Comprehensive Molecular Profiling Research Articles

Abstract PO-016: Test-the-test: Clinical utility of comprehensive whole exome sequencing (WES) and RNA-seq for lymphoma patients

Abstract Due to the rapidly evolving landscape of targeted therapies, there is an unmet need for comprehensive molecular profiling to guide treatment decisions for lymphoma patients. Here, we designed a pilot study to assess the feasibility and turnaround time (TAT) of a comprehensive clinical whole exome sequencing (WES) and transcriptome sequencing (RNA-seq) assay in large B-cell lymphoma (LBCL) patients for clinical decision-making (NCT05464823). Patients aged ≥18 years with histologically documented LBCL requiring therapy were eligible. Formalin-fixed paraffin-embedded tissues underwent WES, RNA-seq, and copy number analysis with concomitant peripheral blood or saliva for germline DNA sequencing. Samples with <20% tumor purity and median coverage of <142x for tumor and/or <95x for normal samples were labeled as quantity not sufficient (QNS). Genomic and transcriptomic data were profiled to uncover clinically relevant biomarkers and match patients with clinical trials on ClinicalTrials.gov. Among 84 patients analyzed to date, 71 received WES and RNA-seq, yielding 67 reports with clinical information (treatment options and clinical trial matching) and 4 QNS reports without actionable findings. The remaining 13 samples were rejected as they failed pathology quality control for next-generation sequencing. Cell of origin (COO, n=62), LymphGen (n=62), and lymphoma microenvironment (LME, n=64) classifications were applied. The median TAT was 8 days for custom reports, and 70.4% of custom reports had a TAT ≤10 days. WES identified frequent TP53 (n=22) and MYD88 (n=11) alterations. COOs were designated as activated (n=26, 41.94%) or germinal center (n=36, 58.06%) LBCL. The LymphGen classifier (Wright et al. Cancer Cell. 2020) showed subtypes EZB MYC- (n=10, 16.13%), MCD (n=8, 12.9%), and A53 (n=6, 11.5%) as the most prevalent. LME classification revealed most LMEs as mesenchymal (n=46, 71.88%). Immune-depleted (n=12, 18.75%), immune-inflamed (n=4, 6.25%), and germinal center-like (n=2, 3.13%) LMEs were less prevalent, but the immune-depleted LME was more common in patients with relapse. Clinically significant findings, such as activated COO subtype (n=27), TP53 loss (n=21), DHITsig+ (n=9), and CARD11 mutations associated with ibrutinib resistance (n=3), were identified in 52 samples. On average, 8 clinical trials were identified per full report. Our results show the clinical utility and acceptable TAT of using a comprehensive WES and RNA-seq assay on a cohort of lymphoma patients. The full test reports included findings on significant alterations, LymphGen and LME subtypes, COOs, treatment options, and clinical trial matches. These robust findings, coupled with rapid TAT, demonstrate the feasibility of using integrated WES and RNA-seq to guide clinical decision-making for lymphoma patients. Citation Format: Dai Chihara, Kumudha Balakrishnan, Gita Masand, Anna Novokreshchenova, Alexander Bagaev, Nikita Kotlov, Ekaterina Postovalova, Eduardo Shugaev-Mendosa, Yuliya Gracheva, Kelley Lauziere, Leznath Kaneunyenye, Brianna McKenna, Amber Berlinski, McKenna Walsh, Linnea Larson, Mariam Kotsinyan, Sheila T Yong, Krystle Nomie, Nathan Fowler, Christopher R. Flowers, Jason Westin. Test-the-test: Clinical utility of comprehensive whole exome sequencing (WES) and RNA-seq for lymphoma patients [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-016.

Consensus statement on extracellular vesicles in liquid biopsy for advancing laboratory medicine.

Extracellular vesicles (EVs) represent a diverse class of nanoscale membrane vesicles actively released by cells. These EVs can be further subdivided into categories like exosomes and microvesicles, based on their origins, sizes, and physical attributes. Significantly, disease-derived EVs have been detected in virtually all types of body fluids, providing a comprehensive molecular profile of their cellular origins. As a result, EVs are emerging as a valuable addition to liquid biopsy techniques. In this collective statement, the authors share their current perspectives onEV-related research and product development, with a sharedcommitment to translating this newfound knowledge into clinical applications for cancer and other diseases, particularly as disease biomarkers. The consensus within this document revolves around the overarching recognition of the merits, unresolved questions, and existing challenges surrounding EVs. This consensus manuscript is a collaborative effort led by the Committee of Exosomes, Society of Tumor Markers, Chinese anti-Cancer Association, aimed at expediting the cultivation of robust scientific and clinically applicable breakthroughs and propelling the field forward with greater swiftness and efficacy.

