysis from BRCA carriers is ongoing, but based on preliminary data, we anticipate a higher burden of pathogenic TP53 mutations, indicating that predisposition to HGSC in BRCA carriers may be related to an excess of TP53 somatic evolution in the fallopian tube. Altogether, our research demonstrates that TP53 somatic evolution is a normal aging phenotype in the fallopian tube, with more and larger pathogenic clones occurring later in life. We expect to determine whether this effect is more prominent in BRCA carriers, which would provide a much needed biological explanation to understand the increased risk of HGSC in these patients, opening new venues for better cancer prediction and prevention. Citation Format: Coleen Ann Coombes, Elizabeth U. Parker, Brendan F. Kohrn, Jeanne Uy Fredrickson, Elena Latorre-Esteves, Emma Hazard, Shreya Suresh, Marc R. Radke, Anh P. Vo, Barbara S. Norquist, Rosana Risques. Characterizing somatic TP53 mutations in non-cancerous and high-risk fallopian tubes using ultra-deep sequencing [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B101.

Abstract A057: Inter-tumor heterogeneity revealed by multi-site whole genome sequencing of high grade serous ovarian cancer

Abstract A057: Inter-tumor heterogeneity revealed by multi-site whole genome sequencing of high grade serous ovarian cancer

Abstract A004: TP53 mutational landscape in non-cancerous endometrium during the lifespan of Black and White individuals

Abstract Background TP53 somatic mutations accumulate and clonally expand in histologically normal tissues with aging, however, little is known about this process and how it influences cancer development. Within endometrial carcinoma, TP53 mutations tend to be found in aggressive, non-endometrioid histologies associated with poor survival outcomes, more commonly affecting Black women. We seek to understand the TP53 mutational burden in benign endometrium by characterizing mutations acquired throughout a lifespan with the integration of social determinants of health in Black and White women. Methods Endometrial tissue was collected at autopsy or hysterectomy, confirmed with H&E staining, and macrodissected. Ultra-deep duplex sequencing (~10,000x) of isolated DNA was used to sequence TP53somatic mutations with high resolution. Each individual’s TP53 mutation frequency was calculated as the number of unique TP53 variants divided by the total duplex nucleotides sequenced. Mutations were classified as pathogenic if insertion-deletion, splice, or nonsense in addition to all pathogenic substitutions identified by the Seshat TP53 database. Socioeconomic metrics were acquired using zip codes and the 2021 US Census Bureau American Community Survey. Individuals with a history of endometriosis, prior malignancy, or prior chemotherapy were excluded. Results Benign endometrial tissue was collected from 69 individuals (24 Black, 45 White), including 22 aged 0-19 years, 25 aged 20-39 years, 17 aged 40-59 years, and 5 aged over 60 years. 174 TP53 variants were identified. Variant types included 66.7% missense (n=116), 13.2% insertion-deletion (n=23), 12.1% synonymous (n=21), 4.6% nonsense (n=8), and 3.4% splice (n=6). 102 TP53 mutations (58.6%) were pathogenic. TP53 mutation frequency increased linearly with age (Spearman correlation p<0.01). 12.6% of mutations were identified in more than one duplex mutant read (large clone) beginning at age 29 through age 81. The frequency of large TP53 mutant clones increased exponentially with age. In this limited dataset, there was no statistical association between socioeconomic metrics and the mutational burden of Black and White individuals. Conclusions The number and size of TP53 mutant clones in benign endometrium are associated with older age, indicating clonal evolution through life. Most somatic mutations identified were pathogenic substitutions or loss-of-function variants, confirming positive selection of cancer-like mutations. Additional data are needed to determine the role of socioeconomic factors in TP53 genomic burden. Citation Format: Eric Rios-Doria, Elizabeth U. Parker, Brendan F. Kohrn, Elena Latorre-Estevez, Jeanne Fredrickson, Elizabeth M. Swisher, Kemi M. Doll, Rosana Risques. TP53 mutational landscape in non-cancerous endometrium during the lifespan of Black and White individuals [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A004.

BRCA1, BRCA2, and TP53 germline and somatic variants and clinicopathological characteristics of Brazilian patients with epithelial ovarian cancer.

Approximately 3/4 of ovarian cancers are diagnosed in advanced stages, with the high-grade epithelial ovarian carcinoma (EOC) accounting for 90% of the cases. EOC present high genomic instability and somatic loss-of-function variants in genes associated with homologous recombination mutational repair pathway (HR), such as BRCA1 and BRCA2, and in TP53. The identification of germline variants in HR genes in EOC is relevant for treatment of platinum resistant tumors and relapsed tumors with therapies based in synthetic lethality such as PARP inhibitors. Patients with somatic variants in HR genes may also benefit from these therapies. In this work was analyzed the frequency of somatic variants in BRCA1, BRCA2, and TP53 in an EOC cohort of Brazilian patients, estimating the proportion of variants in tumoral tissue and their association with progression-free survival and overall survival. The study was conducted with paired blood/tumor samples from 56 patients. Germline and tumoral sequences of BRCA1, BRCA2, and TP53 were obtained by massive parallel sequencing. The HaplotypeCaller method was used for calling germline variants, and somatic variants were called with Mutect2. A total of 26 germline variants were found, and seven patients presented germline pathogenic or likely pathogenic variants in BRCA1 or BRCA2. The analysis of tumoral tissue identified 52 somatic variants in 41 patients, being 43 somatic variants affecting or likely affecting protein functionality. Survival analyses showed that tumor staging was associated with overall survival (OS), while the presence of somatic mutation in TP53 was not associated with OS or progression-free survival. Frequency of pathogenic or likely pathogenic germline variants in BRCA1 and BRCA2 (12.5%) was lower in comparison with other studies. TP53 was the most altered gene in tumors, with 62.5% presenting likely non-functional or non-functional somatic variants, while eight 14.2% presented likely non-functional or non-functional somatic variants in BRCA1 or BRCA2.

