Somatic Oncogenic Mutations Research Articles

Feasibility and tissue concordance of genomic sequencing of urinary cytology in upper tract urothelial carcinoma

BackgroundThere is limited ability to accurately diagnose and clinically stage patients with upper tract urothelial carcinoma (UTUC). The most easily available and widely used urinary biomarker is urine cytology, which evaluates cellular material yet lacks sensitivity. We sought to assess the feasibility of performing next-generation sequencing (NGS) on urine cytology specimens from patients with UTUC and evaluate the genomic concordance with tissue from primary tumor. MethodsIn this retrospective study, we identified 48 patients with a diagnosis of UTUC treated at Memorial Sloan Kettering Cancer Center (MSK) between 2019 and 2022 who had banked or fresh urine samples. A convenience cohort of matching, previously sequenced tumor tissue was used when available. Urine specimens were processed and the residual material, including precipitated cell-free DNA, was sequenced using our tumor-naïve, targeted exome sequencing platform that evaluates 505 cancer-related genes (MSK-IMPACT). The primary outcome was at least 1 detectable mutation in urinary cytology specimens. The secondary outcome was concordance to matched tissue (using ANOVA or Chi-Square, as indicated). ResultsGenomic sequencing was successful for 45 (94%) of the 48 urinary cytology patient samples. The most common mutations identified were TERT (62.2%), KMT2D (46.7%), and FGFR3 (35.6%). All patients with negative urine cytology and low-grade tissue had successful cytology sequencing. Thirty-six of the 45 patients had matching tumor tissue available; concordance to matched tissue was 55% overall (131 of the total 238 oncogenic or likely oncogenic somatic mutations identified). However, in 94.4% (n = 34/36) of patients, the cytology had at least 1 shared mutation with tissue. Eleven (30.6%) patients had 100% concordance between cytology and tissue. ConclusionsSequencing urinary specimens from selective UTUC cytology is feasible in nearly all patients with UTUC. Prospective studies are underway to investigate a clinical role for this promising technology.

EML4::ALK fusions in complex lymphatic malformations.

Gorham-Stout disease (GSD) and generalized lymphatic anomaly (GLA) are subtypes of complex lymphatic malformations (CLMs) with osseous involvement that cause significant complications, including pain and pathologic fractures. As with other vascular anomalies, somatic mosaic mutations in oncogenes are often present, and the mTOR inhibitor sirolimus alleviates symptoms in some, but not all, patients. We describe two patients, one with GSD and one with GLA, found to have EML4::ALK fusions. This report of a targetable, oncogenic fusion in vascular malformations expands our understanding of the genetic basis for CLMs and suggests additional targeted therapies could be effective.

The essential molecular requirements for the transformation of normal cells into established cancer cells, with implications for a novel anti-cancer agent.

Normal adult mammalian cells can respond to oncogenic somatic mutations by committing suicide through a well-described, energy dependent process termed apoptosis. Cancer cells avoid oncogene promoted apoptosis. Oncogenic somatic mutations are widely acknowledged to be the cause of the relentless unconstrained cell proliferation which characterises cancer. But how does the normal cell with the very first oncogenic mutation survive to proliferate without undergoing apoptosis? The phenomena of malignant transformation by somatic mutation, apoptosis, aneuploidy, aerobic glycolysis and Cdk4 upregulation in carcinogenesis have each been extensively discussed separately in the literature but an overview explaining how they may be linked at the initiation of the cancer process has not previously proposed. A hypothesis is presented to explain how in addition to the initial oncogenic mutation, the expression of certain key normal genes is, counter-intuitively, also required for successful malignant transformation from a normal cell to a cancer cell. The hypothesis provides an explanation for how the cyclic amphiphilic peptide HILR-056, derived from peptides with homology to a hexapeptide in the C-terminal region of Cdk4, kill cancer cells but not normal cell by necrosis rather than apoptosis.

Respective roles of Pik3ca mutations and cyproterone acetate impregnation in mouse meningioma tumorigenesis.

Despite their rarity, PIK3CA mutations in meningiomas have raised interest as potentially targetable, ubiquitous mutations owing to their presence in sporadic benign and malignant tumors but also in hormone-related cases. Using new genetically engineered mouse models, we here demonstrate that Pik3ca mutations in postnatal meningeal cells are sufficient to promote meningioma formation but also tumor progression in mice. Conversely, hormone impregnation, whether alone or in association with Pik3ca and Nf2 mutations, fails to induce meningioma tumorigenesis while promoting breast tumor formation. We then confirm in vitro the effect of Pik3ca mutations but not hormone impregnation on the proliferation of primary cultures of mouse meningeal cells. Finally, we show by exome analysis of breast tumors and meninges that hormone impregnation promotes breast tumor formation without additional somatic oncogenic mutation but is associated with an increased mutational burden on Pik3ca-mutant background. Taken together, these results tend to suggest a prominent role of Pik3ca mutations over hormone impregnation in meningioma tumorigenesis, the exact effect of the latter is still to be discovered.

