Cytogenetic and pathologic characterization of MYC-rearranged B-cell lymphomas in pediatric and young adult patients.

MYC-rearranged B-cell lymphoma (BCL) in the pediatric/young adult (YA) age group differs substantially in disease composition from adult cohorts. However, data regarding the partner genes, concurrent rearrangements, and ultimate diagnoses in these patients is scarce compared to that in adult cohorts. We aimed to characterize the spectrum of MYC-rearranged (MYC-R) mature, aggressive BCL in the pediatric/YA population. A retrospective study of morphologic, immunophenotypic, and fluorescence in situ hybridization (FISH) results of patients age ≤ 30 years with suspected Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) or high-grade B-cell lymphoma (HGBCL), and a MYC-R by FISH between 2013-2022 was performed. Two-hundred fifty-eight cases (129 (50%) pediatric (< 18 years) and 129 (50%) YA (18-30 years)) were included. Most MYC-R BCL in pediatric (89%) and YA (66%) cases were BL. While double-hit (DH) cytogenetics (MYC with BCL2 and/or BCL6-R, HGBCL-DH) was rare in the pediatric population (2/129, 2%), HGBCL-DH increased with age and was identified in 17/129 (13%) of YA cases. Most HGBCL-DH had MYC and BCL6-R, while BCL2-R were rare in both groups (3/258, 1%). MYC-R without an IG partner was more common in the YA group (14/116 (12%) vs 2/128 (2%), p = 0.001). The pediatric to YA transition is characterized by decreasing frequency in BL and increasing genetic heterogeneity of MYC-R BCL, with emergence of DH-BCL with MYC and BCL6-R. FISH to evaluate for BCL2 and BCL6 rearrangements is likely not warranted in the pediatric population but should continue to be applied in YA BCL.

High-grade B-cell lymphoma with a quadruple-hit genetic profile including concurrent MYC, BCL2, BCL6, and CCND1 gene rearrangements.

Several reports of concurrent MYC, BCL2, BCL6, and CCND1 rearrangements in high-grade B-cell lymphoma (HGBL) have been recently described. Herein, we aimed to delineate the scope of this entity through a review of HGBL with a "quadruple-hit" genetic profile identified at our institution. We performed a retrospective review (2015-2023) at our institution of B-cell lymphoma (BCL) cases that were evaluated with concurrent MYC, BCL2, and BCL6 break-apart and IGH::MYC and IGH::CCND1 dual-color dual-fusion fluorescence in situ hybridization studies. Of 203 cases meeting inclusion criteria, 2 (1%) with a quadruple-hit genetic profile were identified. Case 1 represented a 59-year-old female with widespread lymphadenopathy and a diagnosis of HGBL who exhibited primary refractoriness to dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R) chemotherapy. Case 2 represented a 58-year-old male with mediastinal and abdominal lymphadenopathy and a diagnosis of large BCL who died from disease after 1 cycle of DA-EPOCH-R chemotherapy. Similarly, a literature review of 7 previously reported cases of HGBL with a quadruple-hit profile also demonstrated aggressive disease behavior. Our study adds 2 new cases to the rarely encountered quadruple-hit HGBL, and a brief meta-analysis of the 9 available cases indicates aggressive disease behavior conferred by this constellation of genetic events.

Section E6.1-6.6 of the American College of Medical Genetics and Genomics (ACMG) Technical Laboratory Standards: Cytogenomic studies of acquired chromosomal abnormalities in neoplastic blood, bone marrow, and lymph nodes.

Cytogenomic analyses of acquired clonal chromosomal abnormalities in neoplastic blood, bone marrow, and/or lymph nodes are instrumental in the clinical management of patients with hematologic neoplasms. Cytogenetic analyses assist in the diagnosis of such disorders and can provide important prognostic information. Furthermore, cytogenetic studies can provide crucial information regarding specific genetically defined subtypes of these neoplasms that may have targeted therapies. At time of relapse, cytogenetic analysis can confirm recurrence of the original neoplasm, detect clonal disease evolution, or uncover a new unrelated neoplastic process. This section deals specifically with the technical standards applicable to cytogenomic studies of acquired clonal chromosomal abnormalities in neoplastic blood, bone marrow, and/or lymph nodes. This updated Section E6.1-6.6 supersedes the previous Section E6 in Section E: Clinical Cytogenetics of the American College of Medical Genetics and Genomics Technical Standards for Clinical Genetics Laboratories.

Superior detection rate of plasma cell FISH using FACS-FISH.

