TP53 Alterations Research Articles

Current status of molecular diagnostic approaches using liquid biopsy

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers, and developing an efficient and reliable approach for its early-stage diagnosis is urgently needed. Precancerous lesions of PDAC, such as pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasms (IPMN), arise through multiple steps of driver gene alterations in KRAS, TP53, CDKN2A, SMAD4, or GNAS. Hallmark mutations play a role in tumor initiation and progression, and their detection in bodily fluids is crucial for diagnosis. Recently, liquid biopsy has gained attention as an approach to complement pathological diagnosis, and in addition to mutation signatures in cell-free DNA, cell-free RNA, and extracellular vesicles have been investigated as potential diagnostic and prognostic markers. Integrating such molecular information to revise the diagnostic criteria for pancreatic cancer can enable a better understanding of the pathogenesis underlying inter-patient heterogeneity, such as sensitivity to chemotherapy and disease outcomes. This review discusses the current diagnostic approaches and clinical applications of genetic analysis in pancreatic cancer and diagnostic attempts by liquid biopsy and molecular analyses using pancreatic juice, duodenal fluid, and blood samples. Emerging knowledge in the rapidly advancing liquid biopsy field is promising for molecular profiling and diagnosing pancreatic diseases with significant diversity.

Unraveling the genetics of transformed splenic marginal zone lymphoma.

The genetic mechanisms associated with splenic marginal zone lymphoma (SMZL) transformation are not well defined. We studied 41 patients with SMZL that eventually underwent large B-cell lymphoma transformation. Tumor material was obtained either only at diagnosis (9 patients), at diagnosis and transformation (18 patients), and only at transformation (14 patients). Samples were categorized in 2 groups: (1) at diagnosis (SMZL, n= 27 samples), and (2) at transformation (SMZL-T, n= 32 samples). Using copy number arrays and a next-generation sequencing custom panel, we identified that the main genomic alterations in SMZL-T involved TNFAIP3, KMT2D, TP53, ARID1A, KLF2, 1q gains, and losses of 9p21.3 (CDKN2A/B) and 7q31-q32. Compared with SMZL, SMZL-T had higher genomic complexity, and higher incidence of TNFAIP3 and TP53 alterations, 9p21.3 (CDKN2A/B) losses, and 6p gains. SMZL and SMZL-T clones arose by divergent evolution from a common altered precursor cell that acquired different genetic alterations in virtually all evaluable cases (92%, 12 of 13 cases). Using whole-genome sequencing of diagnostic and transformation samples in 1 patient, we observed that the SMZL-T sample carried more genomic aberrations than the diagnostic sample, identified a translocation t(14;19)(q32;q13) present in both samples, and detected a focal B2M deletion due to chromothripsis acquired at transformation. Survival analysis showed that KLF2 mutations, complex karyotype, and International Prognostic Index score at transformation were predictive of a shorter survival from transformation (P= .001; P= .042; and P= .007; respectively). In summary, SMZL-T are characterized by higher genomic complexity than SMZL, and characteristic genomic alterations that could represent key players in the transformation event.

Genetic alterations of KRAS and TP53 in intrahepatic cholangiocarcinoma associated with poor prognosis.

The aim of this study is to investigate certain genetic features of intrahepatic cholangiocarcinoma (ICCA). A total of 12 eligible ICCA patients were enrolled, and tumor tissues from the patients were subjected to next-generation sequencing of a multi-genes panel. Tumor mutation burden (TMB), mutated genes, copy number variants (CNVs), and pathway enrichment analysis were performed. The median TMB was 2.76 Mutation/Mb (range, 0-36.62 Mutation/Mb) in ICCA patients. The top two most commonly mutated genes in ICCA were KRAS (33%) and TP53 (25%). The co-mutations of KRAS and TP53 were 16.7% (2/12) in ICCA patients. Notably, patient P6 with the highest TMB did not have KRAS and TP53 mutations. Additionally, TP53 and/or KRAS alterations were significantly associated with poor progression-free survival than those with wild type (1.4 months vs 18 months). DNA damage repair and homologs recombinant repair deficiencies were significantly associated with high TMB in ICCA cases. In conclusion, we found that certain genetic mutations of TP53 and KRAS could predict poor prognosis in ICCA patients.

Machine learning for cryosection pathology predicts the 2021 WHO classification of glioma

Timely and accurate intraoperative cryosection evaluations remain the gold standard for guiding surgical treatments for gliomas. However, the tissue-freezing process often generates artifacts that make histologic interpretation difficult. In addition, the 2021 WHO Classification of Tumors of the Central Nervous System incorporates molecular profiles in the diagnostic categories, so standard visual evaluation of cryosections alone cannot completely inform diagnoses based on the new classification system. To address these challenges, we develop the context-aware Cryosection Histopathology Assessment and Review Machine (CHARM) using samples from 1,524 glioma patients from three different patient populations to systematically analyze cryosection slides. Our CHARM models successfully identified malignant cells (AUROC= 0.98± 0.01 in the independent validation cohort), distinguished isocitrate dehydrogenase (IDH)-mutant tumors from wild type (AUROC= 0.79-0.82), classified three major types of molecularly defined gliomas (AUROC= 0.88-0.93), and identified the most prevalent subtypes of IDH-mutant tumors (AUROC= 0.89-0.97). CHARM further predicts clinically important genetic alterations in low-grade glioma, including ATRX, TP53, and CIC mutations, CDKN2A/B homozygous deletion, and 1p/19q codeletion via cryosection images. Our approaches accommodate the evolving diagnostic criteria informed by molecular studies, provide real-time clinical decision support, and will democratize accurate cryosection diagnoses. Supported in part by the National Institute of General Medical Sciences grant R35GM142879, the Google Research Scholar Award, the Blavatnik Center for Computational Biomedicine Award, the Partners' Innovation Discovery Grant, and the Schlager Family Award for Early Stage Digital Health Innovations.