Increased SPRY1 expression activates NF-κB signaling and promotes pancreatic cancer progression by recruiting neutrophils and macrophages through CXCL12-CXCR4 axis.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a high mortality rate, in which about 90% of patients harbor somatic oncogenic point mutations in KRAS. SPRY family genes have been recognized as crucial negative regulators of Ras/Raf/ERK signaling. Here, we investigate the expression and role of SPRY proteins in PDAC. Expression of SPRY genes in human and mice PDAC was analyzed using The Cancer Genome Atlas and Gene Expression Omnibus datasets, and by immunohistochemistry analysis. Gain-of-function, loss-of-function of Spry1 and orthotopic xenograft model were adopted to investigate the function of Spry1 in mice PDAC. Bioinformatics analysis, transwell and flowcytometry analysis were used to identify the effects of SPRY1 on immune cells. Co-immunoprecipitation and K-ras4B G12V overexpression were used to identify molecular mechanism. SPRY1 expression was remarkably increased in PDAC tissues and positively associated with poor prognosis of PDAC patients. SPRY1 knockdown suppressed tumor growth in mice. SPRY1 was found to promote CXCL12 expression and facilitate neutrophil and macrophage infiltration via CXCL12-CXCR4 axis. Pharmacological inhibition of CXCL12-CXCR4 largely abrogated the oncogenic functions of SPRY1 by suppressing neutrophil and macrophage infiltration. Mechanistically, SPRY1 interacted with ubiquitin carboxy-terminal hydrolase L1 to induce activation of nuclear factor κB signaling and ultimately increase CXCL12 expression. Moreover, SPRY1 transcription was dependent on KRAS mutation and was mediated by MAPK-ERK signaling. High expression of SPRY1 can function as an oncogene in PDAC by promoting cancer-associated inflammation. Targeting SPRY1 might be an important approach for designing new strategy of tumor therapy.

Abstract 251: Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids

Abstract The promise of precision medicine is based on the idea that genetic alterations present only in tumor cells create vulnerabilities that can be targeted for therapeutic intent. In some instances, somatically mutated driver genes such as KRAS, BRAF or PIK3CA can be targeted with small molecule inhibitors specific for mutant oncoproteins. In other cases therapeutic vulnerability is an indirect consequence, such as response to immune checkpoint inhibitors caused by somatic inactivation of mismatch repair genes and subsequent creation of neo-antigens in tumors. Synthetic lethal vulnerabilities dependent on common somatic alterations would be highly specific therapeutic targets applicable to a large number of cancer patients. We sought to identify potential therapeutic vulnerabilities for colon cancer by performing a genome wide CRISPR knockout screen in a colon cancer organoid in both TP53-Wild-Type and TP53-Knockout backgrounds. We identified 1784 gene knockouts with TP53-dependent effects on Darwinian fitness. Examples include MDM2 and PPM1D knockouts, both of which selectively harmed the TP53 WT organoids. 250 gene knockouts selectively harmed the TP53 KO organoids and represent novel avenues for development of TP53 synthetic lethal targeted therapeutics. We also identified ~1000 gene knockouts under significant negative Darwinian selection in both TP53 WT and TP53 KO organoids which are not known to be common essential genes. Several of these organoid model specific vulnerabilities are in pathways that are downstream of the somatic mutations present in the tumor and are therefore candidates for highly-specific targeted therapeutic intervention. Examples include Werner’s pathway dependencies (WRN, EME1, MUS81) which are known to result from mismatch repair deficiency, WNT pathway dependencies (CTNNB1, PORCN) which result from RNF43 inactivating somatic mutation, and BRAF dependency resulting from its own activating oncogenic somatic mutation. In summary, genome-wide CRISPR knockout library screening of human colon cancer organoids can provide a comprehensive overview of dependencies interacting with either natural or engineered mutations and are a promising novel platform for discovering personalized or pathway based therapeutic targets. Future work will focus on target validation and additional primary screens using our biobank of tumor-normal organoid pairs. Citation Format: Phillip J. Buckhaults, Sana Khalili, Carolyn Banister, Prashanth R. Gokare, Dave Pocalyko, Kurtis Bachman. Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 251.

Localized heterochrony integrates overgrowth potential of oncogenic clones.

Somatic mutations occur frequently and can arise during embryogenesis, resulting in the formation of a patchwork of mutant clones. Such mosaicism has been implicated in a broad range of developmental anomalies; however, their etiology is poorly understood. Patients carrying a common somatic oncogenic mutation in either PIK3CA or AKT1 can present with disproportionally large digits or limbs. How mutant clones, carrying an oncogenic mutation that often drives unchecked proliferation, can lead to controlled and coordinated overgrowth is unknown. We use zebrafish to explore the growth dynamics of oncogenic clones during development. Here, in a subset of clones, we observed a local increase in proportion of the fin skeleton closely resembling overgrowth phenotypes in patients. We unravel the cellular and developmental mechanisms of these overgrowths, and pinpoint the cell type and timing of clonal expansion. Coordinated overgrowth is associated with rapid clone expansion during early pre-chondrogenic phase of bone development, inducing a heterochronic shift that drives the change in bone size. Our study details how development integrates and translates growth potential of oncogenic clones, thereby shaping the phenotypic consequences of somatic mutations.