Abstract B016: Recapitulation of gene expression-based immune subtypes of breast cancer using immunohistochemical staining

Abstract Using gene expression profiling data, we previously identified three immune subtypes (low-TIL, high-TIL, high-ISG) of breast cancer (BC) among Chinese BC patients in Hong Kong (HK) (Zhu et al., Breast Cancer Res, 2017). To investigate if standard in situ immunohistochemical (IHC) staining could capture the BC immune landscape, we analyzed archival tissue sections from 148 HKBC patients who were previously characterized based on gene expression. Dual IHC staining for 8 immune markers (CD3, CD8, CD20, FOXP3, CD68, CD163, PDL1, and IDO1) and PanCK was performed. Digitized whole slide images for each marker were analyzed using high-accuracy machine learning algorithms trained to quantify the percentage of positively stained regions. Strong correlations were found between IHC immune markers and gene expression-based immune and intrinsic subtypes. Compared to patients with lower level of tumor-infiltrating lymphocytes (low-TIL) classified based on gene expression data, those in the high-TIL subtype (characterized by high T cell and immune checkpoint gene expression) showed higher proportions of CD3, CD8, CD20, FOXP3, CD68, and PDL1 IHC positively stained cells. Similarly, the high-ISG subtype (with increased interferon-stimulated gene expression and TP53 somatic mutations) had higher proportions of CD3, CD8, CD20, FOXP3, and CD68 stained cells than the low-TIL subtype and in particular exhibited high CD163 staining, indicative of a proinflammatory tumor microenvironment. CD8, CD20, CD163, PDL1, and IDO1 expression varied significantly by PAM50 subtypes, with luminal (A and B) subtypes showing lower staining than non-luminal (HER2 and Basal) subtypes. A distinctive feature, however, of luminal B tumors was the higher infiltration by CD163-expressing cells when compared with the luminal A subtype. Except for FOXP3, all examined IHC markers showed significantly higher expression in patients with somatic TP53 mutations (CD3: P= 0.003, CD8: P= 0.005, CD20: P= 0.003, CD68: P= 0.002, CD163: P= 0.002, PDL1: P= 0.003, and IDO1: P= 0.013). On the other hand, expression of these markers did not significantly vary by PIK3CA mutation or germline APOBEC3A/B deletion polymorphism, which is more prevalent among East Asians than other populations and was previously linked to BC risk and immune environment. Of the BC risk factors assessed, only body mass index (BMI) demonstrated associations with immune cell densities, with higher BMI associated with lower expression of CD3, CD8, and CD20. This study sheds further light on the in situ immune landscape of gene expression-based BC subtypes, suggesting that standard IHC assessment of immune markers could refine BC subtype classification for clinical and epidemiological use. Citation Format: Mustapha Abubakar, Hela Koka, Priscilla Lee, Scott Lawrence, Karun Mutreja, Difei Wang, Bin Zhu, Shelly Tse, Xiaohong R Yang. Recapitulation of gene expression-based immune subtypes of breast cancer using immunohistochemical staining [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B016.

Abstract A040: Cellular ecosystem of breast tumor microenvironment in Chinese breast cancer patients

Abstract Background: Current evidence has demonstrated the important role of tumor microenvironment (TME) in cancer initiation and progression. However, the dynamic interactions among various cells and their communities in TME remain largely unknown. Recently, EcoTyper, was developed to discover different cell states and their ecosystems in silico using bulk gene expression data. The application of this tool in the Pan-Cancer analysis identified distinct cell states and ecotypes (co-association patterns between cell states) that act as strong classifiers for cancer types and subtypes. In this study, we aimed to discover the TME ecosystems in breast tumors from an East Asian population and associate them with well annotated clinical features and breast cancer (BC) risk factors. Methods: We applied the EcoTyper framework to tumor RNA-Seq data from 242 Hong Kong BC patients to identify carcinoma ecotypes (CEs) and associated them with intrinsic subtype (PAM50), driver gene mutations (based on whole genome/exome sequencing analyses), and well-known BC risk factors. Additionally, we used The Cancer Genome Atlas BC (TCGA-BRCA) data, which was part of the initial EcoTyper Pan-Cancer analysis, as a quality control and comparison dataset. The TCGA patients were predominantly of European ancestry and had similar age distribution (median=58 years) compared to HK patients (median=60 years). The distribution of PAM50 subtype was also similar between the two datasets, with HK having a slightly higher proportion of luminal A (38% vs. 33% in TCGA) and a lower proportion of basal (11% vs. 20% in TCGA). Results: Among all ten ecotypes, the frequency of each CE was very similar between HK and TCGA. In both datasets, the prevalence of these CEs varied by tumor subtype. CE1 and CE6 were enriched in luminal A and rare in basal, while CE9 was rarely found in luminal A but showed high prevalence in HER2-enriched and basal subtypes. CE7 was enriched in luminal B (especially in HK, where among tumors with CE7, 82% of them were luminal B), CE10 was enriched in HER2-enriched (especially in HK, where 41% of tumors with CE10 were HER2-enriched), and CE2 was enriched for basal subtype. CE8 was enriched for luminal A in HK and luminal B in TCGA. The presence of CE2, CE9, or CE10 was associated with higher prevalence of TP53 somatic mutations, particularly in HK. Among the BC risk factors examined, we did not find any statistically significant associations with any CEs, probably due to the small sample size. The work of assessing the associations with the abundance of each cell state and CE is currently ongoing. Conclusions: We replicated cellular ecosystems and their associations with tumor subtypes and driver gene mutations in an East Asian population, demonstrating its generalizability and potential utility in refining BC subtypes. Next, we plan to extend the analysis to paired adjacent normal tissue to further assess the role of these ecosystems in shaping TME heterogeneity and tumor evolution. Citation Format: Hela Koka, Xin Li, Difei Wang, Difei Wang, Kristine Jones, Kristine Jones, Aurelie Vogt, Aurelie Vogt, Bin Zhu, Amy Hutchinson, Amy Hutchinson, Belynda Hicks, Belynda Hicks, Priscilla Ming Yi Lee, Lap Ah Tse, Xiaohong R Yang. Cellular ecosystem of breast tumor microenvironment in Chinese breast cancer patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A040.