Mathematical modeling the order of driver gene mutations in colorectal cancer.

Tumor heterogeneity is a large obstacle for cancer study and treatment. Different cancer patients may involve different combinations of gene mutations or the distinct regulatory pathways for inducing the progression of tumor. Investigating the pathways of gene mutations which can cause the formation of tumor can provide a basis for the personalized treatment of cancer. Studies suggested that KRAS, APC and TP53 are the most significant driver genes for colorectal cancer. However, it is still an open issue regarding the detailed mutation order of these genes in the development of colorectal cancer. For this purpose, we analyze the mathematical model considering all orders of mutations in oncogene, KRAS and tumor suppressor genes, APC and TP53, and fit it on data describing the incidence rates of colorectal cancer at different age from the Surveillance Epidemiology and End Results registry in the United States for the year 1973-2013. The specific orders that can induce the development of colorectal cancer are identified by the model fitting. The fitting results indicate that the mutation orders with KRAS → APC → TP53, APC → TP53 → KRAS and APC → KRAS → TP53 explain the age-specific risk of colorectal cancer with very well. Furthermore, eleven pathways of gene mutations can be accepted for the mutation order of genes with KRAS → APC → TP53, APC → TP53 → KRAS and APC → KRAS → TP53, and the alternation of APC acts as the initiating or promoting event in the colorectal cancer. The estimated mutation rates of cells in the different pathways demonstrate that genetic instability must exist in colorectal cancer with alterations of genes, KRAS, APC and TP53.

Distinct genomic landscapes in radiation-associated angiosarcoma compared with other radiation-associated sarcoma histologies.

MYC amplifications have been frequently detected in radiation (RT)-associated angiosarcomas (ASs) by low-resolution molecular methods. However, large-scale next-generation sequencing (NGS) studies to investigate the genomic landscape of RT-AS are scarce, particularly compared with other RT-associated sarcomas. We performed a detailed comparative genomic investigation of RT-AS versus other RT-associated histotypes, as well as sporadic sarcomas with similar histologies. Our institutional targeted DNA-NGS assay database was searched for RT-associated sarcomas. Clinical outcome data, pathologic diagnosis, and the types and frequencies of genomic alterations, including single nucleotide variants (SNVs) and copy number alterations (CNAs), were analyzed. The cohort consisted of 82 patients, 68 (83%) females and 14 (17%) males, aged 37-88 (mean 64) years. Forty-four RT-ASs (38 from breast) and 38 RT sarcomas of other histologies, including 12 malignant peripheral nerve sheath tumors (RT-MPNSTs), 14 undifferentiated pleomorphic sarcomas (RT-UPSs), and 12 osteosarcomas (RT-OSs), were included. Median time intervals from radiation to initial diagnosis in RT-AS (8.0 years) were significantly lower than those in RT-MPNST and RT-UPS (12.5 and 18.5 years), respectively. Each RT-sarcoma histotype harbored distinct mutations and CNAs. RT-associated AS had more frequent MYC, FLT4, CRKL, HRAS, and KMT2D alterations than sporadic AS (enriched in TP53, KDR, ATM, ATRX), whereas the mutational landscapes of MPNST, UPS, and OS were similar in both RT and non-RT settings. CDKN2A/B deletions and TP53 alterations were infrequent in RT-AS compared with other RT sarcomas. Among RT sarcomas, RT-AS harbored the lowest fraction of genome altered (FGA), while RT-MPNST showed the highest FGA. RT-AS had the lowest insertion:SNV and deletion:SNV ratios, while RT-UPS had the highest. The predominant mutational signatures were associated with errors in DNA repair and replication. In conclusion, RT-AS has a distinct genomic landscape compared with other RT sarcomas and sporadic AS. Potential molecular targets for precision medicine may be histotype-dependent. © 2023 The Pathological Society of Great Britain and Ireland.

Plasma cell-free tumor DNA, PIK3CA and TP53 mutations predicted inferior endocrine-based treatment outcome in endocrine receptor-positive metastatic breast cancer

PurposeHow to factor both tumor burden and oncogenic genomic mutations as variables to predict the outcome of endocrine-based therapy (ET) in ER-positive/HER2-negative metastatic breast cancer patients (MBC) remains to be explored.MethodBlood samples prospectively collected from 163 ER-positive/HER2-negative female MBC patients, before ET, were used for cell-free tumor DNA (cfDNA) analysis. cfDNA was subjected to next-generation sequencing (NGS) to interrogate oncogenic PIK3CA hotspot and TP53 DNA-binding domain (DBD) mutations, including single nucleotide variants (SNVs) or small insertions and deletions (InDels). The variant calling threshold was set at 0.5%. Progression-free survival (PFS) was measured from the start of the ET treatment to the time of disease progression of the same treatment regimen.ResultsOverall, the median PFS was 8.3 months (95% CI 5.7–11.1 months). The median cfDNA was 38.5 ng (range 4.4–1935 ng). The proportion of patients with PIK3CA and TP53 alterations were 25.1 and 15.3%, respectively. Patients with high total cfDNA (HR 1.74, p = 0.003), PIK3CA mutation (HR 1.74, p = 0.007), and TP53 mutation (HR 1.64, p = 0.047) in liquid biopsy conferred worse outcome after ET. Even for patients with low tumor burden, the detrimental effect of PIK3CA or TP53 mutation remained significant (p < 0.001). For patients with either PIK3CA (p < 0.001) or TP53 mutation (p = 0.004), there was significant positive correlation between allele frequency (AF) and total cfDNA.ConclusionAfter adjustment of cfDNA level, PIK3CA and TP53 mutations observed in liquid biopsy exerted detrimental effects on the outcome of ET-based regimens. The AF of PIK3CA or TP53 may be a surrogate marker for PFS.