Assessment of the Molecular International Prognostic Scoring System (IPSS-M) Risk Stratification Model in Therapy-Related Myelodysplastic Syndromes

Background Therapy-related myelodysplastic syndromes (t-MDS) represent a heterogenous group of myeloid neoplasms with distinctive clinical, molecular, and cytogenetic features associated with less favorable clinical outcomes relative to their primary counterpart (p-MDS). The original International prognostic scoring system (IPSS) and its revised version IPSS-R development cohorts did not include t-MDS patients. The Molecular IPSS (IPSS-M) is a newly developed clinical-molecular risk stratification model for myelodysplastic syndromes (MDS) that expands on the original IPSS-R by including data on somatic oncogenic mutations. Here we aimed to assess the validity of this tool in the risk stratification of t-MDS using a large cohort of patients treated at a tertiary referral center. Methods A total of 475 t-MDS patients with annotated clinical and molecular data treated at Moffitt Cancer Center were analyzed. For estimation of the IPSS-M score, R version 4.2.1 was used to run batch-calculation using syntax code provided by the Papaemmanuil lab at MSKCC. The mean IPSS-M score was used as reference. Time-to-event analyses were estimated using the Kaplan-Meier method and groups were compared by the log-rank test. We used Cox proportional hazards models for survival endpoints. Results We identified at least 737 driver point mutations involving up to 98 genes across 475 patients. We identified at least one gene mutation in 81% of patients (n=389), and 2 or more in 43% (n=206). Median overall survival (OS) was 1.5 years (95%CI, 1.3-1.8). Median leukemia-free survival (LFS) was 1.1 years (0.9-1.3). Median follow-up was 3.9 years (2.9-4.8). 475 patients (100%) were classified. The most prevalent mutations were TP53 (n=182, 38%), TET2 (n=79, 17%), DNMT3A (n=67, 14%), SF3B1 (n=43, 9%) and U2AF1 (n=35, 7%). Using the IPSS-M risk stratification schema, pts were classified as Very Low (2%, n=8), Low (13%, n=63), Moderate Low (14%, n=64), Moderate High (10%, n=48), High (22%, n=105) and Very High (39%, n=187) (Fig 1). The IPSS-M categories showed significant separation across all examined endpoints. Median LFS was NR, 3.7, 2.1, 1.7, 1.1 and 0.5 years from Very Low to Very High-risk categories (Fig 1). Conversely, median OS was NR, 4.4, 2.9, 3.5, 1.5 and 0.9 years from VL to VH subgroups respectively (Fig 1). A five-to-five mapping between the IPSS-R and IPSS-M risk subgroups (by merging moderate-low (ML) and moderate-high (MH) into moderate), resulted in the restratification of 45% of patients (n=209) (Fig 2). More than 3/4 of patients classified as IPSS-R very-low (VL) were upstaged (majority to IPSS-M low). More than half of patients classified as IPSS-R intermediate shifted: 50% (n=38) were reclassified as IPSS-M high/very high (H/VH). Similarly, 40% of patients classified as IPSS-R high were upstaged to IPSS-M VH. More than 20% of VH risk patients were downstaged (majority to high risk). Most significant was the reclassification of 9.4% (n=44) of the cohort from IPSS-R lower and intermediate risk categories into IPSS-M higher risk strata. Contextually, the restratification of patients from IPSS-R intermediate to higher risk strata in IPSS-M was associated with notable differences in survival outcomes which were as poor as 0.9 yrs and as high as 4.4 yrs for those reclassified as LR (relative to 2.1 yrs). There was significance across all IPSS-M risk strata between t-MDS and p-MDS. Stage to stage t-MDS was associated with worse outcomes compared to p-MDS, except for the very low risk IPSS-M subgroup. Conclusion To our knowledge this is the first external validation of IPSS-M in a large cohort of t-MDS patients. Use of the IPSS-M model for the risk stratification of patients with t-MDS led to reclassification of almost half of MDS patients and refined prognosis estimates. In general, outcomes of t-MDS are worse than p-MDS even when compared stage to stage using IPSS-M. The model can clearly discriminate patients with more indolent or aggressive disease course compared to the IPSS-R and better guide treatment decisions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Validation of the Molecular International Prognostic Scoring System (IPSS-M) Risk Stratification Model for Myelodysplastic Syndromes

