Williamson, Cynthia M.

AbstractAcute 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.

Abstract Rearrangements involving TCF3 (E2A) at 19p13.3 are a common cytogenetic abnormality in B-lymphoblastic leukemia/lymphoma (B-LL) across all age groups accounting for at least 5% of B-LL. The most common translocation partner, PBX1 at 1q23, is associated with an unbalanced der(19) or a balanced t(1;19) in 80% vs. 20% patients, respectively, and results in a PBX1/TCF3 fusion transcriptional activator. Several other rearrangement partners have been described, and as such TCF3 is approaching the dubious status of a "promiscuous" gene partner. Clinically, our laboratory uses a dual-color, dual fusion PBX1/TCF3 FISH probe to characterize the most common gene fusion; however, this FISH strategy also allows for the identification of atypical disruptions of the TCF3 gene region, and may suggest a gene partner based on abnormal metaphases/metaphase-FISH. Interrogation of our pediatric leukemia database (ages 0-30) identified 219 patients with abnormal TCF3 FISH results. We selected 23 samples for mate-pair evaluation to test the methodology, characterize novel rearrangements and identify new TCF3 fusion partners. In total, 19 patient samples were successfully sequenced by Mate-Pair, including 13 samples with variants of the 1;19 translocation. Of the samples with TCF3/PBX1 fusion, 3 patients demonstrated novel insertional translocation events characterized by mate-pair. A total of 6 samples with undefined/unknown TCF3 partner genes were evaluated. The mate-pair results identified 5 samples with a t(12;19)(p13.31;p13.3), resulting in TCF3/ZNF384 fusion. Interestingly, two of the ZNF384 rearrangements were observed in the unbalanced form, suggesting a similar mechanism related to the der(19)t(1;19) regularly observed in the common PBX1-TCF3 rearrangement. From a karyotype perspective, since these rearrangements result in only a small portion of chromosome 12p translocated onto distal 19p, this rearrangement would "cryptic" by conventional karyotyping. One patient sample demonstrated a new TCF3 gene fusion partner (TEF), associated with a t(19;22)(p13.3;q13.2). Overall, our results demonstrate the utility of mate-pair evaluation as an adjunct methodology in the identification of novel TCF3 rearrangements in B-LL. We have successfully expanded the application of mate-pair to characterize other novel/cryptic abnormalities in various hematologic and oncologic neoplasms encountered in our laboratory. Disclosures No relevant conflicts of interest to declare.

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.

AbstractT‐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.

AbstractBackgroundKaryotype determination has a central role in the genetic workup of pregnancy loss, as aneuploidy (trisomy and monosomy) and polyploidy (triploidy and tetraploidy) are the cause in at least 50% of first trimester, 25% of second trimester, and 11% of third trimester miscarriages. There are several limitations with the current approaches of obtaining a karyotype using traditional cytogenetics, fluorescence in situ hybridization with a limited number of probes, and chromosomal microarray. These include culture failure, incomplete results, lower sensitivity, and longer reporting time.MethodsTo overcome current limitations, a novel molecular assay is developed with a Standard Resolution Interphase Chromosome Profiling probe set which is a variation of the recently developed High Resolution probe set. It generates a molecular karyotype that can detect all major changes commonly associated with pregnancy loss. Initial familiarization of signal patterns from the probe set was used, followed by validation of the method using 83 samples from miscarriages in a blind study from three different laboratories. Finally, the clinical utility of the method was tested on 291 clinical samples in two commercial reference laboratory settings on two different continents.ResultsThe new molecular approach not only identified all the chromosome changes observed by current methods, but also significantly improved abnormality detection by characterizing derivative chromosomes and finding subtle subtelomeric rearrangements, balanced and unbalanced. All Robertsonian translocations were also detected. The abnormality rate was 54% on clinical samples from commercial laboratory 1 and 63% from laboratory 2.ConclusionThe attributes of this method make it an ideal choice for the genetic workup of miscarriages, namely (1) near 100% successful results, (2) greater sensitivity than conventional chromosome analysis or FISH panels, (3) rapid reporting time, and (4) favorable comparisons with chromosomal microarray.