Targeted Next-Generation Sequencing Improves the Prognostication of Patients with Disseminated Appendiceal Mucinous Neoplasms (Pseudomyxoma Peritonei).

Appendiceal mucinous neoplasms (AMNs) with disseminated disease (pseudomyxoma peritonei) are heterogeneous tumors with variable clinicopathologic behavior. Despite the development of prognostic systems, objective biomarkers are needed to stratify patients. With the advent of next-generation sequencing (NGS), it remains unclear if molecular testing can improve the evaluation of disseminated AMN patients. Targeted NGS was performed for 183 patients and correlated with clinicopathologic features to include American Joint Committee on Cancer/World Health Organization (AJCC/WHO) histologic grade, peritoneal cancer index (PCI), completeness of cytoreduction (CC) score, and overall survival (OS). Genomic alterations were identified for 179 (98%) disseminated AMNs. Excluding mitogen-activated protein kinase genes and GNAS due to their ubiquitous nature, collective genomic alterations in TP53, SMAD4, CDKN2A, and the mTOR genes were associated with older mean age, higher AJCC/WHO histologic grade, lymphovascular invasion, perineural invasion, regional lymph node metastasis, and lower mean PCI (p < 0.040). Patients harboring TP53, SMAD4, ATM, CDKN2A, and/or mTOR gene alterations were found to have lower OS rates of 55% at 5 years and 14% at 10 years, compared with 88% at 5 years and 88% at 10 years for patients without the aforementioned alterations (p < 0.001). Based on univariate and multivariate analyses, genomic alterations in TP53, SMAD4, ATM, CDKN2A, and/or the mTOR genes in disseminated AMNs were a negative prognostic factor for OS and independent of AJCC/WHO histologic grade, PCI, CC score, and hyperthermic intraperitoneal chemotherapy treatment (p = 0.006). Targeted NGS improves the prognostic assessment of patients with disseminated AMNs and identifies patients who may require increased surveillance and/or aggressive management.

METB-08. GENOMIC AND MOLECULAR PROFILING OF PEDIATRIC CENTRAL NERVOUS SYSTEM TUMORS: A SINGLE INSTITUTION ANALYSIS

Abstract BACKGROUND Molecular characterization has revolutionized management of pediatric central nervous system (CNS) tumors, with genomic sequencing for diagnosis and determination of actionable variants now largely considered standard of care. We report a single institution’s experience performing genomic sequencing for pediatric CNS tumors over nearly five years. METHODS Nucleic acid isolated from tumor tissue and paired normal specimens from patients at Children’s Hospital of Philadelphia (CHOP) undergoes paired next generation sequencing (NGS) and copy number analyses of 237 cancer genes and a fusion panel of more than 700 exons of 117 cancer genes (tumor only). All variants are tiered 1-4 according to known clinical significance. Descriptive statistics summarize clinical and molecular data and ensemble-based methods will be used to correlate molecular and clinical characteristics. RESULTS Between January 1, 2016 and June 30, 2020, 497 patients ≤ 21 years old with median age of 9.5 years (n=243 females; 49%) contributed to 539 cases across 25 CNS tumor subtypes. The most common diagnoses were pilocytic astrocytoma (n=118), medulloblastoma (n=49), and diffuse midline glioma (DMG; n=39). Clinically significant variants (tier 1,2) were detected in 91% of the patients. Most tumors demonstrated tier 1,2 single nucleotide variants (SNV)/insertions/deletions (n=263, 53%), most commonly in TP53&amp;gt;BRAF&amp;gt;H3F3A, and/or CNVs (n=310, 62%), most commonly gains of 7q&amp;gt;7p&amp;gt;1q. Thirty percent of tumors demonstrated tier 1,2 fusions (n=149), most commonly involving BRAF, NTRK2, or FGFR. Average tumor mutation burden in patients without germline predispositions was 3.5/Mbp (standard deviation 3.8). Germline/presumed germline pathogenic variants were found in 18% (90/497) of patients tested with most common germline alterations in TP53 followed by NF1 and APC. CONCLUSION A large, single site cohort of molecularly characterized CNS tumors demonstrate the clinical and biologic utility of real-time, institution-based genomic sequencing. Molecular findings in correlation with clinical characteristics and outcomes are underway.