Background The Molecular International Prognostic Scoring System (IPSS-M) is a newly developed risk stratification model for myelodysplastic syndromes (MDS) that expands on the original IPSS-R by including data on somatic oncogenic mutations across 31 gene families and weighing their impact on prognosis and outcomes. The discovery model demonstrated improved prognostic discrimination relative to the IPSS-R in regard to overall survival (OS), leukemia-free survival (LFS) and leukemic transformation, by substratifying pts into six risk categories, and led to restratification of 46% of cases. Adequate risk stratification in MDS is critical for therapeutic decision-making. Here we aimed to externally validate the findings of the original study using a large cohort of pts treated at a tertiary referral center. Methods A total of 2355 MDS pts treated at Moffitt Cancer Center with available clinical and molecular data were analyzed. For estimation of the IPSS-M score, R version 4.2.1 was used to run batch-calculation using syntax code provided by the Papaemmanuil lab at MSKCC. The mean IPSS-M score was used as reference. Correlative analysis between IPSS-R and mean IPSS-M scores and outcome predictions was performed on LFS, OS and leukemic transformation. Time-to-event analyses were estimated using the Kaplan-Meier method and groups were compared by the log-rank test. We used Cox proportional hazards models for survival endpoints. For model discrimination we used Harrell's C concordance index. Results We identified at least 4,327 driver point mutations involving up to 170 genes across 2355 pts. We identified at least one gene mutation in 83% of pts (1948). Median follow-up was 4.6 yrs (4.4-4.9). 2333 pts (99%) were classified. Table 1 shows baseline clinical and molecular characteristics between validation and discovery cohorts. Using the IPSS-M risk stratification schema, pts in the validation cohort were classified as Very Low (96, 4% vs 14% in the discovery cohort), Low (560, 24% vs 32%), Moderate Low (327, 14% vs 11%), Moderate High (258, 11% vs 11%), High (454, 19% vs 14%) and Very High (660, 28% vs 18%) (Fig. 1A). The validation cohort was comprised of sicker pts with higher risk disease per IPSS-M relative to the discovery cohort (47% vs 31%). The IPSS-M score resulted in improved discrimination relative to the IPSS-R regarding LFS, OS and leukemic transformation as evidenced by a 2.3-, 2.0- and 1.5-point increase in C-index. The IPSS-M categories showed significant separation across all examined endpoints. Median LFS and OS estimates were equivalent between validation and discovery cohorts. Median LFS was 12.3 (vs 9.7), 6.9 (6.0), 3.6 (4.1), 2.2 (2.3), 1.4 (1.7) and 0.5 (0.7) yrs from Very Low to Very High-risk categories (Fig. 1B-1C). Conversely, median OS was 11.7 (vs. 10.6), 7.1 (6.0), 4.4 (4.6), 3.1 (2.8), 2.3 (1.7) and 1.3 (1.0) yrs from VL to VH subgroups (Fig.1). A five-to-five mapping between the IPSS-R and IPSS-M risk subgroups (from combining moderate-low (ML) and moderate-high (MH) into moderate), resulted in the restratification of 45% of pts (1056), equivalent to the discovery cohort (46%). Almost 3/4 of pts classified as IPSS-R very-low (VL) were upstaged (majority to IPSS-M low and ML/MH). Almost half of pts classified as IPSS-R intermediate shifted: 39% (174) were reclassified as IPSS-M high/very high (H/VH). Similarly, about 42% of pts classified as IPSS-R high were upstaged to IPSS-M VH. Almost 20% of VH risk pts were downstaged (majority to high risk). Most significant was the reclassification of 10.5% (244) of the validation cohort from IPSS-R lower and intermediate risk categories into IPSS-M higher risk strata. Contextually, the restratification of pts from IPSS-R intermediate to higher risk strata in IPSS-M was associated with notable differences in survival outcomes which were as poor as 0.5 yrs and as high as 6.9 yrs for those reclassified as LR (relative to 1.9 yrs). Conclusion To our knowledge this is the first external validation of IPSS-M in a large cohort. Use of the IPSS-M model for the risk stratification of pts with MDS resulted in improved discrimination of LFS, OS and risk of leukemic transformation over the IPSS-R. Furthermore, it led to restratification of almost half of MDS pts and exposed important differences in survival outcomes. Generalized use of this tool will likely result in more accurate prognostic assessment and optimize therapeutic decision-making among MDS pts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Increased osteoclastogenesis contributes to bone loss in the Costello syndrome Hras G12V mouse model.