OBJECTIVES: Fluorescence in situ hybridization (FISH) for plasma cell neoplasms (PCNs) requires plasma cell (PC) identification or purification strategies to optimize results. We compared the efficacy of cytoplasmic immunoglobulin FISH (cIg-FISH) and fluorescence-activated cell sorting FISH (FACS-FISH) in a clinical laboratory setting. METHODS: The FISH analysis results of 14,855 samples from individuals with a suspected PCN subjected to cytogenetic evaluation between 2019 and 2022 with cIg-FISH (n = 6917) or FACS-FISH (n = 7938) testing were analyzed. RESULTS: Fluorescence-activated cell sorting-FISH increased the detection rate of abnormalities in comparison with cIg-FISH, with abnormal results documented in 54% vs 50% of cases, respectively (P < .001). It improved the detection of IGH::CCND1 (P < .001), IGH::MAF (P < .001), IGH::MAFB (P < .001), other IGH rearrangements (P < .001), and gains/amplifications of 1q (P < .001), whereas the detection rates of IGH::FGFR3 fusions (P = .3), loss of 17p (P = .3), and other abnormalities, including hyperdiploidy (P = .5), were similar. Insufficient PC yield for FISH analysis was decreased between cIg-FISH and FACS-FISH (22% and 3% respectively, P < .001). Flow cytometry allowed establishment of ploidy status in 91% of cases. In addition, FACS-FISH decreased analysis times, workload efforts, and operating costs. CONCLUSIONS: Fluorescence-activated cell sorting-FISH is an efficient PC purification strategy that affords significant improvement in diagnostic yield and decreases workflow requirements in comparison with cIg-FISH.

Detection of an MN1::ETV6 Gene Fusion in a Case of Acute Myeloid Leukemia with Erythroid Differentiation: A Case Report and Review of the Literature.

The MN1::ETV6 gene fusion resulting from t(12;22)(p13;q12) has been rarely reported in myeloid neoplasms. We describe a 69-year-old male with newly diagnosed acute myeloid leukemia (AML) with erythroid differentiation and t(12;22)(p13;q12) demonstrated by conventional chromosome studies. Subsequent fluorescence in situ hybridization studies demonstrated a balanced ETV6 gene rearrangement (at 12p13). To further characterize this translocation, whole-genome sequencing was performed which confirmed t(12;22) with breakpoints involving the MN1 and ETV6 genes. Herein, we describe our case and review the literature to summarize the clinical and laboratory findings in patients with this rare but recurrent MN1::ETV6 gene fusion observed in myeloid neoplasms. Importantly, this case expands the clinical spectrum associated with the MN1::ETV6 gene fusion to include AML with erythroid differentiation. Lastly, this case demonstrates the importance of moving toward more comprehensive molecular testing to fully characterize the driver events in neoplastic genomes.

Impact of cytogenetic abnormalities on the risk of disease progression in solitary bone plasmacytomas.

Most patients with solitary bone plasmacytomas (SBP) progress to multiple myeloma (MM) after definitive radiation therapy as their primary treatment. Whether the presence of high-risk (HR) cytogenetic abnormalities by fluorescence in situ hybridization (FISH) in the clonal plasma cells, obtained either directly from the diagnostic SBP tissue or the corresponding bone marrow examination at the time of diagnosis, is associated with a shorter time to progression (TTP) to MM is unknown. This study evaluated all patients diagnosed with SBP at the Mayo Clinic from January 2012 to July 2022. The presence of del(17p), t(14;16), t(4;14), or +1q (gain or amplification) by FISH in clonal plasma cells was defined as HR. A total of 114 patients were included in this cohort, and baseline FISH was available for 55 patients (48%), of which 22 were classified as HR (40%). The median TTP to MM for patients with SBP and HR FISH was 8 months (95% confidence interval [CI], 6.3-26) compared with 42 months (95% CI, 25-not reached [NR]) in patients with SBP without HR FISH (P < .001). In a multivariate analysis, only HR FISH was a significant predictor for shorter TTP to MM, independent of minimal marrow involvement and an abnormal serum free light chain ratio at diagnosis. Deletion (17p) and gain 1q abnormalities were the most common FISH abnormalities responsible for the short TTP to MM. Thus, assessing for HR FISH abnormalities in clonal plasma cells derived from either the diagnostic SBP tissue or the staging bone marrow examination of patients with newly diagnosed SBP is feasible and prognostic for a shorter TTP to MM.

Conditional survival in multiple myeloma and impact of prognostic factors over time.