DIPG-51. ABDOMINAL METASTASES OF DIFFUSE MIDLINE GLIOMA, H3K27-ALTERED WITH A VENTRICULOPERITONEAL SHUNT

Abstract Extraneural metastases are extremely rare in diffuse midline glioma (DMG), H3K27-altered, despite a tendency toward aggressive local growth and potential neuroaxis dissemination at progression. Ventriculoperitoneal shunt (VPS) placement for cerebrospinal fluid diversion is commonly performed in patients with thalamic tumors and increasingly considered in brainstem disease with developing hydrocephalus. Herein, we present a case of DMG seeding the abdomen presumably via the VPS. This patient was diagnosed at 22 years of age with a bithalamic tumor with extension into the brainstem and cerebellum as well as non-contiguous periventricular spread to the bilateral frontal lobes, with associated hydrocephalus. The patient underwent VPS insertion and biopsy, with pathology consistent with DMG, H3K27-altered. Whole exome sequencing revealed H3-3A and TP53 alterations as well as PDGFRA amplification. Treatment consisted of upfront craniospinal photon irradiation, with subsequent reduction in thalamic disease burden. Adjuvant therapy with an oral PDGFRA inhibitor (avapritinib) was initiated, though discontinued after two weeks due to worsening neurocognitive deficits. The patient presented three months post-completion of radiation, five months from diagnosis, and six weeks following VPS revision with rapidly progressive abdominal distension and altered mental status, with imaging demonstrating omental caking and significant malignant ascites, resulting in shunt malfunction and increased intracranial pressure. With limited therapeutic options, the family decided not to pursue further intervention and the patient died five days later. Autopsy confirmed metastatic disease throughout omentum, in addition to diffuse leptomeningeal dissemination. Comprehensive molecular profiling of diagnostic and post-mortem thalamic and omental tissue is in process to assess temporal as well as spatial genomic heterogeneity, including of metastasis-mediated pathways. In summary, abdominal dissemination of DMG, H3K27-altered is uncommon, but possible, especially in the presence of a VPS. Further research is necessary to determine biologic mechanisms and predictors of extraneural metastases in this disease.

Correlation of Programmed Death-Ligand 1 Expression With Lung Adenocarcinoma Histologic and Molecular Subgroups in Primary and Metastatic Sites

Programmed death-ligand 1 (PD-L1) expression in terms of the tumor proportion score (TPS) is the main predictive biomarker approved for immunotherapy against lung nonsmall cell carcinoma. Although some studies have explored the associations between histology and PD-L1 expression in pulmonary adenocarcinoma, they have been limited in sample size and/or extent of examined histologic variables, which may have resulted in conflicting information. In this observational retrospective study, we identified primary and metastatic lung adenocarcinoma cases in the span of 5 years and tabulated the detailed histopathologic features, including pathological stage, tumor growth pattern, tumor grade, lymphovascular and pleural invasion, molecular alterations, and the associated PD-L1 expression for each case. Statistical analyses were performed to detect associations between PD-L1 and these features. Among 1658 cases, 643 were primary tumor resections, 751 were primary tumor biopsies, and 264 were metastatic site biopsies or resections. Higher TPS significantly correlated with high-grade growth patterns, grade 3 tumors, higher T and N stage, presence of lymphovascular invasion, and presence of MET and TP53 alterations, whereas lower TPS correlated with lower-grade tumors and presence of EGFR alterations. There was no difference in PD-L1 expression in matched primary and metastases, although higher TPS was observed in metastatic tumors due to the presence of high-grade patterns in these specimens. TPS showed a strong association with a histologic pattern. Higher-grade tumors had higher TPS, which is also associated with more aggressive histologic features. Tumor grade should be kept in mind when selecting cases and blocks for PD-L1 testing.

BRAF mutations and concurrent alterations in patients with soft tissue sarcoma.

BRAF alterations, including V600E and non-V600E mutations and fusions, in soft tissue sarcoma (STS) have been identified in a limited case series. Here, we aimed to evaluate the frequency of BRAF mutations and concurrent alterations in STS to understand their therapeutic action. In this retrospective analysis, we included data from 1964 patients with advanced STS who underwent comprehensive genomic profiling tests at hospitals in Japan between June 2019 and March 2023. The prevalence of BRAF and recurrent concurrent gene alterations were also investigated. BRAF mutations were detected in 24 (1.2%) of 1964 STS patients, with a median age of 47 (range 1-69) years. BRAF V600E was detected in 11 (0.6%) of the 1964 patients with STS, BRAF non-V600E mutations in 9 (4.6%), and BRAF fusions were detected in 4 (0.2%). BRAF V600E was identified in 4 (0.2%) cases of malignant peripheral nerve sheath tumors. The most common concurrent alteration was CDKN2A (11 cases, 45.8%), and the frequency was equivalent to that of the BRAF V600E (5/11 cases, 45.5%) and non-V600E (5/9 cases, 55.6%) groups. Recurrent concurrent alterations, such as TERT promoter mutations (7 cases, 29.2%), were detected at the same frequency in the V600E and non-V600E groups. In contrast, TP53 alterations (4/9 cases, 44.4%) and mitogen-activated protein kinase (MAPK)-activating genes, including NF1, GNAQ, and GNA11 (3/9 cases, 33.3%), were identified as relatively higher in the non-V600E group than in the V600E group (each 1/11 case, 9.1%). We identified BRAF alterations at a rate of 1.2% in all patients with advanced STS. Among them, BRAF V600E and BRAF fusions account for 45.8% and 16.7%, respectively. Collectively, our findings support the clinical characteristics and therapeutic strategies for patients with BRAF-altered advanced STS.

Post-operative mortality and recurrence patterns in pancreatic cancer according to KRAS mutation and CDKN2A, p53, and SMAD4 expression.