RAS GTPases are ubiquitous GDP/GTP-binding proteins that function as molecular switches in cellular signalling and control numerous signalling pathways and biological processes. Pathogenic mutations in RAS genes severely affect cellular homeostasis, leading to cancer when occurring in somatic cells and developmental disorders when the germline is affected. These disorders are generally termed as RASopathies and among them Costello syndrome (CS) is a distinctive entity that is caused by specific HRAS germline mutations. The majority of these mutations affect residues 12 and 13, the same sites as somatic oncogenic HRAS mutations. The hallmarks of the disease include congenital cardiac anomalies, impaired thriving and growth, neurocognitive impairments, distinctive craniofacial anomalies, and susceptibility to cancer. Adult patients often present signs of premature aging including reduced bone mineral density and osteoporosis. Using a CS mouse model harbouring a Hras G12V germline mutation, we aimed at determining whether this model recapitulates the patients' bone phenotype and which bone cells are driving the phenotype when mutated. Our data revealed that Hras G12V mutation induces bone loss in mice at certain ages. In addition, we identified that bone loss correlated with an increased number of osteoclasts in vivo and Hras G12V mutations increased osteoclastogenesis in vitro. Last, but not least, mutant osteoclast differentiation was reduced by treatment in vitro with MEK and PI3K inhibitors, respectively. These results indicate that Hras is a novel regulator of bone homeostasis and an increased osteoclastogenesis due to Hras G12V mutation contributes to bone loss in the Costello syndrome.

Identification of key somatic oncogenic mutation based on a confounder-free causal inference model.

Abnormal cell proliferation and epithelial-mesenchymal transition (EMT) are the essential events that induce cancer initiation and progression. A fundamental goal in cancer research is to develop an efficient method to detect mutational genes capable of driving cancer. Although several computational methods have been proposed to identify these key mutations, many of them focus on the association between genetic mutations and functional changes in relevant biological processes, but not their real causality. Causal effect inference provides a way to estimate the real induce effect of a certain mutation on vital biological processes of cancer initiation and progression, through addressing the confounder bias due to neutral mutations and unobserved latent variables. In this study, integrating genomic and transcriptomic data, we construct a novel causal inference model based on a deep variational autoencoder to identify key oncogenic somatic mutations. Applied to 10 cancer types, our method quantifies the causal effect of genetic mutations on cell proliferation and EMT by reducing both observed and unobserved confounding biases. The experimental results indicate that genes with higher mutation frequency do not necessarily mean they are more potent in inducing cancer and promoting cancer development. Moreover, our study fills a gap in the use of machine learning for causal inference to identify oncogenic mutations.

Abstract 1327: Epithelial memory of resolved inflammation cooperates with oncogenic KRAS to limit tissue damage while promoting pancreatic cancer

Abstract The association between tumors and inflammation is a long-established clinical observation. Although many studies demonstrated that the inflammatory microenvironment can promote tumor growth through the activation of survival and proliferation programs in cancer cells, the reason why inflammation, an evolutionarily conserved response to damage aimed at reestablishing tissue integrity upon injury, might be integral to tumorigenesis still remains unknown. Pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by poor prognosis, represents a distinctive example of cooperation between inflammation and activated oncogenes. Frequently developed in a context of chronic pancreatitis, PDAC is always associated with an inflammatory microenvironment. As supported by a substantial body of evidence across a multitude of experimental models, when occurring in the context of pancreatitis, mutations of KRAS, the universal oncogenic driver of pancreatic cancer, lead to accelerated tumor development inducing the appearance of neoplastic precursor lesions, such as acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN), which can evolve into invasive tumors. Interestingly, preneoplastic pancreatic alterations, specifically ADM, have been previously identified in acute and chronic pancreatitis in the absence of oncogene activation. Investigating the effects of inflammation on normal pancreatic epithelial cells, we discovered that long after complete resolution a transient inflammatory event primes cells to cooperate with oncogenic KRAS. Indeed, upon recovery from a single acute inflammation, epithelial cells display an enduring adaptive response associated with sustained epigenetic and transcriptional reprogramming. Such adaptation facilitates the prompt reactivation of acinar-to-ductal metaplasia upon subsequent inflammatory events, representing a physiological mechanism for limiting tissue damage via rapid decrease of zymogen production. Because metaplastic lesions are mediated by the activation of MAPK signaling, we demonstrated that activating mutations of KRAS, maintaining an irreversible ADM, are protective against tissue damage in a contest of pancreatitis. Uncovering a new physiologic role of somatic oncogenic mutations in preserving tissue integrity during repeated damages, we propose that KRAS mutations represent a nearly universal event in pancreatic cancer because beneficial and under strong positive selection in the context of recurrent pancreatitis. Citation Format: I-Lin Ho, Edoardo Del Poggetto, Chiara Balestrieri, Er-Yen Yen, Francesca Citron, Rutvi Shah, Shaojun Zhang, Shan Jiang, Wantong Yao, Haoqiang Ying, Giannicola Genovese, Timothy Heffernan, Anirban Maitra, Linghua Wang, Timothy Wang, Giulio Draetta, Alessandro Carugo, Gioacchino Natoli, Andrea Viale. Epithelial memory of resolved inflammation cooperates with oncogenic KRAS to limit tissue damage while promoting pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1327.

Impact of cancer susceptibility gene (CSG) mutations in advanced NSCLC (aNSCLC).