HGG-32. PNOC008: A PILOT TRIAL TESTING THE CLINICAL BENEFIT OF USING MOLECULAR PROFILING TO DETERMINE AN INDIVIDUALIZED TREATMENT PLAN IN CHILDREN AND YOUNG ADULTS WITH NEWLY DIAGNOSED HIGH-GRADE GLIOMA (EXCLUDING DIFFUSE INTRINSIC PONTINE GLIOMA)

Abstract BACKGROUND Children and young adults diagnosed with high-grade glioma (HGG) face extremely poor prognoses. Despite multiple clinical trials testing new treatments in this population, a survival advantage has yet to be achieved. Herein we assessed, in a single-arm, multi-center pilot trial, the feasibility of molecular profiling of primary HGG tumor tissue to create an individualized treatment plan with up to four FDA approved medications. METHODS Patients aged <21 years with newly diagnosed, localized, hemispheric HGG (Stratum A) or midline HGG (non-DIPG; Stratum B) were eligible. Tumor tissue underwent comprehensive molecular profiling (targeted gene panel, whole exome, and whole transcriptome sequencing). Based on detailed review of the molecular data by a dedicated tumor board, an individualized treatment plan that combined up to four FDA approved drugs was recommended. Circulating tumor DNA (ctDNA), imaging, and quality of life (QOL) measures were collected at multiple timepoints. RESULTS Fifty-five patients enrolled between 2018 and 2023 (median age 11 years [range 2-20], n=31 female, n=29 Stratum A). The most common integrated diagnoses included H3K27-altered diffuse midline glioma (n=17), H3/IDH-wildtype diffuse pediatric-type HGG (n=16), and H3G34-mutant diffuse hemispheric glioma (n=12). Median overall survival (OS) from the time of study enrollment was 26.5 months in Stratum A (lower 95% CI: 18.7) and 21.7 months in Stratum B (lower 95% CI: 16.8), with a median follow-up of 35.4 months for all patients (lower 95% CI: 32.5). The most common grade 3 or 4 treatment-related adverse events were decreased neutrophils (n=28), decreased platelets (n=22), and decreased white blood cells (n=16). As of December 2023, seven patients remain on therapy. Central imaging, ctDNA, and QOL analyses are underway. CONCLUSIONS A personalized treatment recommendation for children and young adults with HGG based on comprehensive transcriptomic and genomic analysis is feasible. Current survival data are encouraging, and molecular subgroup analyses are ongoing.

Application of comprehensive molecular genetic profiling in precision cancer medicine, Hungarian experiences.

The study aims to evaluate the clinical benefit of CGP in our Comprehensive Cancer Center Methods and patients: CGP was introduced into our routine clinical practice in 2021. An NGS-based large (> 500 genes) gene panel was used for cases where molecular genetic testing was approved by the National Molecular Tumor Board. From 2021 until August 2023 163 cases were tested. The majority of them were ECOG 0-1 patients with advanced-stage diseases, histologically rare cancer, or cancers with unknown primary tumours. Seventy-four cases (74 of 163, 45%) had clinically relevant genetic alterations. In 34 patients, the identified variants represented an indication for an approved therapy (approved by the Hungarian authorities, on-label indication), while in 40 cases the recommended therapy did not have an approved indication in Hungary for certain tumour types, but off-label indication could be recommended. Based on our CGP results, 24 patients (24/163; 14.7%) received targeted therapy. Treatment duration was between 1 and 60 months. In total 14 (14/163; 8.5% of the tested cases) patients had a positive clinical response (objective response or stable disease) and were treated for more than 16 weeks. NGS-based CGP was successfully introduced in our institution and a significant number of patients benefited from comprehensive genetic tests. Our preliminary results can serve as the starting point of Drug Rediscovery Protocol (DRUP) studies.

Precision-guided treatment in high-risk pediatric cancers

Recent research showed that precision medicine can identify new treatment strategies for patients with childhood cancers. However, it is unclear which patients will benefit most from precision-guided treatment (PGT). Here we report consecutive data from 384 patients with high-risk pediatric cancer (with an expected cure rate of less than 30%) who had at least 18 months of follow-up on the ZERO Childhood Cancer Precision Medicine Program PRecISion Medicine for Children with Cancer (PRISM) trial. A total of 256 (67%) patients received PGT recommendations and 110 (29%) received a recommended treatment. PGT resulted in a 36% objective response rate and improved 2-year progression-free survival compared with standard of care (26% versus 12%; P = 0.049) or targeted agents not guided by molecular findings (26% versus 5.2%; P = 0.003). PGT based on tier 1 evidence, PGT targeting fusions or commenced before disease progression had the greatest clinical benefit. Our data show that PGT informed by comprehensive molecular profiling significantly improves outcomes for children with high-risk cancers. ClinicalTrials.gov registration: NCT03336931

Comprehensive molecular profiling of newly diagnosed advanced, high-grade ovarian cancer: Unveiling BRCA1/2 mutations and genomic instability scores to advocate for public insurance coverage of genetic testing—Insights from the Hellenic Society of Medical Oncology (HeSMO) national program.