Abstract A044: Tumor-infiltrating lymphocyte density correlates with survival in pancreatic adenosquamous carcinoma

Abstract Background: Pancreatic adenosquamous carcinoma (PASC) is a rare and aggressive form of pancreatic cancer, representing only 1-5% of cases. Histologically, it is defined by the presence of at least 30% squamous differentiation mixed with ductal adenocarcinoma. No standard therapies or prognostic biomarkers have been validated to date in this understudied disease. Methods: All patients diagnosed with pancreatic cancer in the Johns Hopkins Cancer Registry from 2013 to 2018 were queried for adenosquamous histology. Archived slides of available tumor specimens were examined to quantify the percentage of squamous differentiation and tumor-infiltrating lymphocyte (TIL) density. Descriptive statistics were used to summarize clinical, pathologic, and molecular characteristics. Kaplan-Meier estimates and log-rank tests were used for survival analyses. Results: Among a total of 8,908 patients queried, 155 patients with adenosquamous histology were identified. Median overall survival (OS) was 14.2 months and 7.5 months for resectable and unresectable/metastatic disease, respectively. Molecular tumor testing revealed frequent somatic mutations in KRAS, TP53, and SMAD4 in addition to multiple DNA damage repair genes. The percentage of squamous differentiation and TIL density was correlated with survival outcomes in a subset of 28 patients with evaluable tumor specimens. No association between the percentage of squamous differentiation and OS was identified. Of note, compared to those with low TIL density (0-10%), patients with an intermediate/high TIL density (>10%) exhibited significantly improved survival (median OS 36.3 vs 14.2 months, p = 0.011). Conclusions: For the first time, intermediate/high TIL density is associated with prolonged OS in a single-institution cohort of PASC patients. Prospective validation in larger cohorts is needed to establish prognostic biomarkers that can guide therapeutic decision-making in this lethal and under-examined disease subtype. Citation Format: Saurav D. Haldar, Christopher D. Jakubowski, Su Jin Lim, Hao Wang, Richard A. Burkhart, Kelly Lafaro, William R. Burns III, Jin He, Daniel A. Laheru, Elizabeth D. Thompson, Nilofer S. Azad. Tumor-infiltrating lymphocyte density correlates with survival in pancreatic adenosquamous carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A044.

Molecular features of gastrointestinal stromal tumors “wild-type” (<i>KIT/PDGFRA</i> WT)

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract. Their main features are the expression of CD117 (KIT) and mutations of KIT or PDGFRA in 85 % of patients. however, 10–15 % of adult GIST and 85 % of pediatric GIST do not have KIT/PDGFRA mutations (KIT/PDGFRA WT GIST or “wild-type” GIST). The prognosis and clinical course of these tumors and GIST with KIT/PDGFRA mutations differ. “Wild-type” GIST are quite heterogeneous group of tumors in terms of clinical phenotype, genetic etiology, and molecular pathways. Gastrointestinal stromal tumors are divided into SDH-deficient and SDH-competent based on the succinate dehydrogenase (SDH) complex. SDH-deficient GIST occur predominantly in children and young patients with Carney–Stratakis syndrome and Carney triad; there are also sporadic tumors. More than half of SDH-deficient GIST contain mutations in SDHA, SDHB, SDHD or SDHC, while the rest are caused by hypermethylation of the SDHC promoter. SDH-competent “wild-type” GIST include tumors with BRAF, RAS, or NF1 mutations that activate the RAS-RAF-MAPK pathway and KIT/PDGFRA/SDH/RAS-P WT GIST subtype or “quadruple wild type” GIST. The genomic profiles of these tumors and GIST with KIT/PDGFRA mutation or SDH deficiency differ significantly. One of the features of “quadruple wild type” GIST is activation of the FGFR (fibroblast growth factor receptors) signaling pathway due to chimeric FGFR, FGFR mutations, or overexpression of FGF (fibroblast growth factor). Another feature is chimeric genes containing fragments of NTRK, BRAF, FGFR and other genes that behave as oncogene drivers. In “quadruple wild-type” GIST the somatic mutations in TP53, MAX, MEN1, CTNND2, CHD4, ARIDIA and other genes were revealed as well as in the cell cycle genes RB1, CDK4, CDKN1B. There is no specific treatment for patients with “wild-type” GIST; the choice of drug is determined by the genetic disorder. There is a need to improve our understanding of the molecular mechanisms underlying the different GIST subtypes to develop more effective therapeutic approaches.