Alterations in KRAS, CDKN2A (p16), TP53, and SMAD4 genes have been major drivers of pancreatic carcinogenesis. The clinical course of patients with pancreatic cancer in relation to these driver alterations has not been fully characterised in large populations. We hypothesised that pancreatic carcinomas with different combinations of KRAS mutation and aberrant expression of CDKN2A, p53, and SMAD4 might show distinctive recurrence patterns and post-operative survival outcomes. To test this hypothesis, we utilised a multi-institutional cohort of 1,146 resected pancreatic carcinomas and assessed KRAS mutations by droplet digital polymerase chain reaction and CDKN2A, p53, and SMAD4 expression by immunohistochemistry. Multivariable hazard ratios (HRs) and 95% confidence intervals (CIs) for disease-free survival (DFS) and overall survival (OS) were computed according to each molecular alteration and the number of altered genes using the Cox regression models. Multivariable competing risks regression analyses were conducted to assess the associations of the number of altered genes with specific patterns of recurrence. Loss of SMAD4 expression was associated with short DFS (multivariable HR, 1.24; 95% CI, 1.09-1.43) and OS times (multivariable HR, 1.27; 95% CI, 1.10-1.46). Compared to cases with 0-2 altered genes, cases with three and four altered genes had multivariable HRs for OS of 1.28 (95% CI, 1.09-1.51) and 1.47 (95% CI, 1.22-1.78), respectively (ptrend < 0.001). Patients with an increasing number of altered genes were more likely to have short DFS time (ptrend = 0.003) and to develop liver metastasis (ptrend = 0.006) rather than recurrence at local or other distant sites. In conclusion, loss of SMAD4 expression and an increasing number of altered genes were associated with unfavourable outcomes in pancreatic cancer patients. This study suggests that the accumulation of the four major driver alterations can confer a high metastatic potential to the liver, thereby impairing post-operative survival among patients with pancreatic cancer.

An amino acid metabolism-based seventeen-gene signature correlates with the clinical outcome and immune features in pancreatic cancer.

Background: Pancreatic cancer is an aggressive tumor with a low 5-year survival rate and primary resistance to most therapy. Amino acid (AA) metabolism is highly correlated with tumor growth, crucial to the aggressive biological behavior of pancreatic cancer; nevertheless, the comprehensive predictive significance of genes that regulate AA metabolism in pancreatic cancer remains unknown. Methods: The mRNA expression data downloaded from The Cancer Genome Atlas (TCGA) were derived as the training cohort, and the GSE57495 cohort from Gene Expression Omnibus (GEO) database was applied as the validation cohort. Random survival forest (RSF) and the least absolute shrinkage and selection operator (LASSO) regression analysis were employed to screen genes and construct an AA metabolism-related risk signature (AMRS). Kaplan-Meier analysis and receiver operating characteristic (ROC) curve were performed to assess the prognostic value of AMRS. We performed genomic alteration analysis and explored the difference in tumor microenvironment (TME) landscape associated with KRAS and TP53 mutation in both high- and low-AMRS groups. Subsequently, the relationships between AMRS and immunotherapy and chemotherapy sensitivity were evaluated. Results: A 17-gene AA metabolism-related risk model in the TCGA cohort was constructed according to RSF and LASSO. After stratifying patients into high- and low-AMRS groups based on the optimal cut-off value, we found that high-AMRS patients had worse overall survival (OS) in the training cohort (a median OS: 13.1months vs. 50.1 months, p < 0.0001) and validation cohort (a median OS: 16.2 vs. 30.5 months, p = 1e-04). Genetic mutation analysis revealed that KRAS and TP53 were significantly more mutated in high-AMRS group, and patients with KRAS and TP53 alterations had significantly higher risk scores than those without. Based on the analysis of TME, low-AMRS group displayed significantly higher immune score and more enrichment of T Cell CD8+ cells. In addition, high-AMRS-group exhibited higher TMB and significantly lower tumor immune dysfunction and exclusion (TIDE) score and T Cells dysfunction score, which suggested a higher sensitive to immunotherapy. Moreover, high-AMRS group was also more sensitive to paclitaxel, cisplatin, and docetaxel. Conclusion: Overall, we constructed an AA-metabolism prognostic model, which provided a powerful prognostic predictor for the clinical treatment of pancreatic cancer.

Abstract B049: Radiogenomic profiling of prostate tumors prior to external beam radiotherapy (EBRT) converges on a transcriptomic signature of TGF-beta activity driving tumor recurrence

