Comprehensive Genomic Profiling Research Articles

Abstract 7507: Capturing live single circulating tumor cells platform for comprehensive genomic profiling with paired ctDNA in breast cancer patients

Abstract Background: Single circulating tumor cells (sCTC) capture without cell fixation with no blood contaminants like leukocytes are highly challenging. sCTC genome transcriptomics anticipates new avenues in new discovery, targeted therapies, and inclusion of patients in clinical trials where the tissue is inaccessible. We report, an assay to selectively capture live sCTCs at singularity with NGS-based comprehensive mutational profiling in breast cancer (BC) patients. Further, paired ctDNA outcomes using blood with mutational heterogeneity scores of sCTCs adds comprehensive genomics for establishing an accelerated treatment outcomes and resistance signatures. Methods: Retrospectively, sCTC were isolated from 70 breast cancer (BC) patients using OncoRadar platform consisting affinity-based glass beads mediated by anti EpCAM antibody. CTCs were selectively captured and released without fixing and contamination of any leukocytes. Platform possess cleavable disulfide spacer at pH 8.0, between glass beads (diam. 2 mm, area 12.57 mm2) and a ligand. Sensitivity, specificity, positive predictive value (PPV), negative prediction value (NPV), and accuracy were evaluated using MCF-7 cells and CTCs. Assay to isolate CTC has been developed in 96 well plate, for downstream NGS-based comprehensive gene panel analysis using hybridization-based target enrichment. Interestingly, no mutations detected in blood samples (~20%) for CtDNA versus CTC DNA offered non-chip implicated mutations. Results:. Analytical validation using OncoRadar showed higher cell capture efficiency (99%) with spiked MCF-7 cells/ml of blood. Sensitivity and specificity values were 97% and 98% respect, while PPV was 99% and NPV was 7.5% with the accuracy parameter of 99%. Sensitivity, specificity, PPV, and NPV parameters when assessed for CTC isolation from 70 BC patients. NGS-based mutational analysis, sCTC showed the presence of PIK3CA, ESR1, and NF1 mutations, which were absent from paired ctDNA samples. Conclusion: Detection of ctDNA obscure mutations in paired sCTC samples suggests high sensitivity of sCTC-based genomic assays compared to current standard of practice. Heterogeneity scores amongst inter and intra CTCs with MATH scores offer new dimensions in genomics. Combination of ctDNA and sCTC DNA assay offers a comprehensive realistic genomic outcome for more significant therapy decisions, especially when guideline based options may exhausts. Citation Format: Gowhar Shafi, Atul Bharde, Ganesh Khutale, Saloni Andhari, Richa Deshpande, Bhagwat Jadhav, Sangeeta Prajapati, Moubeen Fauzul, Kanchan Hariramani, Madhura Basavalingegowda, Jayant Khandare. Capturing live single circulating tumor cells platform for comprehensive genomic profiling with paired ctDNA in breast cancer patients [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 7507.

Abstract 340: Validation of TSO500 NGS panel for comprehensive genomic profiling from FFPE tumor samples