Heterogeneity of TP53 Mutations in Mantle Cell Lymphoma- Challenges in Risk Stratification and Subclassification

Introduction: Somatic mutations in TP53 occur in approximately 10-20% of mantle cell lymphoma (MCL) patients (pts) at diagnosis. Eskelund and colleagues (Blood 2017) demonstrated poor survival among 20 pts with TP53-mutated MCL following intensive induction and transplant, and reported sequence-level variant data. However, TP53 mutations are heterogeneous, and few studies in MCL provide detailed sequencing data or variant allele frequency (VAF). Furthermore, the clinical impact of TP53 mutations may vary, and there is no algorithm shown to predict outcome for a given variant. We hypothesized that high VAF, or presence of a “disruptive” TP53 mutation as reported in squamous cell carcinoma (SCC; Poeta NEJM 2007), may adversely impact prognosis in MCL. To address this, we analyzed all MCL pts with available TP53 sequencing data at our center, including exact TP53 variant data and VAF, sMIPI score, and classification as disruptive vs not, and impact on survival from time of testing. Methods: Institutional records were analyzed with IRB approval, identifying 65 MCL pts who underwent sequencing for TP53 mutation from any tumor sample, as a single-gene assay or part of a panel. We analyzed VAF, type of mutation, and sMIPI score at time of TP53 testing. We classified TP53 mutations as “disruptive” or not according to method of Poeta and colleagues; these mutations result in a STOP codon, or an amino acid polarity change in the L2-L3 DNA binding domain of the TP53 protein (Poeta NEJM 2017). The Kaplan-Meier method was used to estimate overall survival (OS) and progression-free survival (PFS) from time of TP53 testing to death or last follow up, and the log-rank test was used to compare outcomes based on TP53 mutational status. Among TP53 mutated cases, Cox proportional analyses were performed to explore the impact of the following covariates on survival: disruptive status, VAF, sMIPI score at time of TP53 testing, and presence of complex cytogenetics. Results: Sixty-five pts underwent TP53 sequencing, and 23 (35%) had a TP53 mutation (mTP53+). mTP53+ pts had inferior PFS (p=0.03) compared to TP53 wild-type, and a trend toward inferior OS (p=0.07); median OS was 26 months (95% CI: 8.1, NA) from time of TP53 testing. Ki-67 index, blastoid histology, and age were similar between mTP53+ and wild-type status, but mTP53+ pts had a higher rate of complex cytogenetics (> 3 lesions; 48% vs 26%). SMIPI was also higher in mTP53+ pts (median score 6, interquartile range (IQR) range 4-7) vs TP53 wild-type pts (median 4, IQR range 3-5.2). MTP53+ pts were more likely to have received a BTK-inhibitor (BTK-I) (91 % vs 64%) and CAR-T therapy (39% vs 18%). Details of the mTP53+ subset are shown in Table 1; 4 pts had >1 TP53 mutation, median VAF was 40%, and 30% of pts had a disruptive mutation. Among mTP53+ pts, on univariate analysis, higher sMIPI at time of TP53 testing (p=0.003) was associated with worse OS (Table 2). In a multivariable Cox model including VAF >= 40%, presence of disruptive mutation, sMIPI, and complex cytogenetics, only sMIPI at time of TP53 testing was associated with inferior OS (p=0.02). Conclusion: TP53 variants in MCL are diverse, and in our cohort were largely unique from those reported by Eskelund and colleagues (Blood 2017). Despite now-routine application of sequencing and improvements in therapy, both of which may attenuate the negative prognostic impact of TP53 mutation, we confirmed poor outcomes in mTP53+ MCL. Among mTP53+ pts, only sMIPI predicted inferior OS on both univariate and multivariate analysis. Higher VAF and adoption of a clinical algorithm from SCC failed to discriminate among TP53 variants in terms of prognosis. Larger datasets of uniformly tested MCL pts are needed to elucidate prognosis among TP53 mutation variants, and improve understanding and management of mTP53+ MCL.

Coronado CLL: A Phase Ib/II Trial of Combination Rp-3500 and Olaparib in DNA Damage Repair Pathway Deficient Relapsed/Refract ory Chronic Lymphocytic Leukemia