Abstract Background: In patients who receive definitive therapy for locally advanced prostate cancer, biochemical recurrence is due to local recurrence and/or the presence of undetected metastatic disease at the time of initial diagnosis and treatment. Approximately 25-40% of patients who receive radiotherapy plus androgen deprivation therapy (ADT) with curative intent are expected to recur within 5-10 years. We hypothesized that applying multiparametric (mp) MRI, gene expression, and genomic analyses to prostate tumors prior to EBRT+ADT would identify biomarkers predictive of recurrence (NCT01834001). Methods: At baseline and 6 months after completion of radiotherapy, mpMRI was performed and lesions were manually contoured. MR/ultrasound-fusion biopsies acquired at baseline targeted to up to three distinct MR lesions per patient. 29 patients with intermediate and high risk localized prostate cancer treated with EBRT+ADT with sufficient baseline biopsy tissue were selected for this analysis (median follow-up: 91 months). Tumor tissues were macrodissected to obtain DNA and RNA from 60 distinct biopsy regions (1-4 per patient). DNA was used for generating whole-exome sequencing libraries (92.5 × median coverage); RNA was converted into cDNA and hybridized to Affymetrix Human Exon 1.0 microarrays. Secondary gene expression cohorts with clinical annotation were obtained from the University of Miami and Queen’s University Belfast. Gene expression signatures were processed using Ingenuity Pathway Analysis (IPA) and the Decipher GRID. Results: Five of the 29 patients experienced biochemical failure by the time of data analysis; four of these patients were identified to have distant metastases with no evidence of local recurrence at the time of biochemical failure. Baseline and posttreatment lesion volumes by MRI and posttreatment ADCmax (apparent diffusion coefficient) were positively associated with recurrence (p&amp;lt;0.05). The Decipher signature positively correlated with these three MR features. Comparison of gene expression using a linear mixed-effect model identified 1,120 differentially expressed transcripts. IPA nominated TGF-beta signaling as the topmost enriched upstream regulator (z=3.32). This result was validated in the Miami (z=2.54) and Belfast (z=3.67) cohorts. By network analysis, genes that independently tracked with the baseline and posttreatment volumes and posttreatment ADCmax MR features similarly converged to the TGF-beta pathway. Co-occurrence of biallelic TP53 alterations with 1- or 2-copy PTEN losses was observed exclusively in patients who recurred. Conclusions: Genomic, transcriptomic and radiomic analyses have nominated predictive biomarkers that identify the subset of patients with locally advanced prostate cancer destined to recur after definitive EBRT+ADT. Identification of molecular features predictive of failure suggests that aggressive pre-existing subclones harboring these alterations have metastatic potential. Emerging systemic therapies including TGF-beta ligand traps targeted to these tumors may improve overall outcomes. Citation Format: Adam G. Sowalsky, Anson T. Ku, Uma Shankavaram, Shana Y. Trostel, Houssein A. Sater, Stephanie A. Harmon, Nicole V. Carrabba, Yang Liu, Radka Stoyanova, Elai Davicioni, Alan Pollack, Baris Turkbey, Deborah E. Citrin. Radiogenomic profiling of prostate tumors prior to external beam radiotherapy (EBRT) converges on a transcriptomic signature of TGF-beta activity driving tumor recurrence [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B049.

Prognostic value of TP53 mutation status in metastatic triple-negative and hormone receptor-positive breast cancer using cell-free DNA genomic profiling.

e13099 Background: TP53 is one of the most frequently mutated genes in metastatic breast cancer (MBC); however, its prognostic role remains undetermined. Plasma-based genotyping via cell-free DNA (cfDNA) is a noninvasive means of assessing the genomic landscape of MBC, accounting for heterogeneity between tumors. This study evaluated the association between TP53 alterations detected via cfDNA and clinical outcomes among patients with hormone receptor-positive HER2-negative breast cancer (HR+/HER2- BC) and triple-negative breast cancer (TNBC) in a metastatic setting. Methods: cfDNA analyses were performed on plasma samples collected from patients with TNBC and HR+/HER2- BC treated at the Massachusetts General Hospital Cancer Center and Washington University in St Louis using Guardant360, a 74-gene cfDNA sequencing assay. Progression-free survival (PFS) and overall survival (OS) were compared based on TP53 alteration status in cfDNA. Multivariable Cox regression was performed adjusting for age, de novo metastases, number of prior treatments, and immunotherapy. Results: TP53 alterations were found in 187 out of 540 patients with HR+/HER2- BC (34.6%) and 87 out of 117 patients with TNBC (74.4%). The most frequent co-existing mutations with TP53 mutations in TNBC were PIK3CA (27.6%), EGFR (26.4%), and CCNE1 (20.7%). For HR+/HER2- BC, the top co-existing mutations were PIK3CA (51.3%), ESR1 (30.5%), and EGFR (21.9%). Median PFS for patients with TNBC with and without TP53 mutations was 6.8 and 6.5 months, respectively (HR = 1.1, 95% CI 0.6-2.0, p = 0.66). Median OS for patients with TNBC with TP53 mutations was 14.2 months, whereas patients without TP53 mutations did not reach median OS (HR = 2.0, 95% CI 0.9-4.1, p = 0.08). Median PFS and OS for patients with HR+/HER2- BC were stratified by the type of therapy initiated immediately after cfDNA testing. Median PFS for patients who received endocrine therapy with and without TP53 mutations was 13.1 vs. 15.9 months, respectively (HR = 1.1, 95% CI 0.7-1.6, p = 0.78). Median OS with and without TP53 mutations was 40.6 vs. 55.8 months, respectively (HR = 1.4, 95% CI 0.8-2.3, p = 0.25). For patients with HR+/HER2- BC who received non-endocrine-based therapy following cfDNA testing, median PFS was 4.2 vs 5.6 months for those with and without TP53 mutations, respectively (HR = 1.0, 95% CI 0.6-1.7, p = 0.92). Median OS for those with TP53 mutations was 9.9 months and 17.1 months for those without (HR = 1.8, 95% CI 1-3.3, p = 0.05). Conclusions: TP53 alterations were detectable via cfDNA in patients with metastatic HR+/HER2- BC and TNBC and were associated with poor survival suggesting a potential prognostic role. The analysis did not filter out potential clonal hematopoiesis, a limitation of the study. Further study is warranted to determine the association of these mutations with outcomes in the setting of specific treatment regimens.