Abstract Comprehensive genomic profiling (CGP) of tumor sample using large targeted NGS panels (e.g. MSK-IMPACT) has been shown to allow the detection of biomarkers from hundreds of cancer relevant genes involved in targeted and immunotherapy. Illumina TruSight Oncology 500 (TSO500), a commercial CGP assay, allows concurrent analysis of DNA and RNA to simultaneously detect multiple types of variants across 523 genes. The assay is intended to detect single nucleotide variants (SNV), insertions and deletions (indel), and multinucleotide variants (MNV), copy number gain (CNV), tumor mutational burden (TMB) and microsatellite instability (MSI) in DNA, and fusions and splicing variants (exon skipping) in RNA. Here we aimed to validate TSO500 assay for its analytical performance on FFPE tumor samples. The validation samples include commercial controls, cell lines, clinical FFPE from NSCLC, CRC, Uterus and GIST (tumor content ≥20%). Library preparation was performed from minimum of 40 ng DNA and 40ng RNA. The data analysis is performed using TruSight Oncology 500 Local App software (LRM v2.2). A custom script (v1.0) was used to add SNP annotation for small variants and combine data output into cumulative format. For SNV and indel detection QC, 61 of 64 DNA libraries (95%) passed the QC for library size (median insert size ≥70bp) and coverage (median exon coverage ≥150). The percentage of DNA libraries passing QC for CNV and MSI are 95% and 88%, respectively. All 64 RNA libraries passed QC. The analytical performance of TSO500 was summarized in the Table below. In summary, the results showed that all the acceptance criteria were met and the TSO500 assay can be utilized for genomic profiling of FFPE tumor samples. The study also identified a few limitations of the Illumina LRM software e.g. potential false positive SNV within a indel background, potential false negative of complex indel, and the challenge of long indel detection. Summary of analytical performance of TSO500 Sensitivity and specificity Assay LoD Accuracy by concordance Inter-run reproducibility SNV/Small indel Sensitivity for SNV and indel detection are 99.24% and 99.36%. The LoD for SNV is 3-5%, for DEL is 5-10% and for INS is 3-5%. PPA for SNV detections is 96.64% (115/119; 95% CI: 91.68%, 98.69%) and for small indel is 92.86% (13/14; 95% CI: 68.53%, 98.73%) between TSO500 and comparator assays. The positive call rate across all SNV and small indel variants is 98.7% (42842/43419). Specificity for SNV and small indel detection are 99.9998% and 100.00%, respectively. MSI Sensitivity and Specificity for MSI detection are both 100%. NA MSI status for all samples were detected with 100% of concordance by three assays; MSI concordance for MSI-H is 100% (95% CI: 60.97%, 100.0%) and for MSS is 100% (87.13%, 100.0%). PPA is 100% (95% CI: 51.01%, 100%) and NPA is 100% (95% CI: 34.24%, 100%). TMB NA NA TSO500 reported TMB is well correlated and comparable to exome determined TMB. The CV values for samples are acceptable. CNV NA NA All positive CNVs detected by the comparator assay were detected by the TSO500 assay with similar fold changes; The overall reproducible call rate is 91.5% (43/47). PPA between TSO500 and OCAV3 assays is 100% (95% CI: 51.01%, 100%). Fusion Sensitivity and specificity for fusion detection is 91.94% and 100%, respectively. NA All TSO500 assay detected fusions were confirmed by other assays;PPA is 100% (95% CI: 60.97%, 100%) and NPA is 100% (95% CI: 75.75%, 100%) The reproducibility of fusion call is 92.59% (25/27; 95% CI: 76.63%, 97.94%). Splicing Sensitivity and Specificity for splicing variant detection are 91.67% and 100.00%, respectively. NA The splicing variant detections were confirmed by OCAV3, OFA and Archer assays; The overall reproducibility of positive call is 100% with 95% CI (43.85%, 100%) and negative call reproducibility is 100% with 95% CI (87.13%, 100%). PPA is 100% (95%% CI: 20.65%, 100%) and NPA is 100% (95% CI: 83.18%, 100%) Citation Format: Jin Li, Zhenyu Yan, Weihua Liu, Lin Shi, Cynthia Spittle, Chad Galderisi. Validation of TSO500 NGS panel for comprehensive genomic profiling from FFPE tumor samples [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 340.

The Impact of Prior Single-Gene Testing on Comprehensive Genomic Profiling Results for Patients with Non-Small Cell Lung Cancer.

Tissue-based broad molecular profiling of guideline-recommended biomarkers is advised for the therapeutic management of patients with non-small cell lung cancer (NSCLC). However, practice variation can affect whether all indicated biomarkers are tested. We aimed to evaluate the impact of common single-gene testing (SGT) on subsequent comprehensive genomic profiling (CGP) test outcomes and results in NSCLC. Oncologists who ordered SGT for guideline-recommended biomarkers in NSCLC patients were prospectively contacted (May-December 2022) and offered CGP (DNA and RNA sequencing), either following receipt of negative SGT findings, or instead of SGT for each patient. We describe SGT patterns and compare CGP completion rates, turnaround time, and recommended biomarker detection for NSCLC patients with and without prior negative SGT results. Oncologists in > 80 community practices ordered CGP for 561 NSCLC patients; 135 patients (27%) first had negative results from 30 different SGT combinations; 84% included ALK, EGFR and PD-L1, while only 3% of orders included all available SGTs for guideline-recommended genes. Among patients with negative SGT results, CGP was attempted using the same tissue specimen 90% of the time. There were also significantly more CGP order cancellations due to tissue insufficiency (17% vs. 7%), DNA sequencing failures (13% vs. 8%), and turnaround time > 14days (62% vs. 29%) than among patients who only had CGP. Forty-six percent of patients with negative prior SGT had positive CGP results for recommended biomarkers, including targetable genomic variants in genes beyond ALK and EGFR, such as ERBB2, KRAS (non-G12C), MET (exon 14 skipping), NTRK2/3, and RET . For patients with NSCLC, initial use of SGT increases subsequent CGP test cancellations, turnaround time, and the likelihood of incomplete molecular profiling for guideline-recommended biomarkers due to tissue insufficiency.

