Rearrangements involving ETV6 and ABL1 are more commonly observed in Philadelphia-negative myeloproliferative neoplasms, with rare reports in patients with AML or ALL. We present the evaluation of a 12-year old male who presented with acute fever and a CBC which showed progressive anemia. Bone marrow analysis identified precursor B-cell ALL. Chromosome, fluorescence in situ hybridization (FISH) and SNP CMA analyses were performed on cells from a bone marrow aspirate. Chromosome analysis identified a slightly atypical hyperdiploid ALL clone [51,XY,+X,+15,+17,+21,+21]. Interphase FISH showed two signals for chromosomes 4 and 10, four RUNX1 signals, negativity for KMT2A rearrangement and BCR/ABL1 or ETV6/RUNX1 fusions. However, one ETV6 signal was clearly diminished in addition to a deletion of one ASS1 9q34 probe signal. We have previously associated the latter with either ABL1 or NUP214 9q34 gene fusions typically seen in T-ALL, which can be resolved by microarray, RNA sequencing, or dual-color FISH. A SNP microarray confirmed the chromosome gains observed in the karyotype and revealed that the ASS1 deletion included partial deletion of ABL1 distally but no specific gene proximally, suggesting an ectopic gene insertion, resulting in a probable ABL1 fusion. The array was notable for a contiguous terminal deletion and proximal duplication of 12p. Although ETV6 was located in the deleted interval, there was an increased copy number for ∼90% of the gene. This was interpreted as strong evidence of ETV6 insertion into chromosome 9. Since both ETV6 and ABL1 have opposite orientation to the centromeres, an in-frame fusion gene requires three chromosome breaks to be generated (limiting the incidence of this fusion) and the putative insertion mechanism in this clone would be consistent with that etiology. An ETV6/ABL1 fusion within 9q34 was confirmed by metaphase FISH and RNA sequencing. This report highlights the utility of a multidiscipline approach to leukemia evaluation.

High-grade B-cell lymphoma with concurrent MYC, BCL2, and BCL6 rearrangements (i.e. Triple Hit Lymphoma (TH)) is an aggressive lymphoma generally detected by conventional cytogenetics and/or FISH. We present a patient referred for high grade diffuse large B-cell lymphoma with a complex karyotype. The diagnosis was consistent with follicular lymphoma and clonal evolution, based only on the karyotype analysis. Dual fusion and breakapart (BAP) FISH strategies and microarray testing were required to diagnosis TH lymphoma. The karyotype included a t(14;18) with an extra derivative 14 (no normal 14), a t(3;22)(q27;q11.2), a t(2;6) and trisomy 8. IGH/BCL2 FISH identified three fusions, consistent with the novel double derivative (14)t(14;18) and derivative 18. BCL2 -BAP FISH showed one germline fusion, a 3' signal and two 5' signals rather than the typical evolution related extra 3' signal, but consistent with the double derivative 14. MYC-BAP FISH showed three un-rearranged fusions discordant with the double fusion IGH/MYC result. This apparent incongruence could be explained by an IGH insertion into MYC which was confirmed by metaphase FISH. The BCL6 FISH was also atypical with 2 germ line fusions and a small extra 5' BCL6 signal. Since the microarray showed no 3q27 gain, a balanced rearrangement between the 5'BCL6 promotor and the lambda-light gene at 22q11.2 was concluded. Copy neutral LOH (CN-LOH) of 14q32.13->qter in the array analysis (spanning IGH) supports mitotic recombination based clonal evolution for the novel double derivative 14, consistent with a reversal of the common oncogenic fusion derivative. A second CN-LOH region was observed spanning distal 6p, although the driver gene for that evolution is not clear. In summary, the TH would have been missed without multiple disciplinary ancillary testing, highlighting their importance in accurately evaluating complex clones in facilitating optimal patient care and management.