Clinical and genomic characteristics between DDR germline and somatic mutations in pan-cancer.

e15139 Background: The defects DNA-damage repair (DDR) genes would drive tumor formation and are associated with increased genomic instability and tumor mutational burden (TMB) in cancer. Although, alterations of DDR genes are common in tumor, the differences between the germline and somatic alterations are poorly characterized. Methods: The DNA sequencing data of 540 genes from 6174 pan-cancer patients were retrospectively collected and 276 DDR genes were analyzed. The variations were annotated as pathogenic (P), likely pathogenic (LP), and nonpathogenic (NP) according to ACMG (American College of Medical Genetics) guideline. The patients were divided into 3 groups: DDR-germline (P&amp;LP germline variants, N = 766), DDR-somatic group (only P&amp;LP somatic variants, N = 2923) and non-DDR group (NP variants, N = 2485). Results: In pan-cancer, 12.41% patients have germline DDR gene mutations, of which tumors with high germline mutation frequency include ovarian cancer (27/102, 26.47%), glioma (22/136, 16.18%), breast cancer (25/159, 15.72%), genitourinary cancer (17/114, 14.91%), biliary tract cancer (42/284, 14.79%), pancreatic cancer (28/193, 14.51%), colorectal cancer (95/727, 13.07%), head-neck tumors (11/85, 12.94%) and esophagus cancer (16/124, 12.90%). Meanwhile, 47.34% patients have somatic DDR gene mutations, of which tumors with high somatic mutation frequency include esophagus cancer (89/124, 71.77%), colorectal cancer (488/727, 67.13%), endometrial cancer (49/73, 67.12%), head-neck tumors (50/85, 58.82%), pancreatic cancer (111/193, 57.51%), gastric carcinoma (233/415, 56.14%), ovarian cancer (53/102, 51.96%) and biliary tract cancer (138/284, 48.59%). The most commonly germline alterations were found in TP53 (33%), PTEN (14%) and BRCA2 (14%), while in the somatic mutations, TP53 (52%) showed the highest frequency, PTEN (9%) and MSH3 (8%) followed. In pairwise comparisons of the three groups’ actionable mutations, DDR-somatic group was more likely to have alterations in TP53 (OR = 5.68), AKT3 (OR = 5.81) and APC (OR = 1.42) compared with DDR-germline group. Taken non-DDR group as reference, mutations in TP53 (OR = 5.09), PTEN (OR = 7.65), BRCA2 (OR = 5.32) were more common in DDR-germline group, but TP53 (OR = 28.90), PTEN (OR = 7.73), APC (OR = 2.68) in DDR-somatic group. The tumor mutational burden (TMB) was statistically different among the three groups (p &lt; 2.22e−16). The DDR-somatic group exhibits the highest median TMB (5.68) compared with 1.42 in DDR-germline and 2.13 in non-DDR group. Conclusions: The differences in the mutation profile between the DDR-germline and DDR-somatic groups and the distinct actionable genes may indicate the different target-therapy choices. Besides, DDR-somatic group exhibited the highest TMB, which might indicate patients with somatic DDR alterations may better benefit from the immunotherapy.

Electronic health record (EHR) and genomics-based machine learning (ML) to predict therapeutic effectiveness among patients with hormone-receptor positive (HR)+/HER2- advanced breast cancer (aBC).

e13574 Background: Patients (pts) with HR+/HER2- aBC may eventually become resistant to endocrine therapies and CDK4/6 inhibitors (CDK4/6i). ML algorithms on EHR linked with NGS data may enable more accurate predictions of therapeutic resistance and identify clinicogenomic (CG) risk factors. Methods: HR+/HER2- aBC pts treated between Jan 2015 – Dec 2022 with at least one NGS test report before line end were identified in ConcertAI’s Genome360 oncology database, consisting of molecular alterations abstracted from NGS reports linked with deeply curated US-based EHR. XGBoost (XGB) and Cox proportional hazards (CoxPH) models were trained to predict real-world progression-free survival (rwPFS) derived from curated tumor progression (TP), as assessed by the providers based on clinical observations, pathology results, and radiologic evidence. Data was split into 60:20:20 for training/validation/test. Pts in the 1st and 4th quartiles of predicted risk from XGB model were defined as low- and high-risk. Predictors were identified from Shapley Additive exPlanations (SHAP)-derived marginal hazard ratios (HR), derived in the 180 days prior to index date, and included demographics, labs, vitals, comorbidities, concomitant medications, procedures, tumor properties, and molecular alterations as features. Results: Among 624 pts (CDK4/6i treated = 519) with median age = 62.5 years and 10.5% black, 46.8% developed TP. The test cumulative dynamic ROC-AUC in the best-performing XGB model was 0.68. The precision at 180 days (%TP = 27) was 40%. The 180-day cumulative incidence of TP in low- and high-risk groups was 11.6% vs 37.7%. Higher line number, lower hemoglobin (HGB), higher alkaline phosphatase (ALP), presence of liver metastasis, and history of thoracic radiation were clinical risk factors. Genomic risk factors were FGF aberrations, alterations in TP53 and in genes within the MAPK, cell cycle, and ESR pathways, and co-alterations in the PIK3CA+TP53, and MAPK+ESR pathways. ALP-by-TP53 interaction was found. ALP level was a risk factor in TP53-wildtype (wt) but not in TP53-mutant (mut) pts. Conclusions: ML on EHR linked with NGS data enables identification of high-risk pts and multimodal predictors of CDK4/6i resistance among women with HR+/HER2- aBC, identifying both known and undescribed interactions between clinical and genomic risk factors as well as co-alteration risk factors. [Table: see text]

Association of alterations in multiple tumor suppressor genes with poor outcomes in patients with EGFR-mutant lung cancer.