Background: Genes within the DNA damage repair ( DDR) pathways are frequently mutated in relapsed/refractory ( R/R) chronic lymphocytic leukemia ( CLL) and clinically correlate with decreased response rates and shortened progression-free ( PFS) and overall survivals ( OS). The most commonly affected DDR genetic aberrations include deletion 17p13 [ del(17p)], deletion 11q22.3 [ del(11q)], and mutations in TP53, ATM, SF3B1, XPO1 and POT1. Due to their poor clinical outcomes, patients (pts) with DDR-deficient CLL remain an area of unmet need and the development of novel drugs and combinations will be critical to improving their prognosis. Mechanistically, CLL cells with DDR genetic alterations lack double-stranded DNA breakage repair and major cell cycle checkpoint pathways. Therefore, R/R CLL cells are dependent on single-stranded DNA breakage ( SSB) repair and alternative DNA replication arrest machinery to ensure DNA integrity, strand repair and cell survival. DNA damage at the replication fork is primarily recognized by ataxia telangiectasia and rad3 ( ATR) protein, which ensures mitotic arrest and recruitment of poly (ADP-ribose) polymerase ( PARP) based SSB repair mechanisms. In germline DDR-deficient breast and ovarian cancers with BRCA1/BRCA2 mutations, the use of both single agent PARP inhibitors ( PARPi) and ATR inhibitors ( ATRi) have led to significant disease response and improved OS. Furthermore, combination PARPi and ATRi have demonstrated synergistic activity in preclinical data producing increased apoptosis and cell death in BRCA2-mutated ovarian cell lines. In DDR-deficient CLL primary cell and murine models, single agent PARPi and ATRi have demonstrated significant cell death as compared to wildtype CLL and healthy donor lymphocytes. RP-3500 is a highly potent ATRi. In preclinical data generated in SUM149PT DDR-deficient breast cancer cell lines, combining RP-3500 with the PARPi, olaparib, substantially reduced tumor volume at lower doses than the previously determined maximum tolerated dose ( MTD) of each drug when used as monotherapy. Furthermore, intermittent dosing of both drugs (3 days per week) demonstrated equal cell apoptosis as continuous dosing. Therefore, we hypothesize that the combination of RP-3500 and olaparib will be a highly efficacious treatment for R/R DDR-deficient CLL pts and that the lower dosages and intermittent dosing schedule required of each drug when administered in combination will improve safety of the treatment. Study Design and Methods: This is a multi-site (University of Utah and the Ohio State University), phase Ib/II investigator-initiated clinical trial using combination RP-3500 and olaparib in R/R CLL pts with DDR pathway deficiencies to determine the safety and efficacy of the drug combination (NCT05405309). Primary objectives include determining the MTD in phase Ib leading to the recommended phase 2 dose ( RP2D), and overall response rate ( ORR) in phase II. Key secondary objectives include safety and tolerability, PFS, OS and duration of response of the combination. Eligibility: Key inclusion criteria include a diagnosis of CLL according to the 2018 iwCLL guidelines, age ≥18 years, ECOG performance status of ≤2, R/R after at least 2 prior lines of therapy. Primary CLL cells must exhibit any one of the following abnormalities: a) somatic mutation(s) in TP53, ATM, SF3B1, XPO1 or POT1, or b) positive FISH for either del(17p) or del(11q). Statistical Methods: Phase Ib: The phase Ib of the trial will utilizea keyboard design, which is a novel Bayesian method that typically underestimates the MTD. The target toxicity is 30% with an equivalence interval between 25-33% of pts. With an anticipated 3 dose levels ( DL) and an option for a DL-1, up to 18 pts may be required to determine the MTD and RP2D. Phase II: Any pt treated at the RP2D within phase Ib will be rolled into the phase II analysis for efficacy. With a planned 24 evaluable pts in phase II, the target ORR will be 60%, which was selected due to a recent phase I/II study of pirtobrutinib enrolling a similar CLL pt population demonstrating an ORR of the drug ~63%. ORR will be summarized by the observed proportion and an exact one-sided 95% confidence interval (Clopper-Pearson method). With 24 evaluable pts, the lower bound of the confidence interval will be approximately 17-20% below the observed proportion for observed ORR near 60%, which is an acceptable level of precision in a dose expansion cohort.

CD7 Chimeric Antigen Receptor T-Cell Therapy Bridging to Allogeneic Hematopoietic Stem Cell Transplantation Remarkably Improved Long-Term Disease-Free Survival in Refractory/Relapsed T-Cell Acute Lymphoblastic Leukemia/Lymphoma