9131 Background: Next generation sequencing (NGS) is used widely to identify somatic oncogenic driver mutations in aNSCLC to guide treatment. Comprehensive profiling has led to identification of multiple gene mutations of unclear significance, including germline genes associated with cancer risk, termed CSG. Using data from two randomized trials comparing atezolizumab to docetaxel, we investigated the prognostic and predictive values of CSG in aNSCLC. Methods: We used publicly available data from the OAK (NCT02008227) and POPLAR (NCT01903993) trials. At baseline, plasma was analyzed for cfDNA using FoundationOne CDx NGS assay. We defined CSGs as pathogenic variants of APC, BAP1, BRCA1/2, BRIP1, CDH1, CDKN2A, CHEK2, FH, FLCN, MEN1, MET, MSH2/6, MLH1, PMS2, PALB2, PTCH1, PTEN, RAD51, RB1, SDHB, SMARCA4, STK11, TSC1/2 and VHL. Cox models with treatment covariate, CSG status (mutant [mt] vs wild-type [wt]) and their interaction was used to assess the predictive value by treatment of CSG mt for overall survival (OS) and progression free survival (PFS) using univariate and multivariate models. Similar analysis was performed for objective response rate (ORR). Results: Of 1137 patients, 853 with sufficient tumor content formed the analysis population. In total, 295 (35%) had a known/likely pathogenic CSG mt. The variant allele frequencies (vAF) of CSG mt ≥30%, 10-30%, and <10% were 8% (N=23), 25% (N=73), and 67% (N=199) respectively. Patients with CSG mt were more likely to be smokers (89% vs 81%, P=0.005), had squamous tumors (37% vs 26%, P=0.001) with higher blood-based tumor mutation burden (mean 13.8 vs 10.2 per megabase, P<0.001). CSG mt was not predictive of greater OS benefit with atezolizumab over docetaxel (Table). CSG mt was associated with 35% increase risk of death in univariable analysis (HR 1.35, 95% CI 1.15-1.59). CSG mt was associated with inferior OS in multivariable analysis adjusting for performance status, smoking status, tumor histology, age, sex and number of organ sites of metastasis (HR 1.26, 95% CI 1.07-1.48). Conclusions: Plasma CSG mt is an independent poor prognostic factor in two large aNSCLC clinical trial datasets. Majority of vAF of CSG were low, suggesting that very few were potentially germline in origin, but dedicated sequencing for confirmation will be required. If confirmed, CSG status could be used as a stratification factor in future aNSCLC trials. [Table: see text]

Driver gene mutations in micro-invasive cervical squamous cancers have no prognostic significance

ObjectiveTo evaluate the prevalence of somatic gene mutations in different stages of cervical carcinogenesis placing special emphasis on micro-invasive pT1a cervical squamous cell cancers (SCC). MethodsMicro-dissected samples of 32 micro-invasive pT1a and 55 ≥ pT1b SCC were evaluated by next generation sequencing of 50 cancer genes (cancer hot spot panel). ResultsAt primary diagnosis, 8/32 (25%) pT1a SCC, 10/28 (36%) pT1b SCC and 15/27 (56%) pT2/3 SCC carried somatic gene mutations. The most commonly affected gene was the PIK3CA gene in hot spot regions E545K and E453K in 5/8 (62%) pT1a SCC, 7/15 (70%) pT1b SCC and 10/15 (66%) pT2/3 SCC followed by FBXW7 (n = 4), KRAS and RB1 (n = 2 each). ERBB2, APC, ATM, MLP gene mutations occurred only once. Solitary activating oncogenic somatic mutations dominated over tumor suppressor mutation in 88% pT1a, 80% pT1b and 60% pT2/3 SCC. Concomitant mutations involved typically an activating oncogenic mutation and an inactivating tumor-suppressor gene mutation. All patients with pT1a SCC are alive without evidence of disease after surgical treatment, independent of mutational status or lympho-vascular space invasion. ConclusionsActivating oncogenic gene mutations, in particular in the PIK3CA gene, occur early in cervical carcinogenesis. Although driver gene mutations bestow tumor cells with a growth advantage, early detection and complete removal of all cancer cells – with or without somatic gene mutations – are essential for cure. In contrast to advanced inoperable SCC, where PIK3CA driver gene mutations carry an adverse prognosis, the mutational status in surgically treated micro-invasive SCC is prognostically irrelevant.

Sarcomatoid Yolk Sac Tumor Harbors Somatic Mutations That Are Otherwise Rare in Testicular Germ Cell Tumors.