T-cell acute lymphoblastic leukemia (T-ALL) is not as frequently reported as the B-cell counterpart (B-ALL), only occurring in about 15% of pediatric cases with a typically heterogeneous etiology. Approximately 8% of childhood T-ALL cases have rearrangements involving the ABL1 tyrosine kinase gene at 9q34.12; although a t(9;22), resulting in a fusion of ABL1 with the BCR gene at 22q11.23 is a common occurrence in B-ALL, it is not a typical finding in T-ALL. A subset of 10 of 40 documented cases of T-ALL analyzed over a 5-year period is presented, each having gene rearrangements within band 9q34 that resulted in fusions other than BCR/ABL1. These cases included fusions involving ABL1, SET (9q34.11), NUP214 (9q34.13), SPTAN1 (9p34.11), and TNRC6B (22q13.1). Among the 10 cases are: six SET/NUP214 fusions, two ABL1/NUP214 fusions (one of which was associated with episomal amplification) and novel SPTAN1/ABL1 and TNRC6B/ABL1 fusions. The evaluations of these clones were each significantly aided by FISH analysis, which directed subsequent microarray and anchored multiplex PCR testing for fusion confirmations.

The present study reviews a large laboratory’s experience with utilization and efficacy of microarray analysis for prenatal diagnosis to illustrate it’s efficacy as a first tier test. This study addresses 45,000 prenatal patients that were studied by microarray analysis (using an Affymetrix® Cytoscan® HD array) to detect aberrations in both advanced maternal age (AMA) patients and those with ultrasound abnormalities not detected by standard chromosome analysis. Approximately 75% of the specimens were amniotic fluid (AF) and 68% of the specimens were direct (not cultured) analyses. Overall the analyses were successful greater than 99.5% of the time. Referrals for an abnormal ultrasound finding (U/S) were the most frequent indication (∼58%) followed by AMA (∼31%). Referrals for NIPT follow-up have increased over 600% in the past two years. Microarray analysis detected an cytogenetic abnormalities (when chromosomes were normal) for both AMA (1.6%) and abnormal U/S (∼4%). This was even greater when the U/S revealed multiple anomalies which included a heart defect (∼9%). Over 88% of the aberrations detected were smaller than 7 Mb. Variants of unknown significance (VOUS) were detected in only 1.3% of the patients and were familial ∼86% of the time. Mosaicism was more frequent than expected, being found in ∼0.5% of patients studied. The results from the analysis of 45,000 prenatal microarray patients, the largest study to date, has revealed several important findings: (1) It is clear that microarray analysis should be a first-tier test regardless the indication for testing (e.g. AMA or U/S); (2) The majority of specimens have been AF, referred because of abnormal U/S and successfully analyzed in > 99.5% of the patients; (3) The frequency of VOUS can be kept low (by using carefully considered criteria) and the vast majority are familial; (4) The highest frequency of aberrations were in patients ascertained with multiple anomalies and a heart defect (∼9%), with a surprising >90% not involving a 22q deletion; (5) Microarray analysis of mosaicism was more sensitive than chromosome analysis, providing additional key clinical information in numerous patients; (6) Copy-neutral changes, detected by SNP analysis was instrumental in the detection of UPD, GWUPiD and recessive disorders (associated with consanguinity).

Uniparental disomy (UPD) in the context of whole chromosome non-disjunction mediated trisomy or monosomy is well documented with mechanisms largely understood. In contrast, the etiology of segmental UPD (segUPD) existing in only a portion of a chromosome pair, primarily evinced by a terminal absence of heterozygosity, occurs rarely and the etiology is not well documented. In the course of confirming (300) cases of whole chromosome UPD using single nucleotide polymorphism (SNP) microarrays to detect runs of homozygosity (ROH) associated with UPD we identified 62 cases of segUPD in prenatal and postnatal samples. Nearly half of these cases were due to positive selection based Beckwith-Wiedemann syndrome 11p15 imprinting imbalance. We highlight a subset of the remaining cases to show that segUPD occurs secondarily to mitotic recombination based correction of genomic imbalances that include deletions, translocation derivative chromosomes and terminal deletions with contiguous duplications. Non-invasive prenatal testing (NIPT), CVS, placental, amniotic fluid and peripheral blood samples taken from several individuals at multiple times show temporal correction of aberrations during fetal development. Although the various genetic lesions are “repaired”, sustained clinical effects might still be present because the original imbalance may have affected early development, may still be present in some cells or the resulting seg-UPD may unmask a recessive disorder. In the case of genomic rearrangements consisting of terminal deletions with contiguous duplications, we hypothesize that genomic repair processes restore euploidy if initiated proximal to the duplication, but result in a triplication and terminal segUPD if initiated at the “end” of the inverted duplication. Importantly, these studies suggest that the incidence of segUPD mediated correction is underestimated, and may explain the etiology of clinical phenotypes that are undetected by CGH based microarray analysis and whole exome sequencing studies.