9104 Background: Co-occurring alterations in tumor suppressor genes (TSG) have been described as determinants of disease heterogeneity in EGFR-mutant non-small cell lung cancer (NSCLC), however, detailed analyses of their impact on patient outcomes are limited. Methods: Patients with EGFR-mutant NSCLC treated with EGFR tyrosine kinase inhibitors (TKIs) at the Yale Cancer Center who had tumor genomic profiling either before EGFR-TKI therapy (pre-TKI) or at disease progression (post-TKI) were included. Whole exome sequencing (WES) of paired pre- and post-TKI tumor specimens were performed for a subset of patients. The cohort was divided into three subgroups based on alterations in TP53 and five additional TSGs ( RB1, NF1, ARID1A, BRCA1 and PTEN): patients with tumors harboring a TP53 mutation plus a mutation in at least 1 additional TSG ( TP53mut/TSGmut), patients with tumors harboring a TP53 mutation without an additional TSG mutation ( TP53mut/TSGwt), and patients with TP53wt tumors. Clinical characteristics including progression-free (PFS) and overall survival (OS) were assessed. Results: One-hundred-one patients were included in this retrospective study. TP53 mutations were identified in 65 (64%) tumors, of which 23 (35%) and 42 (65%) were classified as TP53mut/TSGmut and TP53mut/TSGwt, respectively. Among those cases with paired WES available (n = 34), frequencies of alterations in the six included TSGs did not significantly differ between pre- and post-TKI tumor specimens. In the full study cohort, the presence of a TP53 mutation was associated with numerically worse PFS (HR 1.46, CI 0.97 – 2.21, p = 0.09) and OS (HR 1.68, CI 1.05 – 2.70, p = 0.04) on first-line EGFR-TKI. Strikingly, after dividing the TP53mut cohort into TP53mut /TSGmut and TP53mut /TSGwt cases, alterations in additional TSG were found to drive the poor outcomes: TP53mut /TSGmut cases had significantly worse PFS and OS on first-line EGFR TKI than TP53mut /TSGwt(mPFS 8.0 vs 10.6 months, p = 0.006; mOS 30.0 vs 33.3 months, p = 0.12) or TP53wt cases (mPFS 8.0 vs 12.6 months, p &lt; 0.0001; mOS 30.0 vs 48.8 months, p = 0.001). There was no significant difference in PFS or OS between patients with TP53mut /TSGwt and TP53wt tumors. Similar outcome differences between the three groups were found in patients who received osimertinib in the second-line setting. Conclusions: The inferior outcomes associated with EGFR/TP53-mutant NSCLC tumors may be due to additional TSG alterations rather than TP53 mutational status alone. Alterations in the investigated TSGs did not appear to emerge under EGFR-TKI therapy, which suggests that they are truncal. Our findings may have implications for understanding the biologic underpinnings of differential outcomes to EGFR TKIs.

First results from the RETgistry: A global consortium for the study of resistance to RET inhibition in RET-altered solid tumors.

9065 Background: Rearranged during transfection ( RET) gene alterations are the oncogenic driver in diverse tumor types, including RET fusions identified in 1-2% of non-small cell lung cancers (NSCLC). While RET-selective tyrosine kinase inhibitors (TKIs) selpercatinib and pralsetinib are effective, acquired drug resistance remains a challenge. Here, we report the initial results from the RETgistry, an international consortium aimed at elucidating mechanisms of resistance to RET TKIs across RET-altered solid tumors. Methods: This was a retrospective analysis performed across 16 institutions. Patients (pts) were eligible if they had advanced solid tumor harboring an oncogenic RET alteration, received ≥1 RET TKI with disease progression, and underwent resistant tumor or liquid biopsy for next-generation sequencing (NGS). Results: A total of 103 time-distinct biopsies (62 tissue, 30 plasma, 11 paired tissue/plasma) were included in analysis, obtained from 88 pts with progression on a RET-selective TKI [selpercatinib (n = 70), pralsetinib (n = 14), selpercatinib and pralsetinib sequentially (n = 4)]. Pts had the following tumor types: 72 NSCLC (69% KIF5B-RET, 21% CCDC6-RET, 10% other RET fusion), 13 medullary thyroid cancer (TC) (54% RET M918T, 46% other RET mutation), and 2 papillary and 1 anaplastic TC (all, RET fusion). Median age was 58 (range, 21-86); 48% were male with 89% never/light smokers. Median duration of RET TKI preceding biopsies [first-line, n = 32 (31%); second-line, n = 42 (41%); third-/greater-line, n = 29 (28%)] was 16.5 months (mos) (95% CI, 14.0-19.6); median PFS was 14.1 mos (95% CI, 9.3-17.0). Resistant biopsies were obtained at median 15.0 mos from TKI initiation (range, 1.8-58.8). Acquired RET mutations were detected in 14 (14%), most common being G810 substitutions in 12 (12%). Potential off-target resistance gene alterations identified in 43 cases (42%) included MET amplification (14%), BRAF V600E or fusion (2%), KRAS gain or mutation (5%), ERBB2 amplification (2%), EGFR amplification (3%), ROS1 fusion (1%), and activating PIK3CA mutation or PTEN loss (4%). No resistant lung cancer biopsy demonstrated small cell transformation. The duration of TKI therapy (HR 0.64, p = 0.11) or PFS (HR 0.75, p = 0.33) did not differ according to the presence of on-target vs off-target resistance. NGS analyses of pre-RET-selective TKI tumors (n = 92) revealed frequent co-occurring alterations in TP53 (29%) and CDKN2A/B (12%). Conclusions: On-target resistance to RET inhibition due to acquired RET mutations was less common than off-target resistance, identified in 14%. The majority of RET TKI resistance is mediated by off-target mechanisms such as bypass receptor tyrosine kinase activation. Further studies are warranted to enable the development of therapeutic strategies to address resistance in pts with RET-altered tumors. The RETgistry accrual and data analyses continue.