Evaluation of the Cytomorphology, Immunophenotype, and Molecular Genetics of Lymphoblastic Lymphoma/Leukemia in Serous Effusion.

Quantitative PCR Detection of t(14;18) bcl-2/JH Fusion Sequences in Follicular Lymphoma Patients : Comparison of Peripheral Blood and Bone Marrow Aspirate Samples

Noncanonical Gene Fusions Detected at the DNA Level Necessitate Orthogonal Diagnosis Methods Before Targeted Therapy.

Background: Advanced sequencing technologies have contributed to identifying genomic and transcriptomic features of various types of tumors. Despite several sequencing data from multiple myeloma (MM) being generated, understanding various properties of MM remained insufficient. Methods: The patients confirmed with MM by bone marrow examination were analyzed in the study. Myeloma cells were enriched from the bone marrow aspirates of the patients using CD138. Whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) were performed for isolated myeloma cells. Mutational signatures were analyzed using somatic mutations detected by WGS. Results: A total of 37 MM patients (median age 67) were enrolled in this study. WGS revealed somatic mutations of an average of 8872 single nucleotide variants (SNVs) and 934 insertions and deletions (Indels) from the patients, respectively. By the analysis of structural variants, 7 chromothripsis and 4 chromoplexy patients were identified. Mutational signature analysis showed that single base substitution (SBS) 9 signature largely varied between patients. SBS9 signature high patients were mostly diagnosed with non-IgG/non-IgA heavy chain and lambda light chain type myeloma. Notably, IgD myeloma patients were solely detected in the SBS9 signature high group compared to the low group (p = 0.001). Gene expression patterns obtained from WTS were well-divided into SBS9 signature high and low groups, and analysis of differentially expressed genes (DEG) between SBS9 signature high and low groups implied IgD myeloma features including high expression of IGHD. As a validation test, we tried mutational signature analysis on whole exome sequencing (WES) data of 784 MM patients from the MMRF database. Of the 38 patients with more than 500 exonic mutations, only the SBS9 signature high group had IgD myeloma candidates, whereas the SBS9 signature low group had no IgD myeloma candidate (p = 0.014). Conclusions: DNA and RNA sequencing of myeloma patients could classify specific subtypes of disease categories. Further study for SBS 9 signature high and IgD myeloma is needed to discover the underlying mechanism of distinct features and to find out their clinical implication. Citation Format: Sheehyun Kim, Hyundong Yoon, Youngil Koh, Sung-Soo Yoon. Distinct subtype of multiple myeloma revealed by whole genome and transcriptome sequencing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6073.

Pediatric acute myeloid leukemia (AML) exhibits distinct genetic characteristics, including unique driver alterations and mutations with prognostic and therapeutic implications. Cytogenetics study, along with Next Generation Sequencing (NGS) panel testing, have long been the standard for molecular diagnosis of AML. While these approaches enable diagnosis and prognosis determination in most cases, they have limitations-particularly in detecting emerging rare, recurrent genetic abnormalities. In this study, we systematically reviewed our real-time clinical experience with the diagnostic workup of pediatric AML using an integrated whole genome and whole transcriptome sequencing (iWGS-WTS) approach and compared the test results obtained from various methodologies, including whole genome sequencing (WGS), whole exome sequencing (WES), whole transcriptome sequencing (WTS), iWGS-WTS, cytogenetics, and targeted panel NGS. Our findings demonstrate that the iWGS-WTS approach improves the identification of clinically relevant genetic alterations, enhancing precise disease classification and risk assessment. Additionally, the iWGS-WTS approach streamlines sample acquisition and reduces testing redundancy, positioning it as a practical and superior alternative to traditional diagnostic methods in pediatric AML management.

Pediatric acute myeloid leukemia (AML) exhibits distinct genetic characteristics, including unique driver alterations and mutations with prognostic and therapeutic implications. Cytogenetics study, along with Next Generation Sequencing (NGS) panel testing, have long been the standard for molecular diagnosis of AML. While these approaches enable diagnosis and prognosis determination in most cases, they have limitations—particularly in detecting emerging rare, recurrent genetic abnormalities. In this study, we systematically reviewed our real-time clinical experience with the diagnostic workup of pediatric AML using an integrated whole genome and whole transcriptome sequencing (iWGS-WTS) approach and compared the test results obtained from various methodologies, including whole genome sequencing (WGS), whole exome sequencing (WES), whole transcriptome sequencing (WTS), iWGS-WTS, cytogenetics, and targeted panel NGS. Our findings demonstrate that the iWGS-WTS approach improves the identification of clinically relevant genetic alterations, enhancing precise disease classification and risk assessment. Additionally, the iWGS-WTS approach streamlines sample acquisition and reduces testing redundancy, positioning it as a practical and superior alternative to traditional diagnostic methods in pediatric AML management.