Introduction: The prognosis of refractory/relapsed T-cell acute lymphoblastic leukemia/lymphoma (r/r T-ALL/LBL) is poor, and salvaged allogeneic hematopoietic stem cell transplantation (allo-HSCT) can only result in 20%-30% disease-free survival (DFS). Our previous clinical trials have shown that the patients with r/r T-ALL have achieved 90% complete remission (CR) with CD7 chimeric antigen receptor T cell (CART) therapy (Pan J. et al. JCO 2021), then quick received allo-HSCT to obtain 57% of 1-year DFS in 12 patients (Li ZH. et al. Transplantation and Cellular Therapy 2022). Objectives : In current study, the long-term outcomes of allo-HSCT in r/r T-ALL/LBL after CD7 CART therapy in larger cohort are investigated, and compared with that chemotherapy only before allo-HSCT simultaneously. The risk factors for prognosis after allo-HSCT in this setting are also analyzed. Methods: Between February 2018 and January 2023, total 90 patients with r/r T-ALL/LBL who underwent allo-HSCT in our hospital were included. The median age was 14 (2-65) years old. Somatic and germline gene mutations were detected by sequencing pre-transplant. Thirty-two (35.6%) patients were sensitive to chemotherapy and achieved CR before transplant (group A), and 58 (64.4%) cases were resistant to chemotherapy and in non-remission (NR) pre-HSCT. Forty-one of 58 patients in NR received CD7 CART before allo-HSCT (group B) and the rest 17 patients in NR underwent salvaged transplant (group C). Donor types included haploidential (60, 66.7%), unrelated (16, 17.8%) and identical sibling (14, 15.6%). Myeloablative conditioning regimens with either total body irradiation (TBI)/fludarabine (51, 56.7%) based or busulfan/fludarabine (39, 43.3%) based were applied. Antithymocyte globulin was used for haploidentical and unrelated transplants. Cyclosporine, mycophenolate mofetil and short-term methotrexate were employed for graft-versus-host disease (GVHD) prophylaxis. Results: All patients achieved durable engraftment. No significant difference was found in the incidences of acute GVHD (aGVHD), chronic GVHD (cGVHD) and infections in 3 groups (p=0.612, p=0.091, p=0.649). With a median follow-up of 25.5 (19.6-32) months, 2-year overall survival (OS) in group B was similar to that in group A (54.4% vs. 69.9%, p=0.33) and much higher than that in group C (35.3%, p=0.0065), and 2-year DFS in group B was similar to that in group A (51.0% vs. 61.1%, p=0.24) and much higher than that in group C (17.6%, p=0.0029). No statistical difference was seen on transplant-related mortality (TRM) (17.3% vs. 9.8% vs. 11.8%, p=0.65) among group B, A, C. Two-year cumulative incidence of relapse in group B (31.7%) was similar to group A (29.0%), and much lower than that in group C (70.5%). The risk for relapse and death decreased remarkably in group B (p=0.0047) and group A (p=0.0003) compared with that in group C. NR before transplant (p=0.0062), aGVHD (p=0.013) and viral infections (p=0.047) were risk factors for relapse-related death, but TBI-based conditioning (p=0.01) was positive impact factor on OS. The most common somatic gene mutations were NOTCH 1(55.7%), JAK 3 (18.0%), TP53 (16.4%), JAK1 (14.8%) and N R AS(14.8%). The most common germline gene mutations were EP300 (10.3%), ATM (8.6%), F7 (8.6%), KIT (8.6%) and N CF2 (8%). The patients with somatic TP53 or MED12 mutation had significantly higher risk for relapse and death (p=0.0018, p=0.0068). Germline gene mutations had no significant influence on prognosis post-transplant. Multivariate analysis had shown that CD7 CART (p=0.037), TBI-based conditioning (p=0.002) and cGVHD (p=0.004) were independent positive impact factors on OS, but aGVHD (p=0.034) and TP53 mutation (p=0.002) were independent negative impact factors for OS. Meanwhile, CD7 CART (p=0.005) and TBI-based conditioning (p=0.024) had positive impact on DFS, but TP53 mutation (p=0.02) also had negative impact on DFS. Conclusion s : Our study has demonstrated that CD7 CART followed by allo-HSCT has remarkably improved long-term DFS for chemotherapy-resistant T-ALL/LBL, and achieved comparable results in safety and efficacy with chemotherapy-sensitive disease post-transplant. Our study has also shown the profiles of somatic and germline gene mutations in r/r T-ALL/LBL, and identified some positive and negative impact factors for prognosis after transplant in this setting.

Higher TP53 somatic mutation prevalence from liquid biopsy analysis in ever smoker non-small-cell lung cancer patients

ObjectiveCigarette smoking is a primary risk factor, linked to 80% of LC deaths. TP53, a key gene, is implicated in various cancers, with TP53 alterations in 36.7% of cancers. This research aims to investigate TP53 mutations detected in NSCLC patients by liquid biopsy and explore the relationship between these mutations and smoking history. Material and methodThe study enrolled a total of 340 patients diagnosed with non-small cell lung cancer (NSCLC). For sequencing, the Illumina NextSeq 500 system was utilized. The oncogenicity of the variants was assessed according to the ClinGen/CGC/VICC SOP and the variants were categorized into four tiers according to AMP/ASCO/CAP. ResultsThe most common mutations were in TP53 (48.7%), followed by EGFR, PIK3CA, and PTEN. Missense mutations were frequent, with TP53 and EGFR having higher rates in ever-smokers. No indels or complex mutations were found in ever-smokers. Patient age ranged from 20 to 86 years. Tier I-II variants were more common in ever-smokers, while Tier III variants were prevalent in never-smokers. TP53 mutations were more frequent in ever-smokers, showing a strong association with smoking. Domain distribution showed differences in PIK3CA. Transversion/transition ratios varied by gene and smoking status. DiscussionThe presence of TP53 mutations is strongly associated with both cigarette smoking and elevated Tv/Ti ratios. The tier status of TP53, EGFR, and PTEN variants does not show a specific domain distribution, but interesting associations are observed between the tier status and domain distribution in PIK3CA variants. Therefore, further comprehensive investigations are needed to explore this entity, as well as the underlying factors contributing to the increased Tv/Ti rates in the TP53 gene. Such research will provide deeper insights into the genetic alterations associated with smoking and tumor heterogeneity, ultimately aiding in the development of targeted therapies.

Biological and genetic characterization of a newly established human external auditory canal carcinoma cell line, SCEACono2

Squamous cell carcinoma of the external auditory canal (EACSCC) is an extraordinarily rare and aggressive malignant disease. Establishment of EACSCC cell line with robust molecular characteristics is essential for the basic and translational research of EACSCC. Here, we show the newly established EACSCC cell line SCEACono2, derived from a patient with well-to-moderately differentiated EACSCC. We analyzed histologic and genetic features of SCEACono2 hiring multiple experiments, including next-generation sequencing (NGS). Immunocytochemical staining of SCEACono2 showed positivity of p53 and SCC1/2. Furthermore, SCEACono2 exhibited a unique characteristic that cytokeratin, vimentin as well as cancer stem cell markers (CD44, CD133, ALP and Oct3/4) were positive. SCEACono2 had an ability to form tumors at the temporal lesion xenograft nude mice model. NGS revealed that SCEACono2 harbored the somatic mutations of TP53 (p.G245S) and NOTCH1 (p.A465T). RNA-seq and downstream bioinformatics analysis revealed significant enrichment of genes involved in inflammation and cell adhesion in SCEACono2 compared to SCC-9 and HSC-4. STR profiling indicated no evidence of cross-contamination. In conclusion, SCEACono2 could serves as a promising and robust research resource of EACSCC in vitro and in vivo.