In testicular germ cell tumors (TGCTs), components with nonspecific sarcomatous features that express keratins and glypican 3 are classified as sarcomatoid yolk sac tumor (SYST). SYST is most frequently seen in metastatic sites after chemotherapy. Like so-called "somatic-type" malignancies arising in TGCTs, SYST is markedly resistant to systemic therapy and has a more aggressive clinical course than conventional types of TGCT. However, the clinicopathologic and molecular features of SYST remain incompletely described. This study evaluated a multi-institutional series of 20 SYSTs using massively parallel sequencing and p53 immunohistochemistry. The histologic and clinical characteristics of the cases were also assessed, including analyses of disease-specific outcomes. DNA sequencing identified somatic mutations in 12/20 cases (60%), including recurrent TP53 and RIF1 mutations (present in 4/20 cases, 20% each). In 3 of the 4 SYST with TP53 mutations, there was molecular evidence of loss of heterozygosity. Immunohistochemistry demonstrated diffuse overexpression of p53 protein in 3/4 (75%) cases with TP53 mutations. The remaining TP53-mutant case demonstrated multifocal overexpression of p53, suggestive of subclonal inactivation of the gene. Overexpression of p53 protein was not seen in any of 15 TP53 wild-type cases evaluated by immunohistochemistry. A subset of 4 cases underwent RNA sequencing (fusion panel), which demonstrated the absence of oncogenic gene fusions. A 2-tiered grading system based on 3 histologic parameters (cellularity, number of mitoses, and necrosis) demonstrated that high-grade SYSTs have a higher risk of disease-specific death compared to low-grade tumors. The risk of disease-specific mortality was also higher in SYSTs with somatic mutations. In conclusion, this study demonstrated that 60% of SYSTs harbor somatic oncogenic mutations that are otherwise rare in TGCTs, and the presence of these mutations is associated with an aggressive clinical course. In addition, the results presented herein suggest that grading SYSTs may be clinically relevant.

Early detection of colorectal cancer somatic mutations using cfDNA liquid biopsies in a murine carcinogenesis model.

Colorectal cancer (CRC) is one of the top five cancers in incidence and mortality worldwide. The early detection of this neoplasm through analysis of circulating free DNA (cfDNA), which carries tumor genetic alterations, as a liquid biopsy, could have a major impact in enhancing early detection and reducing the mortality rate. The aim of this work was to demonstrate the feasibility of using cfDNA as a liquid biopsy for the early detection of CRC. For this purpose, we implemented an azoxymethane and dextran sodium sulfate-induced murine carcinogenesis model to detect oncogenic somatic mutations in Ctnnb1 and Kras during CRC development. To enhance the sensitivity in the detection, E-ice-COLD-PCR was utilized to selectively enrich for mutant alleles, followed by massively parallel sequencing. Driving somatic mutations were detected in Ctnnb1 and Kras in the liquid biopsies of early stages of tumor development, corresponding to the formation of aberrant crypt foci, the first histological alterations that can be identified throughout the formation of CRC. The concentration of cfDNA was increased along the carcinogenic process. Polyclonality in Ctnnb1 was found in tumor samples and cfDNA in this model. On the other hand, the use of cfDNA as a non-invasive test resulted in superior early detection compared to microPET/CT imaging. As a proof-of-principle, this study shows the great potential use of allelic-specific PCR for the detection and enrichment of pathogenic alleles present in cfDNA samples, as a test for early non-invasive detection of CRC. This work provides scientific evidence to set methodological bases that allow early detection of mutations in cfDNA obtained from plasma of CRC in humans.

Shared Positivity in Daily Life of Younger and Older Couples: Links Between We-ness and Positive Emotions

Age is the single major risk factor for human cancer, but naturally occurring cancers are rarely studied in aging models. Like humans, mice spontaneously develop cancer with age, and standard laboratory strains are predisposed for B-cell lymphoma. Here, we uncover how B-cell lymphoma develops as a consequence of the aging immune system. We found that aged B cells acquire somatic mutations in tumor suppressors and oncogenes (e.g. Trp53, Pim1, and Myh11) and undergo monoclonal expansions, with some clones representing 86% of splenic B cells. Clonal B cells had hypermethylated promoters and globally silenced expression, suggesting a role of DNA methylation in clonal selection of premalignant B cells. B-cell size, spleen weight, and a novel population of B cells, which we named Myc+ cells, emerged as convenient markers of malignancy. High-throughput analyses of clonal B cells and the use of genetic mouse models revealed that c-Myc drives B-cell size increase and clonal expansion with age. Phoshoproteome and co-culture experiments revealed that c-Myc is activated by signals from the aging microenvironment. Moreover, single-cell RNA-seq suggested that clonal B cells originate from age-associated B cells, further underlying the importance of aging environment in cancer transformation. Longitudinal analyses demonstrated a negative impact of premalignant B cells on mouse lifespan and linked it to age-related myeloid bias. Together, our study revealed cell-autonomous changes that cooperate with the aging microenvironment to give rise to preneoplastic B cells. This stidy established a novel model to study how aging predisposes cells to cancer transformation.