Background. With technological advances such as next-generation sequencing, recent gains in understanding pediatric cancer can aid in treatment decisions, especially in the setting of relapse. To discover expressed, clinically significant mutations for pediatric patients with relapsed tumors, we performed a pilot trial using a combination of whole exome sequencing (WES) of tumor/normal DNA and whole transcriptome sequencing (WTS) of tumors, complemented by single nucleotide polymorphism (SNP) arrays. Objectives. We identified 48 pediatric and young adult patients with relapsed or refractory solid tumors with matched tumor/normal samples. Our goals were to determine the feasibility of performing comprehensive genomic analyses in this population, to compare the genomic profile of relapsed tumors to prior reports of primary tumors, and to delineate the percentage of patients with actionable mutations. Actionable changes were defined as reportable germ line mutations determined by the American College of Medical Genetics (ACMG), a change in diagnosis, and somatic changes targetable by FDA approved medications or drugs undergoing clinical trials. Methods. WES was performed on matched tumor and normal samples to identify germ line and somatic mutations. WTS was performed on tumor samples to identify fusion genes, gene expression profiling, and expressed variants. SNP arrays were performed to identify copy number changes. Sanger validation confirmed reportable mutations. Results. In the exome, we noted a median of 33 somatic mutations per sample (range 1-375), a higher mutational burden compared to previously reported primary pediatric malignancies. Transcriptome data further refined results to a median of 7 expressed somatic mutations per sample (range 0-95). The majority of patients had one oncogenic driver. Sequencing relapsed tumors at multiple time points showed the continued presence of driver mutations but a shift in passenger mutations. Eleven of the 48 patients (23%) had a targetable mutation, such as ALK, BRAF, GNAQ, GNA11, IDH1, MTOR, PIK3CA, and SKP2. Two patients (4%) had a change in diagnosis due to the presence or absence of diagnostic fusion genes. In the germ line of 5 patients (10%) we discovered mutations in ACMG-reportable genes MUTYH, SCN5A, TP53, and MLH1. A total of 16 patients (33%) had actionable mutations. Conclusions. Our study showed the feasibility of next-generation sequencing in relapsed pediatric solid tumors, with actionable mutations detected in a third of our patients. We demonstrated the utility in using exome and transcriptome sequencing with SNP arrays. Implementation of these techniques has the potential to change the practice of precision medicine. In summary, we have developed a prototype that will be utilized to design a national Pediatric Match trial in collaboration with the Children's Oncology Group. Citation Format: Wen-I Chang, Andrew S. Brohl, Rajesh Patidar, Jack F. Shern, Jun S. Wei, Young K. Song, Hongling Liao, Jimmy Lin, Sivasish Sindiri, Li Chen, Berkley Gryder, Marielle E. Yohe, Shile Zhang, Melinda S. Merchant, Brigitte C. Widemann, Javed Khan. Clinical exome and transcriptome sequencing for identification of actionable cancer targets: A pilot study for children and young adults with relapsed or refractory solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3882. doi:10.1158/1538-7445.AM2015-3882

Gene fusion is one of the mechanisms that promote tumor development. It is also an important cause for the poor prognosis of patients. The detection of gene fusion is crucial for the recognition of tumor biomarker, cancer subtype classification, and clinical medication guidance. Appropriate methods can help the early diagnosis and avoid ineffective medication. Traditional tests include fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), reverse transcription of PCR (RT-PCR), and next generation sequencing (NGS). The next generation sequencing (NGS) mainly includes: whole genome sequencing (WGS), whole transcriptome sequencing (WTS) and target sequencing (hybridization capture method/amplicon method). In clinical concomitant diagnostic applications, some factors such as operability, time/money costs, and the level of expertise required for data analysis should be considered. This article concludes with a discussion of the technical principles of different detection methods and advantages/limitations. Meanwhile, it provides reference opinions for the detection methods of gene fusion.