Developing Novel Genomic Risk Stratification Models in Soft Tissue and Uterine Leiomyosarcoma.

Leiomyosarcomas (LMS) are clinically and molecularly heterogeneous tumors. Despite recent large-scale genomic studies, current LMS risk stratification is not informed by molecular alterations. We propose a clinically applicable genomic risk stratification model. We performed comprehensive genomic profiling in a cohort of 195 soft tissue LMS (STLMS), 151 primary at presentation, and a control group of 238 uterine LMS (ULMS), 177 primary at presentation, with at least 1-year follow-up. In STLMS, French Federation of Cancer Centers (FNCLCC) grade but not tumor size predicted progression-free survival (PFS) or disease-specific survival (DSS). In contrast, in ULMS, tumor size, mitotic rate, and necrosis were associated with inferior PFS and DSS. In STLMS, a 3-tier genomic risk stratification performed well for DSS: high risk: co-occurrence of RB1 mutation and chr12q deletion (del12q)/ATRX mutation; intermediate risk: presence of RB1 mutation, ATRX mutation, or del12q; low risk: lack of any of these three alterations. The ability of RB1 and ATRX alterations to stratify STLMS was validated in an external AACR GENIE cohort. In ULMS, a 3-tier genomic risk stratification was significant for both PFS and DSS: high risk: concurrent TP53 mutation and chr20q amplification/ATRX mutations; intermediate risk: presence of TP53 mutation, ATRX mutation, or amp20q; low risk: lack of any of these three alterations. Longitudinal sequencing showed that most molecular alterations were early clonal events that persisted during disease progression. Compared with traditional clinicopathologic models, genomic risk stratification demonstrates superior prediction of clinical outcome in STLMS and is comparable in ULMS.

Evaluation of pancreatic cancer specimens for comprehensive genomic profiling.

Inadequate specimen quality or quantity hinders comprehensive genomic profiling in identifying actionable mutations and guiding treatment strategies. We investigated the optimal conditions for pancreatic cancer specimen selection for comprehensive genomic profiling. We retrospectively analyzed 213 pancreatic cancer cases ordered for comprehensive genomic profiling and compared results from pancreatic biopsy, liver biopsy of pancreatic cancer metastases, pancreatectomy, liquid, and nonliver metastatic organ specimens. We examined preanalytical conditions, including cellularity (tumor cell count/size). The successfully tested cases were those that underwent comprehensive genomic profiling tests without any issues. The successfully tested case ratio was 72.8%. Pancreatic biopsy had the highest successfully tested case ratio (87%), with a high tumor cell percentage, despite the small number of cells (median, 3425). Pancreatic biopsy, liver biopsy of pancreatic cancer metastases, and non-liver metastatic organ had higher successfully tested case ratios than that for pancreatectomy. Liver biopsy of pancreatic cancer metastases and pancreatectomy cases with tumor size (mm2) × tumor ratio (%) > 150 and >3000, respectively, had high successfully tested case ratios. The success of comprehensive genomic profiling is significantly influenced by the tumor cell ratio, and pancreatic biopsy is a potentially suitable specimen for comprehensive genomic profiling.

An approach for improvement of the accuracy of cancer gene panel testing.

Tissue-based comprehensive genomic profiling (CGP) is increasingly being employed for genotype-directed therapies in patients with advanced cancer. However, tissue availability may limit their potential applications. In Japan, the cost of cancer gene panel tests is covered by public insurance for patients diagnosed with advanced solid tumors once in their lifetime. Therefore, it is essential to improve the success rate (reportability) and accuracy of CGP tests. The purpose of this study was to identify the factors associated with efficient and accurate CGP testing using relevant information obtained from real-world data. This study included 159 samples analyzed using tumor-only panel FoundationOne® CDx cancer genome profiling (F1CDx) and 85 samples analyzed using matched-pair panel OncoGuide™ NCC Oncopanel system (NCCOP) at St. Marianna University Hospital. Sample characteristics (fixation conditions, storage period, histology, tumor cell ratio, and genomic tumor cell content), CGP performance, and quality control status were evaluated across all 244 tested samples. In 237/244 samples (97.1%), CGP testing results were successfully obtained [F1CDx, 99.4% (158/159) and NCCOP, 92.9% (79/85)]. An increased number of fibroblasts, inflammatory cells, and necrotic tumor cells, long-term storage, and/or prolonged fixation of tissue sections were involved in the unreported results and/or qualified CGP results. In addition, a negative correlation between median insert size values and ΔΔCq was observed in the NCCOP system. We identified various factors associated with efficient and accurate CGP testing using relevant information obtained from real-world data, suggesting that thorough selection and preparation of tissue sections could optimize CGP and maximize useful information.