Disclaimer: ACMG Clinical Laboratory Practice Resources are developed primarily as an educational tool for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these practice resources is voluntary and does not necessarily assure a successful medical outcome. This Clinical Laboratory Practice Resource should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient’s record the rationale for the use of a particular procedure or test, whether or not it is in conformance with this Clinical Laboratory Practice Resource . They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures. Noninvasive prenatal screening (NIPS) using cell-free DNA has been rapidly adopted into prenatal care. Since NIPS is a screening test, diagnostic testing is recommended to confirm all cases of screen-positive NIPS results. For cytogenetics laboratories performing confirmatory testing on prenatal diagnostic samples, a standardized testing algorithm is needed to ensure that the appropriate testing takes place. This algorithm includes diagnostic testing by either chorionic villi sampling or amniocentesis samples and encompasses chromosome analysis, fluorescence in situ hybridization, and chromosomal microarray.

Advances in genetic diagnostic technologies has allowed for monumental improvements in the diagnosis of genetic diseases. With newer technologies, offering improved diagnostic results, unexpected findings have also been delineated in some cases. Many groups have addressed this issue by providing guidelines delineating what results should be shared with patients. However, most of the guidelines and scientific thought has involved the study of pediatric patients and have addressed results from sequencing studies. We utilized results of 30,000 prenatal arrays and 6500 oncology arrays to provide information concerning the occurrence and importance of incidental findings in array studies in both of these populations. This experience has allowed us to formulate a protocol regarding how to proceed if an incidental result was encountered, and to postulate what should be reported back to patients.

We describe a 5-day-old male with minor facial anomalies, a congenital laryngeal web, severe laryngomalacia, and prominent fixed flexion of the proximal interphalangeal joints of digits 2 through 5 bilaterally. A whole genome SNP microarray analysis identified a 2.55 Mb interstitial deletion of 22q11.21, typical of that seen in the DiGeorge and Velocardiofacial syndromes. A review of the literature identifies 10 other cases with camptodactyly. Camptodactyly appears to be an associated but rarely reported anomaly in patients with the 22q11.2 microdeletion syndrome. © 2016 Wiley Periodicals, Inc.

Background: Although preliminary reports suggest that ALK gene amplification may occur in inflammatory breast cancer (IBC), data are limited. We performed a comprehensive investigation of the status of ALK gene in IBC.Methods: We used core biopsy (CB) samples from 30 IBC patients for immunohistochemistry (IHC), 25 of these samples for fluorescence in situ hybridization (FISH) of ALK gene rearrangement, 8 for chromosome 2 aneusomy, and 20 microdissected frozen CBs for array comparative genomic hybridization (CGH) and mRNA analysis.Results: All 30 samples were negative for ALK protein expression by IHC. FISH analysis showed no EML4-ALK gene rearrangement in any samples, although 16 of the 25 samples (64%) contained 3 to 4 extra copies of the ALK gene, and chromosome 2 aneusomy was found in 7 of 8 samples that had extra copies of the ALK gene. Only 3 of the 20 samples showed evidence of mild ALK gene amplification by array CGH. mRNA analysis revealed that mRNA expression of ALK was not significantly higher in these samples compared with samples that showed no evidence of ALK gene amplification in CGH analysis, nor was mRNA expression of ALK significantly different in tumor compared with 5 normal breast samples (P > 0.05, t test).Conclusion: Our comprehensive evaluation suggests that ALK gene rearrangement did not occur in the IBC patients studied. The significance of our finding of mildly increased copy numbers of the ALK gene resulting from chromosome 2 aneusomy rather than mild amplification of the ALK gene requires further investigation.