Metastatic urothelial carcinoma (UC) to serous effusion (SE) is extremely rare and its cytomorphological features have only been described in case reports. In this study, we searched the pathology database at University of Michigan for SEs due to metastatic UC in the last 20 years. A total of 25 cases from 20 patients with clinically and pathologically confirmed metastatic UC in SEs were retrieved. The specimens consisted of 15 pleural, 8 peritoneal, and 2 pericardial effusions. Smears were reviewed and evaluated for the following features: cellularity, single cells, cell clusters or short cords, cell wrapping, "windows" between the cells, two-tone cytoplasm, cytoplasmic vacuoles, signet ring cells, nuclear to cytoplasmic (N/C) ratio, nuclear hyperchromasia, irregular nuclear membrane, nuclear centricity, double or multiple nuclei, nucleoli, anaplastic cells and mitosis. Our results showed that UC manifested in SEs predominantly as a single cell population with or without clusters or short cords, and frequently exhibited the "cell wrapping" of two or more cells. Individual UC cell in SEs exhibited nuclear enlargement with increased N/C ratio, irregular nuclear membranes, hyperchromatic coarse chromatin and frequently prominent nucleoli. Double or multinucleated cells, cells with vacuolated cytoplasm or signet ring appearance were also frequently present. Our results demonstrated that while certain features could suggest the diagnosis of UC, the cytomorphological features are not specific and often overlap with those of reactive mesothelium, mesothelioma, metastatic adenocarcinoma, or squamous cell carcinoma in SEs. Accurate diagnosis of UC rests on the combination of clinical history, cytomorphologic features and appropriate immunohistochemical panel.

4629 Background: Genomic profiling has identified KRAS mutations in 88-90% of PC. KRAS WT tumors represent a molecularly heterogeneous group that may harbor targetable alterations (TA). We studied KRAS WT PC using NextGen sequencing (NGS) and whole transcriptome sequencing (WTS) to characterize the molecular landscape of this unique group and to assess the prevalence of TA. Methods: A total of 1164 PC tumors were tested at Caris Life Sciences by NGS (592 genes), WTS (NovaSeq), IHC and fragment analysis. Comparison of KRAS WT vs. mutant (MT) was done by Fisher-Exact or Chi2 and was corrected for multiple tests. Results: The KRAS WT cohort included 144 tumors (12.4%). No differences were seen in gender (female: 46% in both WT & MT) and age (median: 66 & 67) compared to KRAS MT. In KRAS WT tumors, targetable fusions tested by WTS and pathogenic mutations by NGS were seen in 22% (32 of 144) and 52% (75 of 144) respectively; potentially targetable amplifications (amp) were seen in 5 additional tumors. No TA were seen in 22% of WT tumors. Key alterations are in Table. Alterations inducing MAPK activation, including BRAF, RAF1, NF1 and GNAS changes were seen in 38 (26%) tumors. Frequent alterations were seen in FGFR genes (11 tumors), MET (4, including 1 exon 14 skip), and ERBB receptor and ligands (10). Fusions in ALK, ROS1, RET and NOTCH1 were seen (8), largely exclusive of other drivers. Wnt, PI3K, chromatin remodeling (CR) and DDR changes were common and sometimes seen concurrent with other alterations. Compared to KRAS MT, no difference of PD-L1 or TMB-H was seen. BRAF, APC, PBRM1, CTNNB1 mutations, MDM2 amp, gene fusions and MSI-H/dMMR were all more frequent in KRAS WT tumors (corrected p < 0.05). Conclusions: KRAS WT PC is enriched with TA (e.g., BRAF, ALK, ROS1, NRG1, MSI-H). The use of WTS in combination with NGS identifies activated molecular pathways in the majority of KRAS WT tumors. Based on our findings, comprehensive profiling of PC at the DNA and RNA level is recommended to provide patients with therapeutic opportunities beyond standard treatments. [Table: see text]

CD46 Is Amplified in High-Risk Myeloma with Gain of Chromosome 1q and Selectively Targeted By a Novel Anti-CD46 Antibody-Drug Conjugate

Bone Marrow B cell Precursor Number after Allogeneic Stem Cell Transplantation and GVHD Development