3146 Background: Homologous recombination deficiency (HRD) in ovarian cancer, indicative of impaired DNA repair, contributes to genomic instability (GIS). Beyond mutations in BRCA1/2 genes, GIS is particularly significant for identifying patients who may benefit from PARP inhibitors. Equivalence between Myriad Genetics' myChoice CDx and the standard diagnostic tests OncoScan and Amoy Diagnostics has been published, prompting HeSMO to launch a national program addressing GIS and BRCA 1/2 mutations in newly diagnosed advanced high grade ovarian cancer patients, with the utilization of these more economically advantageous tests, in order to guide effective therapeutic strategies, since official reimbursement is not yet applicable in our country. Methods: Patients with locally advanced or metastatic high-grade stage III/IV ovarian cancer were eligible for the HeSMO program, submitting an FFPE tissue tumor sample. The evaluation focused on HRD detection (positive or negative) and GIS assessment (high or low), combining results from OncoScan and Amoy Diagnostics. Results: Between August 2022 and December 2023, 511 patients from Greece underwent testing. 48.14% (n=246) tested HRD positive, 46.58% (n=238) HRD negative. High GIS score was observed in 44.81% (n=229), low GIS in 49.71% (n=254). Potential pathogenic mutations in BRCA1/2 genes were found in 19.96% (n=102). BRCA1 mutations (13.70%, n=70) included key mutations: c.190T>C p.(Cys64Arg), c.3700_3704delGTAAA p.(Val1234GlnfsTer8), c.4065_4068del p.(Asn1355LysfsTer10). BRCA2 mutations (6.26%, n=32) comprised of c.1129G>T p.(Glu377Ter), c.3650_3659del p.(Arg1217IlefsTer8), c.7150C>T p.(Gln2384Ter). In 27 samples (5.28%) no result was produced. Conclusions: HRD and GIS are vital genomic instability biomarkers in high-grade ovarian cancer. These metrics provide valuable insights into tumor aggressiveness, guiding therapeutic decisions, including the use of PARP inhibitors. In this study of 511 patients from 50 centers across Greece, 48.14% tested positive for HRD, emphasizing PARP inhibitors' significance and suggesting hereditary implications. Our commitment to enhance patient access to reliable diagnostic biomarker testing in Greece is unquestionable. Furthermore, we believe, our results will strengthen our efforts towards official state reimbursement, paving the way for local HRD testing solutions. [Table: see text]

IMPRESS-Norway: improving public cancer care by implementing precision medicine in Norway; inclusion rates and preliminary results.

In Norway, comprehensive molecular tumour profiling is implemented as part of the public healthcare system. A substantial number of tumours harbour potentially targetable molecular alterations. Therapy outcomes may improve if targeted treatments are matched with actionable genomic alterations. In the IMPRESS-Norway trial (NCT04817956), patients are treated with drugs outside the labelled indication based on their tumours molecular profile. IMPRESS-Norway is a national, prospective, non-randomised, precision cancer medicine trial, offering treatment to patients with advanced-stage disease, progressing on standard treatment. Comprehensive next-generation sequencing, TruSight Oncology 500, is used for screening. Patients with tumours harbouring molecular alterations with matched targeted therapies available in IMPRESS-Norway, are offered treatment. Currently, 24 drugs are available in the study. Primary study endpoints are percentage of patients offered treatment in the trial, and disease control rate (DCR) defined as complete or partial response or stable disease in evaluable patients at 16 weeks (W16) of treatment. Secondary endpoint presented is DCR in all treated patients. Between April 2021 and October 2023, 1,167 patients were screened, and an actionable mutation with matching drug was identified for 358 patients. By the data cut off 186 patients have initiated treatment, 170 had a minimum follow-up time of 16 weeks, and 145 also had evaluable disease. In patients with evaluable disease, the DCR was 40% (58/145). Secondary endpoint analysis of DCR in all treated patients, showed DCR of 34% (58/170). Precision cancer medicine demonstrates encouraging clinical effect in a subset of patients included in the IMPRESS-Norway trial.

Comprehensive surveillance and molecular profiling of viral infections in cultured olive flounder (Paralichthys olivaceus) across Jeju Island, South Korea

To effectively control viral diseases, ongoing research into virus detection and their genetic characteristics is imperative. We conducted an analysis of the prevalence of five species of viruses in olive flounder (Paralichthys olivaceus) farms in Jeju, specifically viral haemorrhagic septicaemia virus (VHSV), lymphocystis disease virus (LCDV), marine birnavirus (MABV), nervous necrosis virus (NNV), and flounder iridovirus (FLIV). Between July and December 2022, a total of 3282 olive flounders were monitored. VHSV and LCDV were detected in 116 and 173 olive flounders, respectively, confirming detection rates of 3.5% and 5.4%. The highest detection rate for VHSV occurred in December (12.5%), while for LCDV, it was in June (13.8%). MABV, NNV, and FLIV were not detected. Phylogenetic analysis of 19 VHSV isolates collected from 2010 to 2022 revealed that all VHSVs belonged to the IVa genotype based on the nucleoprotein gene. Mutations were identified, indicating genetic variability extending to protein mutations. Additionally, continuous and specific nucleotide mutations of VHSV were confirmed on Jeju Island, a central hub for the aquaculture of olive flounder. These observed mutations in VHSV are considered valuable markers for tracking future mutations in subsequent studies. LCDV isolates were classified under genotype II. The results of the phylogenetic analyses of VHSV, a negative-sense single-stranded RNA virus, and LCDV, a double-stranded DNA virus, provide insights into the mutation patterns of marine viruses.

Comprehensive molecular profiling identifies actionable biomarkers for patients from Thailand and the United Arab Emirates with advanced malignancies.

Comprehensive molecular profiling of tissue samples that can help guide therapy management is not widely available across the globe. Comprehensive molecular profiling through Caris Molecular Intelligence involves the analysis of DNA through next-generation sequencing, chromogenic or fluorescent in situ hybridization, pyrosequencing, and copy number alterations; RNA through whole-transcriptome sequencing and multiplex PCR of RNA; and protein through immunohistochemistry. Here we describe the experience of molecular profiling of tumor tissue samples from patients diagnosed with advanced solid tumors and treated in two countries, the United Arab Emirates and Thailand. Tumor cancer cases submitted to Caris Life Sciences (Phoenix, Arizona, USA) for molecular profiling from the UAE and Thailand were retrospectively analyzed (data accessed between 2019 and 2020) for their molecular alterations and clinical biomarkers, without regard to ethnicity. A total of 451 samples from 35 distinct types of advanced cancers were examined for mutations, amplifications, overexpression, exon copy number alterations, microsatellite instability, deficient mismatch repair, tumor mutational burden, and fusions. Interrogating each step of the biological pathway, from DNA to RNA to distinct protein, identified an alteration with an associated therapy for 75% of these tumor samples. The most common alterations identified included elevated PDL-1 that can be targeted with an immune checkpoint inhibitors and amplification of HER2 for which a variety of anti HER2 therapies are available. Comprehensive molecular profiling in patients with advanced malignancies can help optimize therapeutic management allowing for improved prognostic outcome.