A case of treatment-resistant advanced gastric cancer with FGFR2 gene alteration successfully treated with pemigatinib.

Comprehensive genome profiling (CGP) is expected to widen the scope of cancer drug options by identifying the genes involved in carcinogenesis. However, a few patients can access recommended treatments following CGP. Herein, we report a case in which pemigatinib, a selective fibroblast growth factor receptor (FGFR) inhibitor, was used as last-line therapy to treat a patient with advanced gastric cancer exhibiting FGFR2 genomic alterations, as determined by CGP testing. The patient (male, 52years old) was diagnosed with advanced gastric cancer (cStage IV, cT4aN3M1 [LYM], por, HER2 0, microsatellite stable) and received docetaxel + cisplatin + S-1 (7 cycles), irinotecan + ramucirumab (11 cycles), and nivolumab (3 cycles), but experienced progressive disease (PD). Subsequently, FoundationOne Liquid CDx testing was conducted, revealing FGFR2 rearrangement and amplification; however, no clinical trials on genotype-matched therapies for FGFR2 alterations were available. After three cycles of TAS-102, the patient experienced PD and provided consent for the off-label use of pemigatinib. The Cancer Genomics Medical Committee of our hospital approved the self-funded treatment. The patient had markedly decreased CEA and CA19-9 levels after treatment initiation, but experienced PD after five courses. Over the treatment course, grade 1 hyperphosphatemia and onychomadesis were observed. To the best of our knowledge, this is the first reported case of pemigatinib therapy employed in a patient with advanced gastric cancer exhibiting FGFR2 gene alterations. This case could serve as a notable example of tumor-agnostic therapy to broaden treatment options for gastric cancer patients with rare genetic alterations.

Remarkable response to capmatinib in a patient with intrahepatic cholangiocarcinoma harboring TFG-MET fusion.

Dysregulation of mesenchymal-epithelial transition factor (MET) gene due to amplification, mutation, and fusion has been reported in various types of human cancers. Recently, the efficacy of small-molecule tyrosine kinase inhibitors (TKIs) targeting MET has been demonstrated in a wide range of MET-dysregulated tumors. The majority of biliary tract cancers including intrahepatic cholangiocarcinoma (iCCA) are diagnosed at an advanced stage, and the utility of conventional chemotherapy is limited. Here, we present a case of metastatic iCCA harboring TFG-MET gene fusion, which demonstrated a remarkable response to treatment with capmatinib, a selective MET inhibitor. The patient was a 46-year-old man diagnosed with iCCA with hepatic, intraabdominal lymph nodes, and peritoneal metastases. Comprehensive genomic profiling (CGP) revealed TFG-MET gene fusion in his tumor. After becoming refractory to standard chemotherapy, he received capmatinib, which resulted in a marked shrinkage of the liver masses and lymph node metastases, as well as a drastic decrease in serum CA19-9 level. Our case reinforces the importance of CGP in exploring targeted therapy and supports the potential role of capmatinib in the treatment of tumors harboring MET fusions.

Nationwide data from comprehensive genomic profiling assays for detecting driver oncogenes in non-small cell lung cancer.

Driver oncogenes are investigated upfront at diagnosis using multi-CDx systems with next-generation sequencing techniques or multiplex reverse-transcriptase polymerase chain reaction assays. Additionally, from 2019, comprehensive genomic profiling (CGP) assays have been available in Japan for patients with advanced solid tumors who had completed or were expected to complete standard chemotherapy. These assays are expected to comprehensively detect the driver oncogenes, especially for patients with non-small cell lung cancer (NSCLC). However, there are no reports of nationwide research on the detection of driver oncogenes in patients with advanced NSCLC who undergo CGP assays, especially in those with undetected driver oncogenes at diagnosis. In this study, we investigated the proportion of driver oncogenes detected in patients with advanced NSCLC with undetectable driver oncogenes at initial diagnosis and in all patients with advanced NSCLC who underwent CGP assays. We retrospectively analyzed data from 986 patients with advanced NSCLC who underwent CGP assays between August 2019 and March 2022, using the Center for Cancer Genomics and Advanced Therapeutics database. The proportion of driver oncogenes newly detected in patients with NSCLC who tested negative for driver oncogenes at diagnosis and in all patients with NSCLC were investigated. Driver oncogenes were detected in 451 patients (45.7%). EGFR was the most common (16.5%), followed by KRAS (14.5%). Among the 330 patients with undetected EGFR, ALK, ROS1, and BRAF V600E mutations at diagnosis, 81 patients (24.5%) had newly identified driver oncogenes. CGP assays could be useful to identify driver oncogenes in patients with advanced NSCLC, including those initially undetected, facilitating personalized treatment.