Overall survival estimates from diagnosis are valuable for guiding treatment, but do not consider the years already survived. Conditional survival (CS) provides dynamic survival predictions over time. This study was conducted to estimate CS at 1-8 years from diagnosis and the impact of baseline prognostic factors on CS in multiple myeloma (MM) patients. This is a retrospective study including 2556 MM patients diagnosed between 2004 and 2019. CS (t | s) was defined as the probability of surviving t years given survival of s years. Median age was 64 years. Median follow-up was 6.2 years and median overall survival from diagnosis was 7.5 years. The 5-year CS estimates at s = 0, 1, 2, 3, and 5 years were 0.64, 0.61, 0.61, 0.61, and 0.58, respectively. On multivariate analysis, age ≥ 65 and proteasome inhibitor+immunomodulatory-based induction were associated with decreased survival and increased survival, respectively, retained at 5 years. The adverse impact of 1q gain/amplification, high-risk IgH translocation, and ISS-3 was significant at 1 and 3 years but not 5 years. Chromosome 17 abnormality was associated with decreased survival only at 1 year. Among MM patients, 5-year CS was stable at 1-5 years from diagnosis. The prognostic impact of high-risk cytogenetic factors decreased with additional years survived.

Mass Cytometry reveals unique phenotypic patterns associated with subclonal diversity and outcomes in multiple myeloma.

Multiple myeloma (MM) remains an incurable plasma cell (PC) malignancy. Although it is known that MM tumor cells display extensive intratumoral genetic heterogeneity, an integrated map of the tumor proteomic landscape has not been comprehensively evaluated. We evaluated 49 primary tumor samples from newly diagnosed or relapsed/refractory MM patients by mass cytometry (CyTOF) using 34 antibody targets to characterize the integrated landscape of single-cell cell surface and intracellular signaling proteins. We identified 13 phenotypic meta-clusters across all samples. The abundance of each phenotypic meta-cluster was compared to patient age, sex, treatment response, tumor genetic abnormalities and overall survival. Relative abundance of several of these phenotypic meta-clusters were associated with disease subtypes and clinical behavior. Increased abundance of phenotypic meta-cluster 1, characterized by elevated CD45 and reduced BCL-2 expression, was significantly associated with a favorable treatment response and improved overall survival independent of tumor genetic abnormalities or patient demographic variables. We validated this association using an unrelated gene expression dataset. This study represents the first, large-scale, single-cell protein atlas of primary MM tumors and demonstrates that subclonal protein profiling may be an important determinant of clinical behavior and outcome.

Mate Pair Sequencing: Next-Generation Sequencing for Structural Variant Detection.

Structural variant detection by next-generation sequencing (NGS) methods have a higher molecular resolution than conventional cytogenetic techniques (Aypar et al., Eur J Haematol 102(1):87-96, 2019; Smadbeck et al., Blood Cancer J 9(12):103, 2019) and are particularly helpful in characterizing genomic rearrangements. Mate pair sequencing (MPseq) leverages a unique library preparation chemistry involving circularization of long DNA fragments, allowing for a unique application of paired-end sequencing of reads that are expected to map 2-5 kb apart in the genome. The unique orientation of the reads allows the user to estimate the location of breakpoints involved in a structural variant either within the sequenced reads or between the two reads. The precision of structural variant and copy number detection by this method allows for characterization of cryptic and complex rearrangements that may be otherwise undetectable by conventional cytogenetic methods (Singh et al., Leuk Lymphoma 60(5):1304-1307, 2019; Peterson et al., Blood Adv 3(8):1298-1302, 2019; Schultz et al., Leuk Lymphoma 61(4):975-978, 2020; Peterson et al., Mol Case Studies 5(2), 2019; Peterson et al., Mol Case Studies 5(3), 2019).

A TRIP11:: FLT3 gene fusion in a patient with myeloid/lymphoid neoplasm with eosinophilia and tyrosine kinase gene fusions: a case report and review of the literature.