What Do German Molecular Tumor Boards Recommend in Patients with PIK3CA-Mutated Tumors? Launch and First Results from the German Transsectoral Molecular Tumor Board Exchange Platform Deutschland

Introduction: Comprehensive molecular tumor profiling is widely used in the management of patients with cancer. Molecular tumor boards devise treatment strategies based on testing results. In this setting, the Transsectoral Molecular Tumor Board exchange platform Deutschland (TEAM-D) aims to drive peer-to-peer exchange to connect experts in the field. Methods: During the first virtual TEAM-D meeting, participants from 16 German universities and 5 nonacademic institutions discussed five cases with PIK3CA hotspot mutations. Furthermore, an illustrative case vignette was presented. Results: Overall, German caregivers show restraint in administering off-label PIK3CA inhibitor and favor clinical trials in this setting. Conclusion: In the setting of precision oncology, TEAM-D enables virtual case discussion across the different sectors of the German healthcare system. Based on the example of PIK3CA hotspot mutations, TEAM-D demonstrated the value of integrating knowledge from different healthcare professionals.

Abstract GS03-06: Genomic and transcriptomic profiling of primary tumors from patients with HR+, HER2-, node-positive, high-risk early breast cancer in the monarchE trial

Abstract Background Two years of adjuvant abemaciclib combined with endocrine therapy (ET) resulted in significant and clinically meaningful improvement in invasive disease-free survival (IDFS) and distant relapse-free survival (DRFS) in patients (pts) with HR+, HER2-, node-positive, high-risk early breast cancer in the monarchE trial (NCT03155997). Abemaciclib benefit was sustained beyond the completion of treatment (tx) with deepening magnitude of absolute benefit in IDFS and DRFS at 5 years. Here, we evaluate comprehensive molecular profiling of archived primary tumor tissue and association with clinical outcomes. Methods For biomarker analysis, a proportionally stratified random sampling case-cohort design was utilized to include all patients who experienced an IDFS event at a pre-specified interim analysis with a median follow up of 54 months. A cohort of 895 pts (189 with IDFS event) in the abemaciclib + ET arm was matched 1:1 with 903 pts (270 with IDFS event) in the ET arm. Baseline primary tumor samples underwent exome-capture RNA sequencing (RNAseq; n=1324, 23% intent-to-treat (ITT) population) and paired tumor-normal (germline blood samples) whole-exome sequencing (WES; n=1234, 22% ITT population). Expression-based intrinsic subtypes (i.e., luminal A (LumA), luminal B (LumB), HER2-enriched (HER2E), basal- and normal-like) were characterized using the Absolute Intrinsic Molecular Subtyping model. The 21-gene expression signature score (Oncotype DX test) was inferred from RNAseq; samples were categorized into lower (0-25) and high (26-100) risk groups. To investigate associations of biomarkers with abemaciclib benefit, WES genomic events including oncogenic and hotspot mutations by OncoKB, and copy number events, of incidence >9% were pre-selected. Results The biomarker subset of monarchE was reflective of the ITT population. A total of 1190 tumors (abemaciclib+ET n= 605; ET alone n=585) yielded adequate RNAseq results. Intrinsic subtype distribution was consistent across tx arms (Table A). Low tumor purity limited assessment of the normal-like subtype. The 4-year IDFS benefit of abemaciclib was consistent across all subtypes. LumA cancers had the lowest risk of recurrence while HER2E and basal-like subtypes had the highest. Inferred 21-gene expression signature score showed similar benefits from abemaciclib in both lower and high-risk groups (Table B). A total of 1173 tumors yielded adequate WES results (abemaciclib+ET n=580; ET alone n=593). Consistent abemaciclib benefit was observed across the most frequently altered genes (Table C). In exploratory analysis, lower benefit from abemaciclib was seen in the subset of focal-high level MYC amplified tumors (n=176, HR 1.30, 95% CI, 0.77, 2.20) compared to MYC non-amplified tumors (n=997, HR 0.62, 95% CI, 0.47, 0.80, nominal interaction p=0.014). The treatment benefit of abemaciclib was observed across all subpopulations of altered genes based on gene expression data. Conclusions Adjuvant abemaciclib+ET maintained IDFS benefit compared to ET alone across all molecular subtypes as measured by RNAseq. Benefit was consistent across most altered genes assessed by WES, except for the subset of tumors with MYC amplification. Additional research is necessary to confirm these findings. Table. Citation Format: Nicholas Turner, Jorge Reis-Filho, Matthew Goetz, Christine Desmedt, Sarat Chandarlapaty, Hironobu Sasano, Carlos Arteaga, Sherene Loi, Stephanie Graff, Deli Liu, Vanessa Rodrik-Outmezguine, Anthony Sireci, Cynthia Sandoval Rubenstein, Helen Won, Lacey M. Litchfield, Maria Munoz, Stephen Johnston. Genomic and transcriptomic profiling of primary tumors from patients with HR+, HER2-, node-positive, high-risk early breast cancer in the monarchE trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-06.

