Citations

Abstract CALR mutations are present in 70-84% of JAK2 wild-type myeloproliferative neoplasms (MPN) and 67% and 88% of essential thrombocytopenia (ET) and primary myelofibrosis (PMF) respectively. Most cases of MPN are apparently sporadic, but 7-11% have evidence of familial predisposition. While germline mutations in ET-associated genes, MPL and JAK2, have been described in hereditary thrombocytosis, germline mutations in CALR have not been described in any setting. Two types of CALR mutations are common in MPN: a 52-base pair deletion (bp) and a 5 bp insertion, both in exon 9. With rare exceptions, CALR mutations are generally mutually exclusive with JAK2 or MPL mutations and have very rarely been reported in conjunction with the BCR-ABL1translocation. Here, we report a patient with a germline CALR mutation, thrombocytosis, and subsequent development of BCR-ABL+ CML. A 67-year-old female with no significant medical history presented with severe abdominal pain and nausea. Peripheral blood analysis revealed a marked leukocytosis composed of 66% neutrophils, 16% myelocytes, 6.5% monocytes, 3.5% basophils, 2.5% promyelocytes, 2.5% metamyelocytes, 1.5% lymphocytes, 1.5% blasts, and no eosinophils. The patient was non-anemic and had a normal platelet count (340,000/mm3). Bone marrow biopsy revealed a hypercellular marrow with myeloid predominant trilineage hematopoiesis and 1-2% blasts with morphology consistent with chronic myelogenous leukemia (CML). Fluorescence in-situ hybridization analysis of peripheral blood identified a BCR-ABL1fusion in 98.5% of interphase cells. After 3 months of standard imatinib therapy, quantitative RT-PCR showed a reduction of BCR-ABL1/ABL1 in the peripheral blood, however platelet count was elevated at 539,000/mm3. Thrombocytosis persisted over 2 years with a maximal platelet count of 584,000/mm3. Given the patient's thrombocytosis, her peripheral blood was subjected to a next generation sequencing of JAK2, MPL, and CALR genes. A 52-bp out-of-frame deletion in exon 9 of the CALR gene was detected (52% allele frequency) in peripheral blood. In addition, the same 52-bp CALR deletion (63% allele frequency) was present at the time of diagnosis and within a buccal specimen (47% allele frequency) when the BCR-ABL1 transcript was 1% in the peripheral blood. Immunostain of the buccal sample was strongly positive for cytokeratin (CK) AE1/AE3 but CD45 was not detected indicating no leukocyte contamination. This case reports the first instance of a germline CALR mutation associated with thrombocytosis and is the fourth report of the co-occurrence of BCR-ABL1 and CALR mutation in a single patient. Evolution to BCR-ABL1+ CML suppressed the CALR-mutant thrombocytosis phenotype, emphasizing the effect of these genes on lineage determination in abnormal myeloid proliferation. 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.

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.

AbstractTrisomies of odd numbered chromosomes are seen in nearly half of patients with multiple myeloma (MM) and typically correlate with a hyperdiploid state and better overall survival (OS). We compared DNA ploidy of monoclonal plasma cells (as a surrogate for the presence of trisomies) assessed simultaneously by PCPRO (plasma cell proliferative index), a novel method that estimates DNA index by multi‐parametric flow cytometry to fluorescence in situ hybridization (FISH) in 1703 patients with plasma cell disorders. The distribution of ploidy was hyperdiploid: 759 (45%), diploid 765 (45%), hypodiploid: 71 (4%), tetraploid/near‐tetraploid: 108 (6%). FISH identified trisomies in 82% (621/756) of patients with hyperdiploidy by PCPRO and no trisomy by FISH was observed in 88% (730/834) of patients without hyperdiploidy. 95% (795/834) of patients without hyperdiploidy on PCPRO had one or less trisomy by FISH. Sensitivity and specificity of PCPRO for detecting hyperdiploidy was 86% (621/725) and 84% (730/865), respectively. Sensitivity increased to 94% (579/618) for patients with more than one trisomy. Newly diagnosed MM patients with hyperdiploidy on PCPRO (147/275) had better OS compared to nonhyperdiploid patients (median not reached vs 59 months, P = 0.008) and better progression free survival (median: 33 vs 23 months, P = 0.03). Within the hyperdiploidy group, patients with high‐hyperdiploidy (DNA index: 1.19‐1.50) versus those with low‐hyperdiploidy (DNA index: 1.05‐1.18) had superior OS (3 year OS of 88% vs 68% P = 0.03). Ploidy assessment by flow cytometry can provide rapid, valuable prognostic information and also reduces the number of copy number FISH probes required and hence the cost of FISH.

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.