Aging Predisposes B cells to Malignancy by Activating c-Myc and Perturbing the Genome and Epigenome

Age is the single major risk factor for human cancer, but naturally occurring cancers are rarely studied in aging models. Like humans, mice spontaneously develop cancer with age, and standard laboratory strains are predisposed for B-cell lymphoma. Here, we uncover how B-cell lymphoma develops as a consequence of the aging immune system. We found that aged B cells acquire somatic mutations in tumor suppressors and oncogenes (e.g. Trp53, Pim1, and Myh11) and undergo monoclonal expansions, with some clones representing 86% of splenic B cells. Clonal B cells had hypermethylated promoters and globally silenced expression, suggesting a role of DNA methylation in clonal selection of premalignant B cells. B-cell size, spleen weight, and a novel population of B cells, which we named Myc+ cells, emerged as convenient markers of malignancy. High-throughput analyses of clonal B cells and the use of genetic mouse models revealed that c-Myc drives B-cell size increase and clonal expansion with age. Phoshoproteome and co-culture experiments revealed that c-Myc is activated by signals from the aging microenvironment. Moreover, single-cell RNA-seq suggested that clonal B cells originate from age-associated B cells, further underlying the importance of aging environment in cancer transformation. Longitudinal analyses demonstrated a negative impact of premalignant B cells on mouse lifespan and linked it to age-related myeloid bias. Together, our study revealed cell-autonomous changes that cooperate with the aging microenvironment to give rise to preneoplastic B cells. This stidy established a novel model to study how aging predisposes cells to cancer transformation.

Discovery of a novel potentially transforming somatic mutation in CSF2RB gene in breast cancer.

The colony stimulating factor 2 receptor subunit beta (CSF2RB) is the common signaling subunit of the cytokine receptors for IL‐3, IL‐5, and GM‐CSF. Several studies have shown that spontaneous and random mutants of CSF2RB can lead to ligand independence in vitro. To date, no report(s) have been shown for the presence of potentially transforming and oncogenic CSF2RB mutation(s) clinically in cancer patients until the first reported case of a leukemia patient in 2016 harboring a germline‐activating mutation (R461C). We combined exome sequencing, pathway analyses, and functional assays to identify novel somatic mutations in KAIMRC1 cells and breast tumor specimen. The patient’s peripheral blood mononuclear cell (PBMC) exome served as a germline control in the identification of somatic mutations. Here, we report the discovery of a novel potentially transforming and oncogenic somatic mutation (S230I) in the CSF2RB gene of a breast cancer patient and the cell line, KAIMRC1 established from her breast tumor tissue. KAIMRC1 cells are immortalized and shown to survive and proliferate in ligand starvation condition. Immunoblot analysis showed that mutant CSF2RB signals through JAK2/STAT and PI3K/mTOR pathways in ligand starvation conditions. Screening a small molecule kinase inhibitor library revealed potent JAK2 inhibitors against KAIMRC1 cells. We, for the first time, identified a somatic, potentially transforming, and oncogenic CSF2RB mutation (S230I) in breast cancer patients that seem to be an actionable mutation leading to the development of new therapeutics for breast cancer.

Identification and clinical significance of somatic oncogenic mutations in epithelial ovarian cancer

ObjectiveEpithelial ovarian cancer (EOC) is a heterogeneous disease with diverse clinicopathological features and behaviors, and its heterogeneity may be concerned with the accumulation of multiple somatic oncogenic mutations. The major goals of this study are to systematically perform the comprehensive mutational profiling in EOC patients, and investigate the associations between somatic mutations and clinicopathological characteristics.MethodsA total of 80 surgical specimens were obtained from EOC patients who had previously undergone primary debulking surgery, and genomic DNAs were extracted from fresh-frozen tissues. We investigated mutational status in hot spot regions of 50 cancer-related genes by targeted next-generation sequencing using an Ion AmpliSeq Cancer Hotspot Panel v2 Kit.ResultsValidated mutations were detected in 66 of the 80 tumors (82.5%). The five most frequently mutated genes were TP53 (43.8%), PIK3CA (27.5%), KRAS (23.8%), PTEN (10%) and CTNNB1 (10%). PTEN and CTNNB1 mutations were associated with younger age. PIK3CA1, KRAS and CTNNB1 mutations were observed in early-stage, whereas TP53 mutations were more common in advanced stage. Significant associations were observed between TP53 mutation and serous carcinoma, and between KRAS mutation and mucinous carcinoma. Both PIK3CA mutation and CTNNB1 mutation were also significantly associated with endometrioid and clear cell carcinoma. The patients with PIK3CA and KRAS mutations were significantly associated with favorable progression free survival (PFS). In particular, PIK3CA mutations had more significant associations with favorable PFS than PIK3CA wild-type in the endometrioid subtype (P = 0.012). Patients with mutations only in TP53 were significantly associated with worse PFS.ConclusionEOCs were heterogeneous at the genomic level and harbored somatic oncogenic mutations. Our molecular profiling may have the potential for becoming a novel stratification within histological subtypes of EOC. Further studies are needed to define molecular classification for improved clinical outcomes and treatment of EOC patients in future.