Abstract PO1-02-08: Comprehensive Molecular Profiling of Breast Cancer: A real-time PCR assay for identifying ESR1, PGR, ERBB2, MKI67 and a novel proliferative signature in core needle biopsies and resected invasive breast cancer tissues

Abstract Background: Accurate status determination of breast cancer (BC) biomarkers, including estrogen receptor (ER/ESR1), progesterone receptor (PR/PGR), human epidermal growth factor receptor 2 (HER2/ERBB2), and marker of proliferation Ki67 (MKI67), is crucial for guiding therapeutic decisions. However, current “gold standard” methods such as immunohistochemistry (IHC) for assessing these biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues face challenges in standardization and exhibit substantial inter- and intra-laboratory variability, particularly for Ki67. To address these limitations, APIS Breast Cancer Subtyping Kit has been developed, utilizing reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to evaluate the status of ER, PR, HER2, KI67, and a novel proliferative signature in breast cancer core needle biopsy (CNB) and resected specimens. The APIS Breast Cancer Subtyping Kit offers enhanced accuracy, improved reproducibility, and the ability to provide results in a timely manner. Herein we report the development and validation of the APIS Breast Cancer Subtyping Kit, assessing clinical performance using retrospective and prospective sample collection. Furthermore, reproducibility and reliability across three testing sites were evaluated. By comparing the results of the kit with those obtained through standard IHC, we demonstrate the reliability and utility of the APIS Breast Cancer Subtyping Kit for precise breast cancer subtyping and guiding personalized patient management decisions. Methods: FFPE tissue sections (n=652) taken by CNB or resection underwent histological analysis in accordance with laboratory’s standard of care methods. Samples with a HER2 score of 2+ were referred to fluorescence in situ hybridization (FISH) to determine ERBB2 amplification, with the FISH result replacing the IHC result in determining the HER2 status (positive/negative) of the sample. Tumors with greater than 1% positive stained nuclei for ER/PR were scored positive. A cut-off of 20% Ki67 staining was used for positive/negative status. The performance was assessed as the agreement with IHC/ISH status and reported as OPA (overall percent agreement) with corresponding 95% confidence intervals (95% CI). To assess the precision of the assay, a panel of RNA pools (samples) was repeatedly measured on different days across three independent laboratories. Reproducibility was evaluated using variance components analysis. Results: The APIS Breast Cancer Subtyping Kit showed high concordance (OPA) with IHC: 93.1% (95% CI: 90.9-94.8) for ER, 86.8% (95% CI: 84.0-89.2) for PR, 94.2% (95% CI: 92.2-95.8) for HER2 (IHC/FISH), 78.3% (95% CI: 75.0-81.3) for Ki67, and 80.1% (95% CI: 76.8-83.1) for proliferative signature (assessed against MKI67 IHC status). Inter-site reproducibility demonstrated excellent agreement in quantitative measurements, with a total SD ranging from 0.00 to 0.22. Binary single-marker status (positive/negative) calling achieved 100% reproducibility for ER, PR, and HER2 for all tested samples (negative/low positive/medium positive), as well as for most Ki67 samples (87% agreement for negative samples). Conclusions: APIS Breast Cancer Subtyping Kit demonstrated a high level of concordance with standard of care IHC/FISH in assessing breast cancer biomarker status. These findings suggest that the APIS Breast Cancer Subtyping Kit provides highly precise and reproducible quantitative assessment of BC biomarkers and molecular subtypes. Inclusion and exclusion criteria Citation Format: Anna Gasior, Joanna Gorniak, Sara Rollinson, Leanne Gough, Anne-Sophie Wegscheider, Axel Niendorf. Comprehensive Molecular Profiling of Breast Cancer: A real-time PCR assay for identifying ESR1, PGR, ERBB2, MKI67 and a novel proliferative signature in core needle biopsies and resected invasive breast cancer tissues [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-02-08.

Comprehensive Genomic Studies on the Cell Blocks of Pancreatic Cancer.

Pancreatic cancer is one of the deadliest malignancies, characterized by late-stage diagnosis and limited treatment options. Comprehensive genomic profiling plays an important role in understanding the molecular mechanisms underlying the disease and identifying potential therapeutic targets. Cell blocks (CBs), derived from EUS-FNA, have become valuable resources for diagnosis and genomic analysis. We examine the molecular profile of pancreatic ductal adenocarcinoma (PDAC) using specimens obtained from CB EUS-FNA, across a large gene panel, within the framework of next-generation sequencing (NGS). Our findings revealed that over half (55%) of PDAC CB cases provided adequate nucleic acid for next-generation sequencing, with tumor cell percentages averaging above 30%. Despite challenges such as low DNA quantification and degraded DNA, sequencing reads showed satisfactory quality control statistics, demonstrating the detection of genomic alterations. Most cases (84.6%) harbored at least one gene variant, including clinically significant gene mutation variants such as KRAS, TP53, and CDKN2A. Even at minimal concentrations, as long as the extracted DNA is of high quality, performing comprehensive molecular profiling on PDAC samples from cell blocks has remained feasible. This strategy has yielded valuable information about the diagnosis, genetic landscape, and potential therapeutic targets, aligning closely with a precision cytopathology approach.