Durable Remissions in Advanced Stage and Molecularly High Risk Untreated Classic Hodgkin Lymphoma with Pembrolizumab + AVD

Introduction We previously reported results of a frontline study of pembrolizumab + AVD (Lynch et al Blood 2023) in any stage classic Hodgkin lymphoma (CHL). Despite finding surprisingly high rates of positive interim and EOT PET/CT compared to historical data, this study revealed a 2-year PFS of 97%. Separately, recent data (Alig et al. ICML 2023) suggest that CHL is characterized by two dominant genetic subtypes (H1 vs. H2). H2 tumors harbor higher levels of genomic instability, are enriched for somatic mutations in TP53, KMT2D, and BCL2, and have increased risk of upfront treatment failure with conventional regimens. To better evaluate our findings in higher risk patients, we expanded the study to include 20 additional patients with advanced stage disease. Herein we present updated clinical data for our full 50-patient cohort, and report genotype associations including novel genetic subtypes of cHL. Methods We performed a single arm study (NCT03331341) combining concurrent pembrolizumab with AVD in untreated CHL of any stage. Pembrolizumab 200 mg IV was administered every 21 days concurrently with AVD chemotherapy. AVD was administered at the standard dosing and schedule. With the cohort expansion, total planned accrual was 50 patients. The objective of the expanded study was to evaluate longer term efficacy based on concerns with the utility of PET/CT. We defined success as a 1-year PFS in advanced stage patients of at least 85%. Samples were analyzed for ctDNA at baseline, post cycle 1 (if available), post cycle 2, and end of treatment. ctDNA levels were quantified as haploid genome equivalents/mL plasma using CAPP-Seq (Newman et al Nat Biotech 2016) and PhasED-Seq (Kurtz et al. Nat Biotech 2021). Using baseline CAPP-Seq genotypes from plasma ctDNA, patients were classified as H1 vs H2 cHL by a previously validated probabilistic Latent Dirichlet Allocation (LDA) algorithm (Alig et al, 2023, in review). Results 50 patients, enrolled between Feb 1, 2019, and Apr 13, 2023, were evaluable for updated safety, response and/or exploratory analyses (one patient withdrew from study treatment prior to any response assessment). With a median follow up of 2.3 years, 2-year PFS and OS were 98% and 100%, respectively. Among advanced stage patients (n=38), 2-year PFS and OS were 97% and 100% respectively. PET2 was performed in 49 patients, with a CR rate of 73% in the early stage patients, and 58% in those with advanced stage. EOT PET was evaluable in 41 patients, with a CR rate of 91% in early stage, and 73% in those with advanced stage. To date, only one patient with a positive EOT PET has developed biopsy-proven recurrence. Four EOT PET+ patients are now > 3 years post treatment without relapse. Four additional EOT PET+ patients are less than 1 year post therapy, and one has resolved FDG uptake on repeat scan with the others undergoing surveillance. Eight (16%) patients experienced SAEs, including sepsis (one each G3 and G4), G3 Guillain Barre syndrome that resolved after permanent pembrolizumab discontinuation, and five (10%) G3 febrile neutropenia. Non-invasive genotyping using ctDNA was available in 25 patients. Among 6 patients with H2 genotypes (24%), all had advanced stage, and this group included the only patient experiencing disease recurrence. The remaining patients including all early-stage patients were classified as H1 (76%). All patients will have completed treatment and safety evaluations by October 2023, and updated safety, efficacy, and exploratory data including ctDNA will be presented at the meeting. Conclusion With longer follow up, including patients who started therapy over 4 years ago, concurrent pembrolizumab + AVD represents a highly effective frontline therapy for CHL, including for advanced stage patients and those with high risk H2 genotypes. PET2 demonstrated limited clinical utility, and no late relapses have been seen to date.

Targeting Nucleotide Biosynthesis Ameliorates Neutropenia in a Zebrafish Model of Dnajc21-Mutant Shwachman-Diamond Syndrome

