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.

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.

Comparative study of therapy-related and de novo adult b-cell acute lymphoblastic leukaemia.

We report a comparative analysis of patients with therapy-related acute lymphoblastic leukaemia (tr-ALL) vs de novo ALL. We identified 331 patients with B-ALL; 69 (21%) were classified as tr-ALL. The most common prior malignancies were breast (23·2%) and plasma cell disorders (20·3%). Patients with tr-ALL were older (median 63·2 vs. 46·2 years, P < 0.001), more often female (66·7% vs. 43·5%, P < 0·001), and more likely to have hypodiploid cytogenetics (18·8% vs. 5·0%, P < 0·001). In multivariable analysis, patients with tr-ALL were less likely to achieve complete remission [odds ratio (OR) = 0·16, P < 0·001] and more likely to be minimal residual disease-positive (OR = 4·86, P = 0·01) but had similar OS after diagnosis and allo-haematopoietic cell transplantation.

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.

Increased complexity of t(11;14) rearrangements in plasma cell neoplasms compared with mantle cell lymphoma.

Plasma cell neoplasms (PCN) and mantle cell lymphoma (MCL) can both harbor t(11;14)(q13;q32) (CCND1/IGH), usually resulting in cyclin D1 overexpression. In some cases, particularly at low levels of disease, it can be morphologically challenging to distinguish between these entities in the bone marrow (BM) since PCN with t(11;14) are often CD20-positive with lymphoplasmacytic cytology, while MCL can rarely have plasmacytic differentiation. We compared the difference in CCND1/IGH by fluorescence in situ hybridization (FISH) in PCN and MCL to evaluate for possible differentiating characteristics. We identified 326 cases of MCL with t(11;14) and 279 cases of PCN with t(11;14) from either formalin-fixed, paraffin-embedded tissue or fresh BM specimens. The "typical," balanced CCND1/IGH FISH signal pattern was defined as three total CCND1 signals, three total IGH signals, and two total fusion signals. Any deviation from the "typical" pattern was defined as an "atypical" pattern, which was further stratified into "gain of fusion" vs "complex" patterns. There was a significantly higher proportion of cases that showed an atypical FISH pattern in PCN compared with MCL (53% vs 27%, P < .0001). There was also a significantly higher proportion of cases that showed a complex FISH pattern in PCN compared with MCL (47% vs 17%, P < .0001). We confirmed these findings using mate-pair sequencing of 25 PCN and MCL samples. PCN more often have a complex CCND1/IGH FISH pattern compared with MCL, suggesting possible differences in the genomic mechanisms underlying these rearrangements in plasma cells compared with B cells.

Identification of a novel KMT2A/GIMAP8 gene fusion in a pediatric patient with acute undifferentiated leukemia.

Acute undifferentiated leukemia (AUL) is a very rare hematologic neoplasm that expresses no markers specific for either myeloid or lymphoid lineages. While commonly observed in several acute leukemias, KMT2A rearrangements in AUL have been rarely reported in the literature. We report the third case to our knowledge of AUL harboring a KMT2A rearrangement. Furthermore, the KMT2A/GIMAP8 gene fusion identified in this case represents a novel KMT2A rearrangement.

Clinical utility of next generation sequencing to detect IGH/IL3 rearrangements [t(5;14)(q31.1;q32.1)] in B-lymphoblastic leukemia/lymphoma.

The t(5;14)(q31.1;q32.1) associated with B-lymphoblastic leukemia/lymphoma (B-ALL/LBL) is a rare, recurrent genetic abnormality recognized as a distinct entity by the 2017 World Health Organization (WHO) classification. In these cases, the IGH enhancer region (14q32.1) is juxtaposed to the vicinity of the IL3 gene (5q31.1), resulting in increased production of interleukin-3 (IL3) and subsequently a characteristic reactive eosinophilia. B-ALL with t(5;14)(q31.1;q32.1) may have a low lymphoblast count that can complicate detection of t(5;14)(q31.1;q32.1) by conventional chromosome studies. We have identified four patients with IGH/IL3 rearrangements despite normal conventional chromosome studies in each case [one patient had a non-clonal t(5;14)(q31;q32) finding]. Fluorescence in situ hybridization utilizing a laboratory-developed IGH break-apart probe set identified IGH rearrangements in three of four cases, and a next generation sequencing (NGS) based assay, mate-pair sequencing (MPseq), was required to characterize the IGH/IL3 rearrangements in each case. Three patients demonstrated a balanced t(5;14)(q31.1;q32.1) while one patient had a cryptic insertion of the IL3 gene into the IGH region. These results demonstrate that NGS-based assays, such as MPseq, confer an advantage in the detection of IGH/IL3 rearrangements that are otherwise challenging to characterize by traditional cytogenetic methodologies.

Cryptic and atypical KMT2A-USP2 and KMT2A-USP8 rearrangements identified by mate pair sequencing in infant and childhood leukemia.