Molecular classification and intratumoral heterogeneity of gastric adenocarcinoma.

Gastric cancers frequently harbor striking histological complexity and diversity between lesions as well as within single lesions, known as inter- and intratumoral heterogeneity, respectively. The latest World Health Organization Classification of Tumors designated more than 30 histological subtypes for gastric epithelial tumors, assigning 12 subtypes for gastric adenocarcinoma (GAD). Meanwhile, recent advances in genome-wide analyses have provided molecular aspects to the histological classification of GAD, and consequently revealed different molecular traits underlying these histological subtypes. Moreover, accumulating knowledge of comprehensive molecular profiles has led to establishing molecular classifications of GAD, which are often associated with clinical biomarkers for therapeutics and prognosis. However, most of our knowledge of GAD molecular profiles is based on inter-tumoral heterogeneity, and the molecular profiles underlying intratumoral heterogeneity are yet to be determined. In this review, recently established molecular classifications of GAD are introduced in the aspect of pathological diagnosis and are discussed in the context of intratumoral heterogeneity.

39 A multi-centre analysis of the performance of EBUS-TBNA testing for comprehensive molecular profiling in non-small cell lung cancer (NSCLC)

39 A multi-centre analysis of the performance of EBUS-TBNA testing for comprehensive molecular profiling in non-small cell lung cancer (NSCLC)

Added value of whole-exome and RNA sequencing in advanced and refractory cancer patients with no molecular-based treatment recommendation based on a 90-gene panel.

The objective was to determine the added value of comprehensive molecular profile by whole-exome and RNA sequencing (WES/RNA-Seq) in advanced and refractory cancer patients who had no molecular-based treatment recommendation (MBTR) based on a more limited targeted gene panel (TGP) plus array-based comparative genomic hybridization (aCGH). In this retrospective analysis, we selected 50 patients previously included in the PROFILER trial (NCT01774409) for which no MBT could be recommended based on a targeted 90-gene panel and aCGH. For each patient, the frozen tumor sample mirroring the FFPE sample used for TGP/aCGH analysis were processed for WES and RNA-Seq. Data from TGP/aCGH were reanalyzed, and together with WES/RNA-Seq, findings were simultaneously discussed at a new molecular tumor board (MTB). After exclusion of variants of unknown significance, a total of 167 somatic molecular alterations were identified in 50 patients (median: 3 [1-10]). Out of these 167 relevant molecular alterations, 51 (31%) were common to both TGP/aCGH and WES/RNA-Seq, 19 (11%) were identified by the TGP/aCGH only and 97 (58%) were identified by WES/RNA-Seq only, including two fusion transcripts in two patients. A MBTR was provided in 4/50 (8%) patients using the information from TGP/aCGH versus 9/50 (18%) patients using WES/RNA-Seq findings. Three patients had similar recommendations based on TGP/aCGH and WES/RNA-Seq. In advanced and refractory cancer patients in whom no MBTR was recommended from TGP/aCGH, WES/RNA-Seq allowed to identify more alterations which may in turn, in a limited fraction of patients, lead to new MBTR.

Abstract 6399: Real-world experience of an Asian pan-cancer combined DNA/RNA next generation sequencing (NGS) tissue biopsy assay

Abstract Introduction: Comprehensive molecular profiling with combined DNA-RNA next-generation sequencing (NGS) on tumor tissue is now increasingly being used as standard-of-care for sequence therapy selection to improve cancer survival. Combined DNA-RNA NGS is also considered standard-of-care for central nervous system (CNS) tumor classification. Despite the genetic heterogeneity and diversity of cancers, most commercial combined DNA/RNA NGS assays are validated in Western populations, resulting in knowledge gaps in patient outcomes. We report here for the first time a range of actionable outcomes from a comprehensive DNA/RNA tissue NGS assay designed specifically to probe Asian-prevalent cancers and biomarkers in an international multicenter real-world study in Asia. Methods: 1,002 formalin-fixed paraffin-embedded tissue samples from 995 Asian cancer patients across >70 centers in 7 countries and/or territories underwent real-world DNA/RNA NGS testing in a CAP-accredited CLIA-certified laboratory. Genomic alterations including SNVs, INDELS, gene fusions, CNVs, TMB, and MSI were analyzed using an Asian-centric pan-cancer hybrid-capture NGS panel that comprehensively profiles 572 cancer-related genes and 91 RNA fusion partners (UNITED). Biomarkers were considered actionable if the therapeutic indication in any solid tumor or hematological cancer was FDA-approved, guidelines-recommended, or supported by well-powered phase III clinical trials. Results: 27 cancer types were represented in 1,002 tissue samples, with lung, breast, colorectal, and prostate cancers comprising 45.5% of clinical volume. 79.5% of samples harbored ≥1 actionable biomarker, with the most frequently altered genes being TP53, KRAS, PIK3CA, EGFR, and NF1. Overall, 42.6% of samples had ≥1 biomarker with an indication in the patient’s cancer type, including 81.5% of lung, 59.0% of breast, 61.8% of colorectal, and 42.1% of prostate cancers. Across the pan-cancer cohort, 13.3% was TMB-high and 1.9% was MSI-high. 26.3% of samples were from cancer types outside the original assay design; the top 3 were pancreatic cancer (7.3%), sarcoma (3.3%), and CNS tumor (3.1%). Cancers of unknown primary (CUP) also constituted a significant proportion (3.8%). In CNS tumors, 38.7% harbored an actionable mutation in IDH1, BRAF, or H3-3A. In CUP, 78.8% harbored an actionable mutation; 50% of samples harbored biomarkers that additionally informed potential tumor origin, including mutations in EGFR, MET, ALK, ROS1, and BRCA1. Conclusion: The use of an Asian-focused pan-cancer DNA/RNA NGS panel resulted in detection of actionable biomarkers in 79.5% of clinical cases. These findings support the utility of DNA/RNA NGS assays in informing standard-of-care therapeutic decisions in Asian cancer patients for maximizing cancer survival. Citation Format: Jason Yongsheng Chan, Jing Yi Lee, Zi Yi Wan, Abner Herbert Lim, Tun Kiat Ko, Cedric Chuan-Young Ng, Donald Poon, Jens Samol, Tsz Him So, Su Pin Choo, Ravindran Kanesvaran, Cheng-Vai Hui, Joseph Siu-Kie Au, Aya El Helali, Timothy Yip, Vikneswari Rajasegaran, Sandy Lim, Ruifen Weng, Puay Hoon Tan, Bin Tean Teh, Min-Han Tan, Jonathan Poh. Real-world experience of an Asian pan-cancer combined DNA/RNA next generation sequencing (NGS) tissue biopsy assay [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 6399.