Myeloid/lymphoid neoplasms with FLT3 gene fusions have recently been included among myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (MLN-TK) in the World Health Organization classification and International Consensus Classification. As this entity remains remarkably rare, its scope and phenotypic features are evolving. In this report, we describe a 33-yr-old male with MLN-TK. Conventional chromosome analysis revealed a t(13;14)(q12;q32). Further analysis with mate-pair sequencing (MPseq) confirmed a TRIP11::FLT3 gene fusion. A diagnosis of MLN-TK was rendered. To the best of our knowledge, we report the third case of MLN-TK with a TRIP11::FLT3 gene fusion. In contrast to previously described cases, our case exhibited distinctly mild clinical features and disease behavior, emphasizing the diverse spectrum of MLN-TK at primary presentation and variability in disease course. MLN-TK with FLT3 gene fusions are a genetically defined entity which may be targetable with tyrosine kinase inhibitors with anti-FLT3 activity. Accordingly, from diagnostic and therapeutic viewpoints, genetic testing for FLT3 rearrangements using fluorescence in situ hybridization (FISH) or sequencing-based assays should be pursued for patients with chronic eosinophilia.

Optical genome mapping in acute myeloid leukemia: a multicenter evaluation.

Detection of hallmark genomic aberrations in acute myeloid leukemia (AML) is essential for diagnostic subtyping, prognosis, and patient management. However, cytogenetic/cytogenomic techniques used to identify those aberrations, such as karyotyping, fluorescence in situ hybridization (FISH), or chromosomal microarray analysis (CMA), are limited by the need for skilled personnel as well as significant time, cost, and labor. Optical genome mapping (OGM) provides a single, cost-effective assay with a significantly higher resolution than karyotyping and with a comprehensive genome-wide analysis comparable with CMA and the added unique ability to detect balanced structural variants (SVs). Here, we report in a real-world setting the performance of OGM in a cohort of 100 AML cases that were previously characterized by karyotype alone or karyotype and FISH or CMA. OGM identified all clinically relevant SVs and copy number variants (CNVs) reported by these standard cytogenetic methods when representative clones were present in >5% allelic fraction. Importantly, OGM identified clinically relevant information in 13% of cases that had been missed by the routine methods. Three cases reported with normal karyotypes were shown to have cryptic translocations involving gene fusions. In 4% of cases, OGM findings would have altered recommended clinical management, and in an additional 8% of cases, OGM would have rendered the cases potentially eligible for clinical trials. The results from this multi-institutional study indicate that OGM effectively recovers clinically relevant SVs and CNVs found by standard-of-care methods and reveals additional SVs that are not reported. Furthermore, OGM minimizes the need for labor-intensive multiple cytogenetic tests while concomitantly maximizing diagnostic detection through a standardized workflow.

Characterization of unusual iAMP21 B-lymphoblastic leukemia (iAMP21-ALL) from the Mayo Clinic and Children's Oncology Group.

Acute lymphoblastic leukemia (B-ALL) with intrachromosomal amplification of chromosome 21 (iAMP21-ALL) represents a recurrent high-risk cytogenetic abnormality and accurate identification is critical for appropriate clinical management. Identification of iAMP21-ALL has historically relied on fluorescence in situ hybridization (FISH) using a RUNX1 probe. Current classification requires ≥ five copies of RUNX1 per cell and ≥ three additional copies of RUNX1 on a single abnormal iAMP21-chromosome. We sought to evaluate the performance of the RUNX1 probe in the identification of iAMP21-ALL. This study was a retrospective evaluation of iAMP21-ALL in the Mayo Clinic and Children's Oncology Group cohorts. Of 207 cases of iAMP21-ALL, 188 (91%) were classified as "typical" iAMP21-ALL, while 19 (9%) cases were classified as "unusual" iAMP21-ALL. The "unusual" iAMP21 cases did not meet the current definition of iAMP21 by FISH but were confirmed to have iAMP21 by chromosomal microarray. Half of the "unusual" iAMP21-ALL cases had less than five RUNX1 signals, while the remainder had ≥ five RUNX1 signals with some located apart from the abnormal iAMP21-chromosome. Nine percent of iAMP21-ALL cases fail to meet the FISH definition of iAMP21-ALL demonstrating that laboratories are at risk of misidentification of iAMP21-ALL when relying only on the RUNX1 FISH probe. Incorporation of chromosomal microarray testing circumvents these risks.

Single-Cell Proteomics and Tumor RNAseq Identify Novel Pathways Associated With Clofazimine Sensitivity in PI- and IMiD- Resistant Myeloma, and Putative Stem-Like Cells.