Infant leukemias are a rare group of neoplasms that are clinically and biologically distinct from their pediatric and adult counterparts. Unlike leukemia in older children where survival rates are generally favorable, infants with leukemia have a 5-year event-free survival rate of <50%. The majority of infant leukemias are characterized by KMT2A (MLL) rearrangements (~70 to 80% in acute lymphoblastic leukemia), which appear to be drivers of early leukemogenesis. In this report, we describe three cases: a 9-month-old female infant with B-acute lymphoblastic leukemia (B-ALL), an 8-month-old female presenting with B/myeloid mixed phenotype acute leukemia (MPAL), and a 16-month-old male with B-ALL. The first case had a normal karyotype and B-ALL FISH results consistent with an atypical KMT2A rearrangement. The second case had trisomy 10 as the sole chromosomal abnormality and a normal KMT2A FISH result. Case 3 had trisomy 8 and a t(11;15)(q23;q21), an atypical KMT2A rearrangement by FISH studies, and a focal deletion of 15q with a breakpoint within the USP8 gene by chromosomal microarray. Mate pair sequencing was performed on all three cases and identified a KMT2A-USP2 rearrangement (cases 1 and 2) or a KMT2A-USP8 rearrangement (case 3). These recently characterized KMT2A fusions have been described exclusively in infant and pediatric leukemia cases where the incidence varies vary according to leukemia subtype, are considered high-risk, with a high incidence of central nervous system involvement, poor response to initial prednisone treatment, and poor event free survival. Additionally, approximately half of cases are unable to be resolved using standard cytogenetic approaches and are likely under recognized. Therefore, targeted molecular approaches are suggested in genetically unresolved infant leukemia cases to characterize these prognostically relevant clones.

Fluorescence in-situ hybridisation for TP63 rearrangements in T cell lymphomas: single-site experience of 470 patients and implications for clinical testing.

AIMS: The 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 RESULTS: A 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. CONCLUSION: This 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.

Siblings with ETV6/RUNX1-positive B-lymphoblastic leukemia: A single site experience and review of the literature.

Siblings diagnosed with B-lymphoblastic leukemia (B-ALL) that share the same driver abnormality have been rarely described in the literature. Herein, we report three pairs of siblings (one non-identical pair, one maternal half-sibling pair, and one identical pair) all diagnosed with ETV6/RUNX1-positive B-ALL. Considering that ETV6/RUNX1 fusion is thought to represent a prenatal event and necessitates additional genomic alterations to result in leukemia, siblings of patient's with known ETV6/RUNX1-positive B-ALL may be at increased risk of ETV6/RUNX1-positive B-ALL due to common exposures (environmental or infectious) or shared germline polymorphisms.

Detection of cryptic CCND1 rearrangements in mantle cell lymphoma by next generation sequencing.

The accurate detection of recurrent genetic abnormalities for most hematologic neoplasms is critical for diagnosis, prognosis and/or treatment. Rearrangements involving CCND1 are observed in a subset of mature B-cell neoplasms and can be reliably detected by fluorescence in situ hybridization (FISH) in most cases. However, cryptic and complex chromosomal rearrangements may pose a technical challenge for accurate diagnosis. Herein, we describe two patients with suspected mantle cell lymphoma that lacked obvious CCND1 rearrangements by FISH studies. A next generation sequencing (NGS) based assay, mate-pair sequencing (MPseq), was utilized in each case to investigate potential cryptic CCND1 rearrangements and revealed cryptic insertional events resulting in CCND1/IGH and CCND1/IGK rearrangements. These cases demonstrate that NGS-based assays, including MPseq, are a powerful approach to identify cryptic rearrangements of clinical importance that are not detected by current clinical genomics evaluation.

A near-haploid clone harboring a BCR/ABL1 gene fusion in an adult patient with newly diagnosed B-lymphoblastic leukemia.

The 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.

Acute leukemias harboring KMT2A/MLLT10 fusion: a 10-year experience from a single genomics laboratory.

The 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.

B-cell acute lymphoblastic leukemia in an elderly man with plasma cell myeloma and long-term exposure to thalidomide and lenalidomide: a case report and literature review.