11070 Background: A variety of biomarkers are currently used to help guide treatment decisions for patients with non-small cell lung cancer (NSCLC). These include mutation analysis for the EGFR and KRAS genes, along with gene rearrangement analysis for the ALK and ROS1 loci. In this study we have evaluated the clinical and analytical performance features of these assays in a series of formalin-fixed paraffin-embedded (FFPE) tissue samples. Methods: FFPE samples submitted for analysis of the EGFR, KRAS, ALK and ROS1 genes were evaluated using molecular and FISH assays. EGFR mutation analysis was performed using Sanger nucleic acid sequencing methods for exons 18-21. KRAS mutations were detected using allele specific PCR or pyrosequencing methods. Rearrangements involving the ALK gene were detected using break-apart FISH probes (Abbott Molecular). Genetic alterations involving the ROS1 gene were determined using FISH probes (Kreatech Diagnostics). Over 6,200 test results for these 4 markers are included in this analysis. Results: Mutations in the EGFR gene were detected in 10.1% of samples evaluated (n=3,872). A slightly higher percentage of samples from female patients (13%) had a detectable mutation compared to samples from males (7%) (chi-square p<0.0001). Deletions in exon 19 (51%) were the most common alterations detected, followed by point mutations in exon 21 (35%). KRAS mutations were detected in approximately 22% of specimens. ALK gene rearrangements were observed in 3.1% of samples (n=1,524). Specimens from individuals <50y of age were more likely to provide a positive result (11%) compared to samples from individuals >50y of age (2.5%) (chi-square p<0.0001). Gene amplification for the ALK gene was a common finding in the NSCLC samples evaluated. ROS1 alterations were detected in 2.8% of the specimens. In this cohort, no specimens were positive for both an EGFR mutation and an ALK gene rearrangement. Conclusions: Biomarker testing is well established in clinical practice for NSCLC, with results from the tests used to guide important therapy decisions. Assays for biomarkers such as EGFR, KRAS, ALK and ROS1 are robust, allowing for the analysis of multiple analytes in FFPE samples, even when the amount of tissue may be limiting.

We describe 19 unrelated individuals with submicroscopic deletions involving 10p15.3 characterized by chromosomal microarray (CMA). Interestingly, to our knowledge, only two individuals with isolated, submicroscopic 10p15.3 deletion have been reported to date; however, only limited clinical information is available for these probands and the deleted region has not been molecularly mapped. Comprehensive clinical history was obtained for 12 of the 19 individuals described in this study. Common features among these 12 individuals include: cognitive/behavioral/developmental differences (11/11), speech delay/language disorder (10/10), motor delay (10/10), craniofacial dysmorphism (9/12), hypotonia (7/11), brain anomalies (4/6) and seizures (3/7). Parental studies were performed for nine of the 19 individuals; the 10p15.3 deletion was de novo in seven of the probands, not maternally inherited in one proband and inherited from an apparently affected mother in one proband. Molecular mapping of the 19 individuals reported in this study has identified two genes, ZMYND11 (OMIM 608668) and DIP2C (OMIM 611380; UCSC Genome Browser), mapping within 10p15.3 which are most commonly deleted. Although no single gene has been identified which is deleted in all 19 individuals studied, the deleted region in all but one individual includes ZMYND11 and the deleted region in all but one other individual includes DIP2C . There is not a clearly identifiable phenotypic difference between these two individuals and the size of the deleted region does not generally predict clinical features. Little is currently known about these genes complicating a direct genotype/phenotype correlation at this time. These data however, suggest that ZMYND11 and/or DIP2C haploinsufficiency contributes to the clinical features associated with 10p15 deletions in probands described in this study. © 2012 Wiley Periodicals, Inc.

Purpose: The aim of this study was to characterize the clinical phenotype of patients with tetrasomy of the distal 15q chromosome in the form of a neocentric marker chromosome and to evaluate whether the phenotype represents a new clinical syndrome or is a phenocopy of Shprintzen-Goldberg syndrome. Methods: We carried out comprehensive clinical evaluation of four patients who were identified with a supernumerary marker chromosome. The marker chromosome was characterized by G-banding, fluorescence in situ hybridization, single nucleotide polymorphism oligonucleotide microarray analysis, and immunofluorescence with antibodies to centromere protein C. Results: The marker chromosomes were categorized as being neocentric with all showing tetrasomy for regions distal to 15q25 and the common region of overlap being 15q26→qter. Conclusion: Tetrasomy of 15q26 likely results in a distinct syndrome as the patients with tetrasomy 15q26 share a strikingly more consistent phenotype than do the patients with Shprintzen-Goldberg syndrome, who show remarkable clinical variation. Genet Med 2012:14(9):811–818