Citations

Purpose: B-cell acute lymphoblastic leukemia/lymphoma (B-ALL/LBL) represents the most common childhood malignancy. Classification of recurrent genetic abnormalities in B-ALL/LBL is essential for risk stratification and patient management. Observed in approximately 5% of B-ALL/LBL cases, hypodiploidy is considered a poor prognostic finding and can be further characterized as near-haploidy (24-31 chromosomes), low hypodiploidy (32-39 chromosomes) and high hypodiploidy (40-43 chromosomes). Importantly, masked hypodiploid clones that have undergone endoreduplication can be mischaracterized as hyperdiploidy, a favorable prognostic finding in B-ALL/LBL. These "doubled" hypodiploid clones are termed pseudo-hyperdiploid and their accurate detection is critical. Mate-pair sequencing (MPseq) is a next-generation sequencing (NGS) technology designed to sequence larger DNA fragments (2000-5000bp) than paired-end sequencing. This allows for the detection of large genetic rearrangements (e.g. translocations, inversions, etc.) and copy number variants (CNVs) at a high resolution with low base coverage. It has demonstrated particular clinical utility for genetic characterization of hematologic malignancies. To expand the role of MPseq in the evaluation of hematologic neoplasms, we aimed to improve the analysis of MPseq results to allow for the detection of copy-neutral loss of heterozygosity (cnLOH) that would enable the differentiation between hyperdiploid and pseudo-hyperdiploid clones. Methods: A new algorithm was applied that statistically analyzes common SNP locations in MPseq data in order to detect both cnLOH regions, and provides global heterozygosity levels at a resolution that allows for the detection of hypodiploidy. This method accounts for the low base-coverage of MPseq data (<10x) in order to provide the highest possible accuracy and precision without increasing cost by requiring changes to the input or sequencing requirements. The algorithm was applied to 17 B-ALL samples classified by conventional chromosome analysis, FISH, and/or chromosomal microarray as either pseudo-hyperdiploidy (n=8) or hyperdiploidy (n=9). The cases were sequenced using MPseq with 101bp read lengths across a range of base coverages (1-10x; 30-135 million fragments) and tumor percentages (50-100%). Results: We demonstrate the method's ability to differentiate between hyperdiploidy and pseudo-hyperdiploidy in all cases without any changes to the MPseq input or sequencing requirements. We detected an average of 11.5 whole-chromosome cnLOH (range 8-20) in the pseudo-hyperdiploidy cases, compared to an average of 0.67 whole-chromosome cnLOH (range 0-2) in the hyperdiploidy cases. Additionally, the method allows for the detection of cnLOH regions across the genome. For example, we observed a recurrent cnLOH of chromosome 9 in the hyperdiploid subtype (3/9 hyperdiploid cases), which is a recurrent abnormality in the disease. The method provides an easy to read visual output that compares favorably to the output of chromosomal microarray, increasing its utility for clinical application. Conclusions: This advancement in the analysis of low-coverage sequencing data makes MPseq available for the genetic characterization of B-ALL/LBL, where hypodiploidy is a primary cytogenetic abnormality and is sometimes missed by conventional cytogenetic techniques. Additionally, the algorithm allows for the detection of cnLOH regions across the genome, which is important across many disease types for diagnosis, prognosis, and the detection of potential therapeutic targets. The addition of the algorithm to the MPseq analysis pipeline makes it a more complete genetic characterization tool and brings it closer to its promise as a potential single assay replacement for conventional cytogenetic techniques. Disclosures No relevant conflicts of interest to declare.

AimsThe aims of this study were to review our 5‐year experience with clinical FISH testing for TP63 rearrangements using both TP63 break‐apart (BAP) and TBL1XR1/TP63 dual‐fusion (D‐FISH) probes to evaluate the frequency of TP63 rearrangements and the distribution of TBL1XR1 vs. alternate partner loci, and to assess whether both probe sets are necessary in all cases undergoing FISH testing.Methods and resultsA retrospective review of the Mayo Clinic cytogenetic database identified 470 patients evaluated by FISH testing for TP63 rearrangements in formalin‐fixed paraffin‐embedded (FFPE) tissue using both BAP and D‐FISH probes. Of these, 25 (5.3%) had TP63 rearrangements. All samples were being investigated for anaplastic large‐cell lymphoma or other T cell lymphoma subtypes. A TBL1XR1 partner was identified by D‐FISH in 12 (48%) of 25 cases. All cases positive by TBL1XR1/TP63 D‐FISH were also positive by TP63 BAP FISH.ConclusionThis is the largest series of TP63 rearrangements to date. The frequency of positive results among cases referred to a large reference laboratory for TP63 FISH testing was 5.3%. Approximately half of TP63 rearrangements have a TBL1XR1 partner. TP63 BAP FISH testing is sufficient for up‐front testing of FFPE tissue samples. However, because of the genomic proximity of the TP63 and TBL1XR1 loci, we recommend reflex TBL1XR1/TP63 D‐FISH testing in positive and equivocal cases.