Multiple myeloma (MM) is an incurable plasma cell malignancy with dose-limiting toxicities and inter-individual variation in response/resistance to the standard-of-care/primary drugs, proteasome inhibitors (PIs), and immunomodulatory derivatives (IMiDs). Although newer therapeutic options are potentially highly efficacious, their costs outweigh the effectiveness. Previously, we have established that clofazimine (CLF) activates peroxisome proliferator-activated receptor-γ, synergizes with primary therapies, and targets cancer stem-like cells (CSCs) in drug-resistant chronic myeloid leukemia (CML) patients. In this study, we used a panel of human myeloma cell lines as in vitro model systems representing drug-sensitive, innate/refractory, and clonally-derived acquired/relapsed PI- and cereblon (CRBN)-negative IMiD-resistant myeloma and bone marrow-derived CD138+ primary myeloma cells obtained from patients as ex vivo models to demonstrate that CLF shows significant cytotoxicity against drug-resistant myeloma as single-agent and in combination with PIs and IMiDs. Next, using genome-wide transcriptome analysis (RNA-sequencing), single-cell proteomics (CyTOF; Cytometry by time-of-flight), and ingenuity pathway analysis (IPA), we identified novel pathways associated with CLF efficacy, including induction of ER stress, autophagy, mitochondrial dysfunction, oxidative phosphorylation, enhancement of downstream cascade of p65-NFkB-IRF4-Myc downregulation, and ROS-dependent apoptotic cell death in myeloma. Further, we also showed that CLF is effective in killing rare refractory subclones like side populations that have been referred to as myeloma stem-like cells. Since CLF is an FDA-approved drug and also on WHO's list of safe and effective essential medicines, it has strong potential to be rapidly re-purposed as a safe and cost-effective anti-myeloma drug.

Typical, atypical and cryptic t(15;17)(q24;q21) (PML::RARA) observed in acute promyelocytic leukemia: A retrospective review of 831 patients with concurrent chromosome and PML::RARA dual-color dual-fusion FISH studies.

The diagnosis of acute promyelocytic leukemia (APL) relies on the identification of PML::RARA fusion. While the majority of APL cases harbor a typical t(15;17)(q24;q21), atypical genetic mechanisms leading to the oncogenic PML::RARA fusion have been reported yet their frequency and scope remain poorly characterized. We assessed the genetic findings of 831 cases with APL investigated with concurrent chromosome banding analysis and dual-color dual-fusion fluorescence in situ hybridization (D-FISH) analysis at our institution over an 18.5-year timeframe. Seven hundred twenty-three (87%) cases had a typical balanced t(15;17) with both testing modalities. Atypical karyotypic results including complex translocations, unbalanced rearrangements and insertional events occurred in 50 (6%) cases, while 6 (0.7%) cases were cryptic by conventional chromosome studies despite PML::RARA fusion by D-FISH evaluation. Atypical FISH patterns were observed in 48 (6%) cases despite apparently balanced t(15;17) on chromosome banding analysis. Two hundred fifty (30%) cases displayed additional chromosome abnormalities of which trisomy/tetrasomy 8 (37%), del(7q)/add(7q) (12%), and del(9q) (7%) were most frequent. Complex and very complex karyotypes were observed in 81 (10%) and 34 (4%) cases, respectively. In addition, 4 (0.5%) cases presented as an apparently doubled, near-tetraploid stemline clone. This report provides the largest appraisal of cytogenetic findings in APL with conventional chromosome and PML::RARA D-FISH analysis. By characterizing the frequency and breadth of typical and atypical results through the lens of these cytogenetic testing modalities, this study serves as a pragmatic source of information for those involved in the investigation of APL in both the clinical and research laboratory settings.

Identification of EWSR1 rearrangements in patients with immature hematopoietic neoplasms: A case series and review of literature.

Rearrangement of the EWSR1 gene (22q12.2) is a well-recognized genetic lesion in bone and soft tissue tumors. However, few reports have suggested that EWSR1 rearrangements may also occur in the setting of hematopoietic tumors. We herein describe two cases of immature hematopoietic neoplasms presenting with EWSR1 rearrangements. The first occurred in a 41-year-old female diagnosed with mixed-phenotype acute leukemia, B/T/myeloid, in which conventional chromosome analysis revealed a t(2;22)(q35;q12). Further analysis with whole genome sequencing revealed that this rearrangement led to an EWSR1::FEV gene fusion. The second case was identified in an 18-year-old male with a high-grade B-cell lineage malignant neoplasm with immature features in which conventional chromosome analysis revealed a t(17;22)(q25;q12). Mate-pair sequencing, a next generation sequencing-based assay, was performed and revealed three in-frame chimeric gene fusions involving the EWSR1, TEF and STRADA gene regions. This report further expands the repertoire of hematopoietic neoplasms with EWSR1 fusions and partner genes involved in these rearrangements.