Association of Timely Comprehensive Genomic Profiling With Precision Oncology Treatment Use and Patient Outcomes in Advanced Non-Small-Cell Lung Cancer.

Timely biomarker testing remains out of reach for many patients with advanced non-small-cell lung cancer (aNSCLC). Here, we studied the quality-of-care implications of closing the gap in timely receipt of comprehensive genomic profiling (CGP) to inform first-line (1L) decisions. Using a real-world clinicogenomic database, we studied testing and 1L treatment patterns in aNSCLC after the approval of pembrolizumab in combination with pemetrexed and carboplatin (May 10, 2017). To estimate the association of timely CGP results with therapy selection and patient outcomes, we identified patients with no previous genomic testing beyond PD-L1 immunohistochemistry and dichotomized patients by whether CGP results were available before or after 1L therapy initiation. In total, 2,694 patients were included in the 1L therapy decision impact assessment. Timely CGP increased matched targeted therapy use by 14 percentage points (17% with CGP v 2.8% without) and precision immune checkpoint inhibitor (ICPI) use by 14 percentage points (18% with CGP v 3.9% without). Receipt of timely CGP resulted in an estimated 31 percentage point decrease in ICPI use among ALK/EGFR/RET/ROS1-positive patients at an expected per-patient reduction in ineffective ICPI therapy cost of $13,659.37 with timely CGP to inform 1L treatment selection. Patient benefit of CGP extended to real-world time to therapy discontinuation (median time to therapy discontinuation: 3.9 v 10 months [hazard ratio, HR, 0.54 [95% CI, 0.42 to 0.70]; P = 1.9E-06; adjusted hazard ratio [aHR], 0.50 [95% CI, 0.38 to 0.67]; P = 2.0E-06) in 1L driver-positive patients. This effect was not significant for real-world overall survival (median overall survival: 32 v 29 months [HR, 1.2 [95% CI, 0.84 to 1.67]; P = .33; aHR, 1.4 [95% CI, 0.92 to 1.99]; P = .12). Timely CGP is associated with the quality of patient care as measured by 1L matched targeted therapy use, time to therapy discontinuation, and avoidance of ineffective, costly ICPIs.

234P Clinical findings and genomic characterization of NRG-1 alterations with a comprehensive genome profiling (CGP) in advanced non-small cell lung cancer (ANSCLC)

234P Clinical findings and genomic characterization of NRG-1 alterations with a comprehensive genome profiling (CGP) in advanced non-small cell lung cancer (ANSCLC)

Real-World Overall Survival and Treatment Patterns by PTEN Status in Metastatic Castration-Resistant Prostate Cancer.

It is estimated that the PTEN tumor suppressor gene is functionally lost in 40%-50% of patients with metastatic castration-resistant prostate cancer (mCRPC). There is limited information on the prognostic significance of PTEN status identified with genomic testing. This real-world cohort study assessed PTEN as a genetic biomarker using data from US-based oncology practices. This retrospective real-world cohort study used a deidentified US-based metastatic prostate cancer clinicogenomic database linked to longitudinal clinical data derived from electronic health records. Patients were aged 18 years and older and diagnosed with mCRPC between January 1, 2018, and June 30, 2021. Comprehensive genomic profiling (CGP) of tumor specimens was performed using next-generation sequencing. First-line (1L) and second-line (2L) treatment patterns were assessed and stratified by PTEN status. Kaplan-Meier methods and a multivariable Cox model were used to compare the real-world overall survival by PTEN status among patients who received 1L novel hormone therapy or taxanes. In patients with mCRPC who underwent CGP, PTEN loss of function (LOF) was associated with decreased survival compared with intact PTEN (hazard ratio, 1.61 [95% CI, 1.07 to 2.42]; P = .024). The results were not influenced by 1L treatment type. 1L treatment patterns were similar between intact PTEN and PTEN LOF subgroups, with abiraterone and enzalutamide being the two most common treatments in both groups. Patients with PTEN LOF were less likely to receive 2L treatments than patients with intact PTEN. PTEN LOF, identified with genomic testing, was associated with decreased survival and negative prognoses in patients with mCRPC.