Germline mutations in the ribosome maturation factors, SBDS, DNAJC21 and EFL1, lead to Shwachman-Diamond syndrome (SDS), a type of inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency and growth restriction. Further, SDS patients have a 36% increased cumulative risk of developing myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML) by 30 years of age. Given the rarity of SDS, large numbers of primary human samples are not readily available for mechanistic studies, warranting the use of animal models. Mouse and zebrafish models of SBDS mutations exhibit early lethality. In contrast, no in vivo models of DNAJC21 mutations have been published to date. We generated the first in vivo model of dnajc21 loss using zebrafish ( Danio rerio), given their highly conserved hematopoietic program and ease of genetic manipulation. Introduction of a homozygous deletion mutation in the zebrafish dnajc21 gene using CRISPR-Cas9 genomic editing resulted in stunted body growth and neutropenia, recapitulating hallmark SDS patient phenotypes. Whole-mount in situ hybridization for key hematopoietic lineage markers revealed reduced l-plastin +mature leukocytes and hbbe3 + mature erythrocytes in dnajc21 mutant embryos at 48 hours post-fertilization (hpf). A concomitant accumulation of myb + hematopoietic stem and progenitor cells (HSPCs) both in the dorsal aorta (equivalent to aorta-gonad-mesonephros) and the caudal hematopoietic tissue (fetal liver equivalent) was observed. Treatment of embryos with zebrafish granulocyte colony stimulating factor (gcsf) mRNA resulted in a modest expansion of neutrophils in dnajc21 mutant zebrafish compared to wildtype. Further, we observed reduced cell proliferation marked by phosphohistone-H3 (pH3) as well as low ATP:AMP levels measured by liquid chromatography/mass-spectrometry (LC/MS) in the dnajc21 mutants, suggesting a poor bioenergetic profile. At 4 months of age, flow cytometry and histopathological analysis identified reduced mature myelomonocytes and mild erythroid dysplasia in the whole kidney marrow (human bone marrow equivalent) of mutant fish. The link between TP53 somatic mutations and MDS/AML has been established in SBDS-mutant SDS, but has not been explored for DNAJC21-mutant SDS. We hence crossed our dnajc21 mutant zebrafish with a zebrafish line carrying a gain-of-function mutation (R217H) in the tp53 gene. Compound mutant fish had improved neutrophil counts but importantly, this was accompanied by an expansion of myeloid progenitors and significant erythroid dysplasia. To understand the disease mechanisms underlying the neutropenia and hypoproliferation in SDS, we performed bulk RNA sequencing of whole zebrafish larvae at 48 hpf. Biological processes such as oxidative stress response, drug catabolism and pre-mRNA spliceosome assembly were downregulated; whereas, acyl-CoA and nucleotide metabolism were upregulated in the mutants. In parallel, we performed untargeted metabolomics by LC/MS to determine the metabolic changes induced by dnajc21 loss. Upregulated pathways included urea cycle, amino acid and phenylacetate metabolism. Pyrimidine biosynthesis, including both the de novo and salvage pathways were downregulated in the mutants. Remarkably, exogenous supplementation with uridine or thymidine significantly improved neutrophil counts in the mutants. Our findings suggest that in dnajc21-mutant SDS1) neutropenia is caused by a combination of impaired granulocyte differentiation and cell proliferation; 2) the kidney marrow features myelodysplasia-like changes and 3) nucleotide biosynthesis may be a previously unrecognized therapeutic strategy in SDS.

TERT promoter mutations and gene amplification in endometrial cancer

ObjectiveTo assess the clinicopathologic, molecular profiles, and survival outcomes of patients with endometrial carcinomas (ECs) harboring telomerase reverse transcriptase (TERT) hotspot mutations or gene amplification. MethodsECs harboring somatic TERT promoter hotspot mutations or gene amplification (TERT-altered) were identified from 1944 ECs that underwent clinical tumor-normal sequencing from 08/2016–12/2021. Clinicopathologic variables, somatic mutation profiles, and survival outcomes of TERT-alt and TERT-wild-type EC were assessed. ResultsWe identified 66 TERT-altered ECs (43 TERT-mutated and 23 TERT-amplified), representing 3% of the unselected ECs across histologic subtypes. Most TERT-altered ECs were of copy number (CN)-high/TP53abn molecular subtype (n = 40, 60%), followed by microsatellite-unstable (MSI-H) or CN-low/no specific molecular profile (NSMP)(n = 13, 20% each). TERT-amplified and TERT-mutated ECs were molecularly distinct, with TERT-amplified ECs being more genomically instable and more frequently harboring TP53 and PPP2R1A alterations (q < 0.1). Compared to TERT-wild-type ECs, TERT-altered ECs were more commonly of CN-H/TP53abn molecular subtype (31% vs 57%, p = 0.001), serous histology (10% vs 26%, p = 0.004), and were significantly enriched for TP53, CDKN2A/B, and DROSHA somatic genetic alterations (q < 0.1). Median progression-free survival was 18.7 months (95% CI 11.8–not estimable [NE]) for patients with TERT-altered EC and 80.9 months (65.8-NE) for patients with TERT-wild-type EC (HR 0.33, 95% CI 0.21–0.51, p < 0.001). Similarly, median overall survival was 46.7 months (95% CI 30-NE) for TERT-altered EC patients and not reached for TERT-wild-type EC patients (HR 0.24, 95% CI 0.13–0.44, p < 0.001). ConclusionTERT-altered ECs, although rare, are enriched for CN-high/TP53abn tumors, TP53, CDKN2A/B and DROSHA somatic mutations, and independently predict worse survival outcomes.

Advances in translational research of the rare cancer type adrenocortical carcinoma.

Adrenocortical carcinoma is a rare malignancy with an annual worldwide incidence of 1-2 cases per 1 million and a 5-year survival rate of <60%. Although adrenocortical carcinoma is rare, such rare cancers account for approximately onethird of patients diagnosed with cancer annually. In the past decade, there have been considerable advances in understanding the molecular basis of adrenocortical carcinoma. The genetic events associated with adrenocortical carcinoma in adults are distinct from those of paediatric cases, which are often associated with germline or somatic TP53 mutations and have a better prognosis. In adult primary adrenocortical carcinoma, the main somatic genetic alterations occur in genes that encode proteins involved in the WNT-β-catenin pathway, cell cycle and p53 apoptosis pathway, chromatin remodelling and telomere maintenance pathway, cAMP-protein kinase A (PKA) pathway or DNA transcription and RNA translation pathways. Recently, integrated molecular studies of adrenocortical carcinomas, which have characterized somatic mutationsand the methylome as well as gene and microRNA expression profiles, have led to a molecular classification of these tumours that can predict prognosis and have helped to identify new therapeutic targets. In this Review, we summarize these recent translational research advances in adrenocortical carcinoma, which it is hoped could lead to improved patient diagnosis, treatment and outcome.