N Context.-Echinoderm microtubule-associated proteinlike 4-anaplastic lymphoma kinase (EML4-ALK) gene fusions are detected in 3% to 13% of non-small cell lung carcinomas. Accurate testing for detection of EML4-ALK fusions is essential for appropriate therapy selection.Objective.-To compare reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) methodologies for detection of EML4-ALK fusions.Design.-Forty-six pulmonary adenocarcinomas were selected with enrichment for wild-type epidermal growth factor receptor (EGFR) status (wild type, n = 42; mutant, n = 4). Specimens were tested by IHC (Dako; clone ALK1), FISH (Abbott Molecular; LSI ALK break apart), and RT-PCR (variants 1 and 3a/b).Results.-EML4-ALK variant 3a/b was detectable by RT-PCR, FISH, and IHC in 4% (2 of 46) of specimens. Complete agreement among FISH and IHC reviewers was obtained for variant 3a/b. No concordance existed among methodologies for the detection of EML4-ALK variant 1. The RT-PCR method detected variant 1 in 20% (9 of 46) of specimens. Agreement among FISH viewers was poor for variant 1 because only 11% (1/9) of specimens were scored as positive by all 3 viewers. The sensitivity of IHC for detection of variant 1 was also poor because only 1 of 9 samples (11%) was scored as positive. Overall, the frequency of EML4-ALK variants 1 and 3a/b was 24% (11 of 46) in adenocarcinomas enriched for wild-type EGFR status. One EML4-ALK variant 1 fusion was found to coexist with an EGFR exon 21 mutation.Conclusions.-The FISH interpretation demonstrated great variability among observers. The RT-PCR method was the most sensitive and least-subjective methodology for detection of EML4-ALK fusions.

Context.—About 15% of colorectal cancers are characterized by genomic microsatellite instability, and of these, about 1 in 5 (2%–4% overall) are due to Lynch syndrome, a dominantly inherited condition predisposing the patient to cancers of multiple organ systems, including the gastrointestinal tract. Identification of individuals with Lynch syndrome allows for increased surveillance of the affected individual and of potentially affected family members. Objective.—To review the literature on microsatellite instability in colorectal cancer and current laboratory diagnostic testing strategies for the detection of Lynch syndrome. Data Sources.—This review is based on peer-reviewed literature, published guidelines from professional organizations (Evaluation of Genomic Applications in Practice and Prevention Working Group, National Comprehensive Cancer Network), and information from clinical laboratories performing microsatellite instability testing. Conclusions.—Universal screening for Lynch syndrome in all individuals affected with colorectal cancer has been recommended by the Evaluation of Genomic Applications in Practice and Prevention Working Group. Preliminary screening tests can identify individuals unlikely to be affected by Lynch syndrome, thereby reducing the need for full gene analysis. Immunohistochemistry and polymerase chain reaction–based tests for microsatellite instability have similar clinical sensitivity and specificity, and each method has advantages and limitations. BRAF and MLH1 methylation testing are useful reflex tests for those with a defect in MLH1 identified by immunohistochemistry. Emerging technologies, such as high-throughput sequencing, may substantially affect diagnostic algorithms in the future.

Objective To characterize the types of genetic abnormalities and their prevalence in early pregnancy loss at different developmental stages. We hypothesized that the prevalence of genetic abnormalities in pregnancy loss would differ across developmental stages. Methods Women with a pregnancy loss at < 20 weeks’ gestation (n = 86) were enrolled at the time of diagnosis. Maternal tissue without a fetal component was found in 13 samples. Chromosomal microarray analysis (CMA) was performed on 74 samples (including two samples from a twin pregnancy); 15 were pre-embryonic (no visible embryo on ultrasound examination), 31 were embryonic (embryo; 6 + 0 to 9 + 6 weeks’ gestation) and 28 were fetal (fetus; 10 + 0 to 19 + 6 weeks’ gestation) losses. The twin pregnancy was found to be monochorionic diamniotic and was subsequently treated as a single sample in our analysis. Nine samples that underwent CMA were excluded from analysis because of 100% maternal-cell contamination. Results The overall prevalence of genetic abnormalities differed across developmental stages (9.1% pre-embryonic, 69.2% embryonic and 33.3% fetal; P < 0.01). This difference persisted when comparing pre-embryonic with embryonic samples (P < 0.01) and embryonic with fetal samples (P = 0.02) but not pre-embryonic with fetal samples (P = 0.12). Additionally, the prevalence of aneuploidy differed significantly across developmental stages (0.0% in pre-embryonic samples vs 65.4% in embryonic samples vs 25.9% in fetal samples, P < 0.001). Abnormalities were most common in embryonic cases, followed by fetal and then pre-embryonic. Maternal cell contamination (MCC) was noted in 47.4% of 46,XX cases assessed. Conclusions Genetic abnormalities detected by CMA are more likely to occur in the embryonic period than in pre-embryonic or fetal stages. MCC is common in early pregnancy loss and should be excluded when results demonstrate a 46,XX karyotype.

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. K E Y W O R D Sacute myeloid leukemia, molecular cytogenetics, MPseq

Primary aneurysmal bone cyst (ABC) is a benign multiloculated cystic lesion of bone that is defined cytogenetically by USP6 gene rearrangements. Rearrangements involving USP6 are promoter swaps, usually generated by fusion of the noncoding upstream exons of different partner genes with exon 1 or 2 of USP6, thus leading to transcriptional upregulation of full‐length USP6 coding sequence. Testing for USP6 rearrangements is used diagnostically to distinguish it from secondary ABC and other giant cell‐rich primary bone tumors. In this report, we present a case of a 16‐year‐old male with a primary ABC of the left distal femur. USP6 break apart fluorescence in situ hybridization was positive for a rearrangement and conventional chromosome analysis identified a reciprocal X;17 translocation. In order to identify the putative USP6 fusion partner, we performed RNA sequencing and uncovered a novel USP9X‐USP6 promoter swap fusion. This result was confirmed by reverse transcription‐polymerase chain reaction (RT‐PCR) and by mate pair sequencing thus showing the utility of these alternative methodologies in identifying novel fusion candidates. Ubiquitin‐specific protease 9X (USP9X), like USP6, encodes a highly conserved substrate‐specific deubiquitylating enzyme. USP9X is highly expressed in a number of tissue types and acts as both an oncogene and tumor suppressor in several human cancers. We conclude that oncogenic activation of USP6 via USP9X promoter exchange represents a novel driver of primary ABC formation.