Abstract 7037: Comprehensive molecular and immune profiling of triple negative invasive lobular carcinoma

Abstract Background: Triple-negative invasive lobular carcinoma (TN-ILC) is a rare (0.1-1.4%) breast cancer with prognosis worse than ER positive ILC. Currently there are no targeted therapies or clinical trials specifically for TN-ILC. A comprehensive analysis of the molecular and immune landscape can help identify novel targets and pathways for TN-ILC to improve patient outcomes. Here, we characterized the molecular and immune signature of TN-ILC. Methods: 11,760 breast cancer samples (Invasive ductal (ID) TNBC, n=364; TN-ILC, n=31) were analyzed (592, NextSeq; WES, NovaSeq), (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). Tumor mutational burden (TMB) totaled somatic mutations per tumor (high ≥10 mt/MB) was tested by NGS. Microsatellite-instability (MSI) was tested by NGS. Immune cell fractions were calculated by deconvolution of WTS. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q < 0.05). Results: TN-ILC had higher frequency of CDH1 (0.83% vs. 66.67%), ERBB2 (0.84% vs.28.57%), ARID1A (1.65% vs. 23.33%), CBFB (0.55% vs.16.67%), AKT1 (2.22% vs.16.13%) and KMT2C (3.83% vs.13.79%), but lower frequency of TP53 (89.7%5 vs. 38.71%) mutations (all p<0.05) compared to ID-TNBC. TN-ILC had higher frequency of TMB high (23.3% vs. 5.2%, p<0.05) but there was no difference in dMMR/MSI-H (0% vs 1.1%, p=1). TN-ILC had higher AR RNA (FC: 14.2), protein expression (80.6% vs. 24.8%) and higher frequency of fusion variant-AR (16.1% vs 4.9%) (all p < 0.05) compared to ID-TNBC. Analysis of inferred immune cell infiltrates showed that TN-ILC had higher infiltration of M2 macrophages (5.34% vs. 2.94%) and neutrophils (4.83% vs. 2.65%) but lower infiltration of M1 macrophages (3.46% vs 2.17%) and CD8 T cells (0.58% vs. 0.11%) (all p<0.05). TN-ILC had lower T cell inflamed signature (16.1% vs 35.7%, p<0.05), decreased immune checkpoint genes (CD274, CTLA4, FOXP3, LAG3, IDO, FC: 1.2-2.7, all p < 0.05), PD-L1 protein expression (SP142: 30.4% vs. 50.2%, p=0.06; 22c3: 15.4% vs. 42.6%, p = 0.05) and differential expression of BCL2 family genes (upregulation: BIK, FC: 2.2; downregulation: BAX, BAK1, BID, PMAIP1, MCL1, BCL2A1, BCL2L10, FC: 1.0-6.3, all p < 0.05) compared to ID-TNBC. Conclusion: These data suggest that TN-ILC had higher frequency of CDH1, ERBB2, AKT1, ARID1A mutations, higher M2 macrophages and neutrophils and lower M1 macrophages and CD8 T cells infiltration and, lower T cell inflamed signature. High TMB and AR expression can translate into use of immunotherapy (ICI) and AR antagonists in these patients. Additonal analysis to determine the optimal biomarker for ICI response in TN-ILC is needed. Results of our study need to be validated in larger studies with survival correlation. Citation Format: Pooja Advani, Sachin Deshmukh, Sharon Wu, Jacob Andring, Joanne Xiu, Jose Leone, Priya Jayachandran, Stephanie Graff, Mathew Oberley, George Sledge, Asher Chanan-Khan. Comprehensive molecular and immune profiling of triple negative invasive lobular carcinoma [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 7037.