Heterogeneous ERBB gene pathways, their targeted treatment and possible molecular mechanisms of resistance in metastatic gallbladder cancer

BackgroundERBB amplifications and mutations are known in metastatic gallbladder cancer (GBC). We conducted a prospective study on targeted therapy based on comprehensive genomic profiling (CGP). We report molecular pathways of ERBB gene and outcomes and explored the molecular mechanism of resistance. MethodsCGP was done for all consecutive GBC patients aged >18 years. Foundation medicines CDx and TarGT Absolute were used for analysis. Patients were enrolled from Jan 2017 till Dec 2022. Patient recruitment was done after ethics committee approval. Trastuzumab and/ or lapatinib were used as targeted therapy. ResultsTwenty-six patients out of 60 had ERBB1-4 gene alteration (amplification and/or mutation) that resulted in activation of PI3K/AKT/MTOR and RAS/RAF/MEK pathways. ERBB amplification (16%) was the most common genetic aberration. Mutations in TP53, RB1, CDKs, and cyclins lead to dysregulation of cell cycle and evasion of apoptosis. A total of 50% of ERBB2-positive patients carry CDK12 amplifications. The intrinsic resistance to anti-ERBB2 therapy was primarily due to downstream activation in ERBB pathway (PIK3CA) and its crosstalk with cell cycle pathway (TP53 and CDK12), JAK/STAT, VEGF, CTNNB1, and ARID1A that are known to drive MAPK/ERK signaling. Mesenchymal epithelial transition amplification or activating mutations is mechanism of resistance to anti-ERBB2 therapy. Kinase/Juxta membrane domain mutations in ERBB family proteins are also one of the potential mechanisms of resistance. ConclusionGBC is enriched with ERBB gene aberrations. Targeted therapy of ERBB pathway has shown encouraging responses. CDK12 gene amplification is a possible mechanism of resistance identified.

Genomic and global gene expression profiling in pediatric and young adult acute leukemia with PICALM::MLLT10 Fusion.

MLLT10 fusion is a rare but recurrent genetic driver in acute leukemias. To better understand the genomic landscape of PICALM::MLLT10 (PM) positive acute leukemia, we performed genomic profiling and gene expression profiling in twenty PM-positive patients, including AML (n = 10), T-ALL/LLy (n = 8), Mixed-phenotype acute leukemia (MPAL), T/B (n = 1) and acute undifferentiated leukemia (AUL) (n = 1). Besides confirming the known activation of HOXA, differential gene expression analysis compared to hematopoietic stem cells demonstrated the enrichment of genes associated with cell proliferation-related pathways and relatively high expression of XPO1 in PM-AML and PM-T-ALL/LLy. Our study also suggested PHF6 disruption as a key cooperating event in PICALM::MLLT10-positive leukemias. In addition, we demonstrated differences in gene expression profiles as well as remarkably different spectra of co-occurring mutations between PM-AML and PM-T-ALL/LLy. Alterations affecting TP53 and NF1, hallmarks of PM-AML, are strongly associated with disease progression and relapse, whereas EZH2 alterations are highly enriched in PM-T-ALL/LLy. This comprehensive genomic and transcriptomic profiling provides insights into the pathogenesis and development of PICALM::MLLT10 positive acute leukemia.

Genetic landscape of 482 thyroid carcinomas: analysis with the national datacenter for cancer genomic medicine in Japan.

Comprehensive genomic profiling is useful for patients with Thyroid carcinoma (TC) for whom standard treatment has become refractory. We analyzed the clinical and genomic characteristics of patients with TC using the Japanese nationwide Center for Cancer Genomics and Advanced Therapeutics (C-CAT) database. This retrospective observational study used the data obtained from the C-CAT database. Genomic information has been accumulated on representative gene mutations associated with TC. Among the 482 patients, 212 (44%) were male and 270 (56%) were female. According to histological type, 259 (54%), 46 (10%), 16 (3%), 51 (11%), and 110 (23%) patients had papillary TC (PTC), follicular TC, medullary TC, poorly differentiated TC, and anaplastic TC (ATC), respectively. Among the genomic profiling tests, FoundationOne CDx (n = 388; 80%) was the most frequently performed. The frequencies of BRAF, NRAS, HRAS, KRAS, and RET mutations were 259 (54%), 62 (13%), 13 (3%), 16 (3%), and 12 (2%), respectively. The BRAF V600E mutation (n = 257) was the predominant BRAF mutation. TERT promoter mutations, which are associated with tumor aggressiveness, were detected in 308 patients (64%). PTC was the most common histologic type of TC for which genetic profiling was performed in Japan, followed by ATC. Since the most common targetable mutation is the BRAF mutation, practical application of BRAF-targeted therapy can be an important treatment option for Japanese patients with TC.