Acute myeloid leukemia (AML) patients with t(8;16)(p11.2;p13) constitute a small subgroup with a distinct genetic and clinical profile. We present a unique case of a female infant with monocytic AML associated with t(8;19)(p11.2;q13.3), a rarely reported variation of t(8;16)(p11.2;p13). The patient presented with leukemia cutis and demonstrated erythrophagocytosis in the diagnostic bone marrow. She responded well to standard AML chemotherapy and is currently in remission. Here, we highlight her case as the youngest AML patient with t(8;19) described in the literature, discuss the significance and prognostic implications of this genetic variant, and review 8p11.2 fusion proteins in AML.

Key Points This article characterizes a cryptic IGH/CCND1 rearrangement in MCL by NGS. Mate-pair sequencing can help in accurately diagnosing MCL in cases of cyclin-D1–positive B-cell lymphoma with negative CCND1 FISH studies.

AbstractThe detection of recurrent genetic abnormalities in B‐lymphoblastic leukemia (B‐ALL) is critical for risk stratification and therapy‐related decisions. Near‐haploidy (24‐30 chromosomes), a subgroup of hypodiploidy (<46 chromosomes), and BCR/ABL1 gene fusions are both recurrent genetic abnormalities in B‐ALL and are considered adverse prognostic findings, although outcomes in BCR/ABL1‐positive patients have improved with tyrosine kinase inhibitor therapy. While near‐haploid clones are primarily observed in children and rarely harbor structural abnormalities, BCR/ABL1‐positive B‐ALL is primarily observed in adults. Importantly, recurrent genetic abnormalities are considered mutually exclusive and rarely exist within the same neoplastic clone. We report only the second case to our knowledge of a near‐haploid clone that harbors a BCR/ABL1 fusion in an adult with newly diagnosed B‐ALL. Conventional chromosome studies revealed a near‐haploid clone (27 chromosomes) along with a der(22)t(9;22)(q34.1;q11.2) in 17 of 20 metaphases analyzed. Our B‐ALL fluorescence in situ hybridization (FISH) panel confirmed the BCR/ABL1 fusion and monosomies consistent with chromosome studies in approximately 95% of interphase nuclei. Moreover, no evidence of a “doubled” near‐haploid clone was observed by chromosome or FISH studies. This highly unusual case illustrates that while rare, recurrent genetic abnormalities in B‐ALL can exist within the same neoplastic clone.

ABSTRACTThe MLLT10 (formerly AF10) gene is the fourth most common KMT2A fusion partner across all acute leukemias and requires at least 3 breaks to form an in‐frame KMT2A/MLLT10 fusion due to the opposite orientation of each gene. A 10‐year retrospective review was performed to identify individuals from all age groups that harbor KMT2A/MLLT10 fusion obtained by our KMT2A/MLLT10 dual‐color dual‐fusion fluorescence in situ hybridization (D‐FISH) assay. Of the 60 unique individuals identified, 31 were male and 29 were female (M:F ratio, 1.1:1) with ages ranging from 3 days to 86 years (mean 21.5 years, median 5.5 years). The diagnoses included acute myeloid leukemia (AML) (49 patients, 82%), B‐ or T‐lymphoblastic leukemia/lymphoma (7 patients, 12%), myeloid sarcoma (3 patients, 5%), and a single case (2%) of undifferentiated leukemia. Twenty‐seven of 49 patients (55%) with AML were in the infant or pediatric age group. Fifty‐three of 60 patients (88%) had KMT2A/MLLT10 D‐FISH signal patterns mostly consisting of single fusions. In addition, 10 (26%) of 38 patients with conventional chromosome studies had “normal” (5 patients) or abnormal (5 patients) chromosome studies that lacked structural or numeric abnormalities involving chromosomes 10 or 11, implying cryptic cytogenetic mechanisms for KMT2A/MLLT10 fusion. Lastly, mate‐pair sequencing was performed on 4 AML cases, 2 of which had “normal” chromosome studies and cryptic KMT2A/MLLT10 fusion as detected by KMT2A/MLLT10 D‐FISH studies, and verified the multiple breaks required to generate KMT2A/MLLT10 fusion.

ABSTRACT The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell‐released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV‐associated functional activities. Finally, a checklist is provided with summaries of key points.

AbstractObjectiveAcute myeloid leukemia (AML) can be subtyped based on recurrent cytogenetic and molecular genetic abnormalities with diagnostic and prognostic significance. Although cytogenetic characterization classically involves conventional chromosome and/or fluorescence in situ hybridization (FISH) assays, limitations of these techniques include poor resolution and the inability to precisely identify breakpoints.MethodWe evaluated whether an NGS‐based methodology that detects structural abnormalities and copy number changes using mate pair sequencing (MPseq) can enhance the diagnostic yield for patients with AML.ResultsUsing 68 known abnormal and 20 karyotypically normal AML samples, each recurrent primary AML‐specific abnormality previously identified in the abnormal samples was confirmed using MPseq. Importantly, in eight cases with abnormalities that could not be resolved by conventional cytogenetic studies, MPseq was utilized to molecularly define eight recurrent AML‐fusion events. In addition, MPseq uncovered two cryptic abnormalities that were missed by conventional cytogenetic studies. Thus, MPseq improved the diagnostic yield in the detection of AML‐specific structural rearrangements in 10/88 (11%) of cases analyzed.ConclusionUtilization of MPseq represents a precise, molecular‐based technique that can be used as an alternative to conventional cytogenetic studies for newly diagnosed AML patients with the potential to revolutionize the diagnosis of hematologic malignancies.