Abstract 1764: Long read sequencing allows comprehensive molecular profiling of complex karyotype acute myeloid leukemia (CKAML) with 5q deletions

Abstract Background Acute myeloid leukemia with complex karyotype (CKAML) does frequently show del(5q), deletion of chromosome 5q. Previous work in MDS and AML showed that CKAML form clusters distinguishing del(5q) cases with commonly retained regions (CRRs) in the telomeric (≥5q34) and centromeric (≤5q14.2) ends from cases with loss of the telomeric and/or centromeric regions. Interestingly, del(5q) patients with CRRs were shown to have less genomic lesions and were associated with a better prognosis than other 5q-deleted CKAML cases. Aim: Combining the advantages of two different long read sequencing technologies, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), we aimed to more comprehensively characterize the molecular landscape of CKAML with del(5q). Our integrative workflow enabled an in-depth characterization of copy number variations (CNV), genomic rearrangements, transcriptomic and epigenomic changes. Methods: Low coverage whole genome sequencing (WGS) using ONT was performed on 209 patients with CKAML to identify del(5q) cases for further in-depth analysis. For 10 selected cases with known CRR status, genomic deep sequencing using PacBio Revio and ONT PromethION was performed, as well as PacBio Revio MAS-seq for fusion transcript detection. Results: A mean sequencing depth of 3-fold (range 0.4-17.3-fold) was obtained by low coverage WGS of the 209 CKAML cases. With a resolution of 0.1 Mbp n=134 del(5q) cases were identified in our CKAML cohort (64%) and commonly deleted regions on 5q could be resolved at 5q21.2 to 5q23.3 and 5q33.3 to 5q35 (Fisher test, FDR Benjamini-Hochberg, p<10−30). These regions separated del(5q) in n=52 cases showing CRR retention and n=82 showing partially no CRR, (either the centromeric or telomeric regions, n=74, or of both regions, n=8 involved). Subsequent co-occurrence analysis provided evidence of an association between the loss of telomeric 5q region with a 3p12-25 deletion. In-depth analysis (30-50 fold) of ten distinct del(5q) cases by PacBio and ONT revealed additional insights into the complex clonal composition of del(5q) cases. For example, one case showed two distinct subclones with 5q14.2-5q35.1 deletion and with translocation t(3;5)(p14;q14) respectively. Moreover, we could demonstrate that 5q-aberrations are far more complex than initially thought showing, e.g. inverted terminal duplications that resulted in fusion transcripts involving PDE4D and leading to its deregulated expression. Conclusion: Using our long read deep sequencing approach we could further delineate the structural complexity of del(5q) CKAML. While gene expression profiling, methylation analysis and correlation of findings with clinical outcome are ongoing and will be presented at the meeting, our results help to further pinpoint the biology underlying del(5q) and to further refine this heterogeneous CKAML subgroup. Citation Format: Felix Schick, Ekaterina Slonova, Eric Straeng, Sarah Huet, Maurizio Raso, Marlon Tilgner, Frederik Damm, Pierre Sujobert, Olga Blau, Lars Bullinger, Anna Dolnik. Long read sequencing allows comprehensive molecular profiling of complex karyotype acute myeloid leukemia (CKAML) with 5q deletions [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 1764.

Abstract 2695: A higher percentage of M2 macrophages in non-tumor part predict the tumor recurrence in patients with hepatocellular carcinoma after surgical resection

Abstract The primary hurdle in improving outcomes for hepatocellular carcinoma (HCC) patients after curative treatment is the high early recurrence rate. The existing literature indicates the presence of type 2 macrophages (M2), renowned for their immunosuppressive capabilities, has been implicated in creating a microenvironment conducive to tumor growth and recurrence. However, the intricate interplay involving M2 macrophages and the recurrence of HCC is underexplored. Therefore, we conducted an analysis involving HCC patients who underwent hepatectomy at our hospital between 2013 and 2017. We collected samples from both the tumor and non-tumor regions and subjected them to bulk RNA sequencing for comprehensive molecular profiling. The proportion of immune cells was calculated by CIBERSORTx.In our study, we enrolled fifty patients, with sixty percent presenting with cirrhosis and seventy-three percent having viral hepatitis. Twenty-three patients experienced tumor recurrence, with a median time to recurrence of 12.6 months. First, we found no correlation between five TIMR subtypes defined by TIMELASER (Xue R, Nature, 2022) and recurrence. However, as for the proportions of immune cell, a significantly lower NK/Treg ratio (p= 0.0163) and high within the resected tumor section in recurrent group was found. On the other hand, the ratio of M2 macrophages to total macrophages (M2/M) was significantly higher within the non-tumor region in patients experiencing tumor recurrence after hepatectomy (p=0.0357). Furthermore, the expression of the ACGT2 gene in non-tumor part, which strongly correlates with Treg function, was significantly higher (p=0.0056) in recurrent group. In addition, the innate T cells score was increased in non-tumor pat in recurrent group but with borderline significance (p=0.0628). However, in multivariable analysis, only a higher M2/M ratio within the non-tumor region (HR 3.756, p=0.0072) were identified as independent risk factors for tumor recurrence. We also found that patients with a higher M2/M ratio had a lower percentage of cirrhotic patients (p=0.0049) and less association with TIME-IA (immune activation) subtype of HCC by TIMELASER classification (p=0.0030). In conclusion, a higher M2/M ratio within the non-tumor region predict the tumor recurrence in patients with hepatocellular carcinoma. Citation Format: Po-Ting Lin, Wei-Ting Ku, Tsung-Han Wu, Cheng-Heng Wu, Chan-Keng Yang, Wei Teng, Yung-Chang Lin, Chun-Yen Lin. A higher percentage of M2 macrophages in non-tumor part predict the tumor recurrence in patients with hepatocellular carcinoma after surgical resection [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 2695.