Harnessing the potential of reverse-phase protein array technology: Advancing precision oncology strategies.

The last few decades have seen remarkable strides in the field of cancer therapy. Precision oncology coupled with comprehensive genomic profiling has become routine clinical practice for solid tumors, the advent of immune checkpoint inhibitors has transformed the landscape of oncology treatment, and the number of cancer drug approvals has continued to increase. Nevertheless, the application of genomics-driven precision oncology has thus far benefited only 10%-20% of cancer patients, leaving the majority without matched treatment options. This limitation underscores the need to explore alternative avenues with regard to selecting patients for targeted therapies. In contrast with genomics-based approaches, proteomics-based strategies offer a more precise understanding of the intricate biological processes driving cancer pathogenesis. This perspective underscores the importance of integrating complementary proteomic analyses into the next phase of precision oncology to establish robust biomarker-drug associations and surmount challenges related to drug resistance. One promising technology in this regard is the reverse-phase protein array (RPPA), which excels in quantitatively detecting protein modifications, even with limited amounts of sample. Its cost-effectiveness and rapid turnaround time further bolster its appeal for application in clinical settings. Here, we review the current status of genomics-driven precision oncology, as well as its limitations, with an emphasis on drug resistance. Subsequently, we explore the application of RPPA technology as a catalyst for advancing precision oncology. Through illustrative examples drawn from clinical trials, we demonstrate its utility for unraveling the molecular mechanisms underlying drug responses and resistance.

Real-World Clinical Performance of a DNA-Based Comprehensive Genomic Profiling Assay for Detecting Targetable Fusions in Nonsquamous NSCLC.

Genomic fusions are potent oncogenic drivers across cancer types and many are targetable. We demonstrate the clinical performance of DNA-based comprehensive genomic profiling (CGP) for detecting targetable fusions. We analyzed targetable fusion genes in >450 000 tissue specimens profiled using DNA CGP (FoundationOne CDx, FoundationOne). Using a de-identified nationwide (US-based) non-small cell lung cancer (NSCLC) clinico-genomic database, we assessed outcomes in patients with nonsquamous NSCLC (NonSqNSCLC) who received matched therapy based on a fusion identified using DNA CGP. Lastly, we modeled the added value of RNA CGP for fusion detection in NonSqNSCLC. We observed a broad diversity of fusion partners detected with DNA CGP in conjunction with targetable fusion genes (ALK, BRAF, FGFR2, FGFR3, NTRK1/2/3, RET, and ROS1). In NonSqNSCLC with oncogenic ALK, NTRK, RET, and ROS1 fusions detected by DNA CGP, patients treated with a matched tyrosine kinase inhibitor had better real-world progression-free survival than those receiving alternative treatment regimens and benefit was observed regardless of the results of orthogonal fusion testing. An estimated 1.3% of patients with NonSqNSCLC were predicted to have an oncogenic driver fusion identified by RNA, but not DNA CGP, according to a model that accounts for multiple real-world factors. A well-designed DNA CGP assay is capable of robust fusion detection and these fusion calls are reliable for informing clinical decision-making. While DNA CGP detects most driver fusions, the clinical impact of fusion detection is substantial for individual patients and exhaustive efforts, inclusive of additional RNA-based testing, should be considered when an oncogenic driver is not clearly identified.

Pan-cancer Genomic Analysis of AXL Mutations Reveals a Novel, Recurrent, Functionally Activating AXL W451C Alteration Specific to Myxofibrosarcoma.