8045 Background: MagnetisMM-3 (NCT04649359) is an open-label, multicenter, non-randomized phase 2 study of elranatamab monotherapy in patients (pts) with multiple myeloma refractory to at least 1 proteasome inhibitor, 1 immunomodulatory drug and 1 anti-CD38 antibody. This analysis examined molecular correlates of elranatamab response and resistance in pts naïve to B-cell maturation antigen (BCMA)-directed therapy (Cohort A). Methods: Bone marrow aspirate (BMA) samples collected at screening were analyzed by whole exome and whole transcriptome sequencing. To investigate the contribution of the tumor microenvironment (TME) in elranatamab response, the abundance of cell types in the BMA samples collected at screening was estimated using single sample gene set enrichment analysis (ssGSEA) of LM22 cell type signatures. Response was defined as a best overall response of very good partial response or better (partial response was considered non-response for this analysis). Results: TNFRSF17 (BCMA encoding gene) expression correlated with markers of disease burden: levels increased with disease stage (with progressively higher levels in R-ISS stages I, II, and III; p= 0.014), were higher in pts with high-risk cytogenetics ( p= 0.002) and correlated with plasma cell content in BMA samples ( ρ= 0.80; p< 10-10). TNFRSF17 expression trended higher in non-responders ( p= 0.08), but this trend was diminished when adjusting for disease burden. These findings suggest TNFRSF17 expression in bulk BM samples is associated with higher disease burden and likely poorer response. According to ssGSEA of LM22 cell types, plasma cells in BMA were associated with non-responders ( p= 0.03). Further multivariable modeling (controlling for plasma cell content) revealed additional cell types associated with response, including macrophages and monocytes, which were associated with poor outcome. Pts with both low plasma and low myeloid cells were most likely to respond. Genome wide copy number analysis showed that TNFRSF17 locus amplification was associated with non-response ( p= 0.008). Chromosomal alterations associated with non-response were identified, including genomic loci known to define high-risk MM (eg, 1q21+) and loci not known to be associated with high-risk MM (eg, 17q21+ and 6p21+). Conclusions: Genomic analysis of BMA samples from MagnetisMM-3 identified an association between higher TNFRSF17 expression in the TME and unfavorable outcomes, likely due to its surrogacy with increased tumor burden. Features of high-risk disease were also associated with lack of response to elranatamab. Adjusting for tumor cell content revealed additional aspects of the TME associated with poor response, including increased myeloid cell populations. Lastly, alterations in specific genomic loci were also associated with response, consistent with tumor intrinsic features influencing elranatamab response. Clinical trial information: NCT04649359 .

Biomarkers developed from DNA sequencing have improved the accuracy of selecting treatment regimens for oncology patients, such as tumor mutational burden to predict immune checkpoint inhibitor (ICI) response in metastatic non-small cell lung cancer and melanoma. However, mounting evidence demonstrates the need for additional biomarkers to identify patients that could benefit from ICIs. Paired DNA and RNA sequencing have the potential to improve patient diagnosis and treatment selection by providing a more comprehensive view of the tumor biology than DNA sequencing alone. Most next-generation sequencing (NGS) diagnostics have primarily been DNA-based assays with limited, if any, scope in biomarkers derived from RNA. Here, tumor RNA profiling for gene expression and gene fusion detection are evaluated with two Illumina RNA-seq applications: RNA exome enrichment and whole transcriptome sequencing (WTS). RNA exome enriched libraries were prepared with Illumina® TruSeq™ RNA Exome or a modified version of the Illumina TruSight™ Oncology RNA library preparation. WTS libraries were prepared with Illumina TruSeq Stranded Total RNA, a workflow that enables sequencing coding and non-coding transcripts. RNA of variable quality derived from FFPE and fresh frozen tumor tissue, and commercial RNA controls were titrated to determine optimal input quantity. Stranded and non-stranded RNA library preparation workflows were evaluated for differences in gene expression. Gene expression and fusion calling performance were evaluated for each RNA-seq application. A comparison of RNA exome to WTS demonstrated robust performance for tumor RNA profiling. Optimal RNA input was 40ng for RNA Exome and 100ng for WTS, regardless of input type. Gene expression values for exome-enriched and whole transcriptome libraries were reproducible (r > 0.99 for technical replicates), with minimal differentially expressed genes between coding regions of both RNA-seq workflows (r > 0.83). WTS yielded up to 2-fold more transcripts with the addition of non-coding RNAs that were not captured by the RNA coding exome panel. Commercial RNA controls and FFPE tumor RNAs with validated fusions were used for evaluating fusion calling performance from RNA exome and whole transcriptome libraries. Both workflows yielded adequate library diversity for calling clinically relevant fusions. However, RNA exome enrichment fusion calling sensitivity (84.4%) was impacted when one or both fusion partners were not targeted by the capture panel, a caveat that can be resolved with WTS (92.9% sensitivity). Collectively, these data demonstrate the feasibility of RNA-seq for gene expression and fusion calling applications. These assays can be instrumental in the development of novel RNA biomarkers to supplement DNA-derived biomarkers for improved prediction of response to a variety of treatment regimens. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Michael Salmans, Mahdi Golkaram, Joyee Yao, Shile Zhang, Li Liu, Traci Pawlowski. Illumina RNA-sequencing for biomarker analysis from FFPE and fresh frozen tumor specimens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 175.

Alteration of lipids and the transcription of lipid-related genes in myelodysplastic syndromes via a TP53-related pathway

Next-generation sequencing (NGS) of DNA isolated from nail specimens and run in parallel with tumor NGS accurately and efficiently detects germline predisposition variants in myeloid malignancies