BACKGROUND: The advent of the immunomodulatory imide drugs (IMiDs) lenalidomide and thalidomide for the treatment of patients with plasma cell myeloma (PCM), has contributed to more than a doubling of the overall survival of these individuals. As a result, PCM patients join survivors of other malignancies such as breast and prostate cancer with a relatively new clinical problem - second primary malignancies (SPMs) - many of which are a result of the treatment of the initial cancer. PCM patients have a statistically significant increased risk for acute myeloid leukemia (AML) and Kaposi sarcoma. IMiD treatment has also been associated with an increased risk of myelodysplastic syndrome (MDS), AML, and squamous cell carcinoma of the skin. However, within these overlapping groups, acute lymphoblastic leukemia (ALL) is much less common. CASE PRESENTATION: Herein, we describe an elderly man with PCM and a 14-year cumulative history of IMiD therapy who developed persistent pancytopenia and was diagnosed with B-cell acute lymphoblastic leukemia (B-ALL). He joins a group of 17 other patients documented in the literature who have followed a similar sequence of events starting with worsening cytopenias while on IMiD maintenance for PCM. These PCM patients were diagnosed with B-ALL after a median time of 36 months after starting IMiD therapy and at a median age of 61.5 years old. CONCLUSIONS: PCM patients with subsequent B-ALL have a poorer prognosis than their de novo B-ALL counterparts, however, the very low prevalence rate of subsequent B-ALL and high efficacy of IMiD maintenance therapy in PCM should not alter physicians' current practice. Instead, there should be a low threshold for bone marrow biopsy for unexplained cytopenias.

Mate pair sequencing improves detection of genomic abnormalities in acute myeloid leukemia.

OBJECTIVE: Acute 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. METHOD: We 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. RESULTS: Using 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. CONCLUSION: Utilization 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.

KMT2A (MLL) rearrangements observed in pediatric/young adult T-lymphoblastic leukemia/lymphoma: A 10-year review from a single cytogenetic laboratory.

T-lymphoblastic leukemia/lymphoma (T-ALL/LBL) accounts for approximately 15% of pediatric and 25% of adult ALL. While the underlying frequency of KMT2A (MLL) gene rearrangements has been identified in approximately 4-8% of T-ALL/LBL cases, a paucity of literature is available to characterize further the KMT2A rearrangements in pediatric/young adult T-ALL/LBL. A 10-year retrospective review was performed to identify KMT2A rearrangements in specimens sent for T-ALL/LBL fluorescence in situ hybridization studies in patients under the age of 30 years. Of 806 T-ALL/LBL FISH studies performed on unique individuals, 27 (3.3%) harbored KMT2A rearrangements. Nineteen patients were male and eight were female (M:F ratio, 2.4:1) with ages ranging from 1 to 20 years (mean 12, median 12). Of the 27 cases, nine (33%) had KMT2A/MLLT1 fusions, eight (30%) had KMT2A/AFDN fusions, two (7%) had KMT2A/ELL fusions, and one (4%) had a KMT2A/MLLT10 fusion. In addition, five (19%) had KMT2A rearrangements with unidentified gene fusion partners and two (7%) had 3'KMT2A deletions. Our results indicate that MLLT1 and AFDN account for the majority (63%) of KMT2A gene partners in pediatric/young adult T-ALL/LBL, while no KMT2A/AFF1 or KMT2A/MLLT3 fusions were observed despite their common identification in B-ALL and acute myeloid leukemia, respectively. In addition to diagnostic and prognostic value, detecting specific KMT2A fusions may also be of clinical importance in the era of targeted therapies.

Identification of High-risk Cryptic CRLF2 Rearrangements in B-Cell Acute Lymphoblastic Leukemia Utilizing an FGFR3/IGH Dual-Color Dual-Fusion DNA Probe Set.

B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood malignancy with gene rearrangements involving the IGH locus occurring in ∼5% of cases. Fluorescence in situ hybridization (FISH) probes targeting the IGH locus are not included in the standard children's oncology group (COG) fluorescence in situ hybridization panel. At our institute, we incorporated the use of FGFR3/IGH dual-color dual-fusion DNA probes for confirmation of aneuploidy 4 and 14 in diagnostic B-ALL specimens. Subsequently we have identified 4 B-ALL cases with cryptic CRLF2-IGH translocations that would otherwise have gone undetected. Detection of genetic alterations in B-ALL, such as CRLF2 rearrangements, may enhance patient risk stratification and therapy options in pediatric B-ALL.

Craniofacial abnormalities and developmental delay in two families with overlapping 22q12.1 microdeletions involving the MN1 gene.

Deletions spanning the MN1 gene (22q12.1) have recently been proposed as playing a role in craniofacial abnormalities that include cleft palate, as mouse studies have demonstrated that Mn1 haploinsufficiency results in skull abnormalities and secondary cleft palate. We report on four patients (two families) with craniofacial abnormalities and intellectual disability with overlapping microdeletions that span the MN1 gene. Comparative genomic hybridization microarray analysis revealed a 2.76 Mb deletion in the 22q12.1 region, in three family members (Family 1), that contains the MN1 gene. In addition, a complex 22q12 rearrangement, including a 1.61 Mb deletion containing the MN1 gene and a 2.28 Mb deletion encompassing the NF2 gene, has been identified in another unrelated patient (Family 2). Based upon genotype-phenotype correlation among our patients and those previously reported with overlapping 22q12 deletions, we identified a 560 kb critical region containing the MN1 gene that is implicated in human cleft palate formation. Importantly, NF2 was also found within the 22q12 deletion region in several patients which enabled specific clinical management for neurofibromatosis 2.