Myxofibrosarcoma (MFS) is a common soft tissue sarcoma of the elderly that typically shows low tumor mutational burden, with mutations in TP53 and in genes associated with cell cycle checkpoints ( RB1 , CDKN2A ). Unfortunately, no alterations or markers specific to MFS have been identified and, as a consequence, there are no effective targeted therapies. The receptor tyrosine kinase AXL, which drives cellular proliferation, is targetable by new antibody-based therapeutics. Expression of AXL messenger RNA is elevated in a variety of sarcoma types, with the highest levels reported in MFS, but the pathogenic significance of this finding remains unknown. To assess a role for AXL abnormalities in MFS, we undertook a search for AXL genomic alterations in a comprehensive genomic profiling database of 463,546 unique tumors (including 19,879 sarcomas, of which 315 were MFS) interrogated by targeted next-generation DNA and/or RNA sequencing. Notably, the only genomic alterations recurrent in a specific sarcoma subtype were AXL W451C (n = 8) and AXL W450C (n = 2) mutations. The tumors involved predominantly older adults (age: 44 to 81 [median: 72] y) and histologically showed epithelioid and spindle-shaped cells in a variably myxoid stroma, with 6 cases diagnosed as MFS, 3 as undifferentiated pleomorphic sarcoma (UPS), and 1 as low-grade sarcoma. The AXL W451C mutation was not identified in any non-sarcoma malignancy. A review of publicly available data sets revealed a single AXL W451C-mutant case of UPS that clustered with MFS/UPS by methylation profiling. Functional studies revealed a novel activation mechanism: the W451C mutation causes abnormal unregulated dimerization of the AXL receptor tyrosine kinase through disulfide bond formation between pairs of mutant proteins expressing ectopic cysteine residues. This dimerization triggers AXL autophosphorylation and activation of downstream ERK signaling. We further report sarcomas of diverse histologic subtypes with AXL gene amplifications, with the highest frequency of amplification identified in MFS cases without the W451C mutation. In summary, the activating AXL W451C mutation appears highly specific to MFS, with a novel mechanism to drive unregulated signaling. Moreover, AXL gene amplifications and messenger RNA overexpression are far more frequent in MFS than in other sarcoma subtypes. We conclude that these aberrations in AXL are distinct features of MFS and may aid diagnosis, as well as the selection of available targeted therapies.

EORTC-SPECTA Arcagen study, comprehensive genomic profiling and treatment adaptation of rare thoracic cancers

Arcagen (NCT02834884) is a European prospective study aiming at defining the molecular landscape of rare cancers for treatment guidance. We present data from the cohort of rare thoracic tumors. Patients with advanced pleural mesothelioma (PM) or thymic epithelial tumors (TET) underwent genomic profiling with large targeted assay [>300 genes, tumor mutational burden (TMB), microsatellite instability (MSI) status] on formalin-fixed paraffin-embedded (FFPE) or plasma samples. EORTC molecular tumor board (MTB) advised for biomarker-guided treatments. 102 patients recruited from 8 countries between July 2019 and May 2022 were evaluable: 56 with PM, 46 with TET (23 thymomas, 23 thymic carcinomas). Molecular profiling was performed on 70 FFPE samples (42 PM, 28 TET), and 32 cases on ctDNA (14 PM, 18 TET), within a median turnaround time of 8 days from sample reception. We detected relevant molecular alterations in 66 out of 102 patients (65%; 79% PM, 48% TET), 51 of 70 FFPE samples (73%; 90% PM, 46% TET), and 15 of 32 plasma samples (47%; 43% PM, 50% TET). The most frequently altered genes were CDKN2A/B, BAP1, MTAP in PM and TP53, CDKN2A/B, SETD2 in TET. The TMB was low (mean 3.2 Muts/MB), 2 PM had MSI-high status. MTB advised molecular-guided treatment options in 32 situations, for 17 PM and 15 TET patients (75% clinical trial option, 22% off-label drug or compassionate use, 3% early access program). Molecular testing and MTB discussion were feasible for patients with rare thoracic cancers and allowed the broadening of treatment options for 30% of the cases.

Clinical utility of endoscopic ultrasound-guided tissue acquisition for comprehensive genomic profiling of patients with biliary tract cancer, especially with intrahepatic cholangiocarcinoma.

Endoscopic ultrasound-guided tissue acquisition (EUS-TA) is a standard diagnostic method for biliary tract cancer (BTC), and samples obtained in this manner may be used for comprehensive genomic profiling (CGP). This study evaluated the utility of EUS-TA for CGP in a clinical setting and determined the factors associated with the adequacy of CGP in patients with BTC. CGP was attempted for 105 samples from 94 patients with BTC at the Aichi Cancer Center, Japan, from October 2019 to April 2022. Overall, 77.1% (81/105) of the samples were adequate for CGP. For 22-G or 19-G fine-needle biopsy (FNB), the sample adequacy was 85.7% (36/42), which was similar to that of surgical specimens (94%, p=0.45). Univariate analysis revealed that 22-G or larger FNB needle usage (86%, p=0.003), the target primary lesions (88%, p=0.015), a target size ≥30 mm (100%, p=0.0013), and number of punctures (90%, p=0.016) were significantly positively associated with CGP sample adequacy. EUS-TA is useful for CGP tissue sampling in patients with BTC. In particular, the use of 22-G or larger FNB needles may allow for specimen adequacy comparable to that of surgical specimens.