Conventional Cytogenetic Analysis of Hematologic Neoplasms: A 20-Year Review of Proficiency Test Results From the College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee.

CONTEXT.­: One goal of the joint College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee is to ensure the accurate detection and description of chromosomal abnormalities in both constitutional and neoplastic specimens, including hematologic neoplasms. OBJECTIVE.­: To report a 20-year performance summary (1999-2018) of conventional chromosome challenges focusing on hematologic neoplasms. DESIGN.­: A retrospective review was performed from 1999 through 2018 to identify karyotype challenges specifically addressing hematologic neoplasms. The overall performance of participants was examined to identify potential recurring errors of clinical significance. RESULTS.­: Of 288 total conventional chromosome challenges from 1999-2018, 87 (30.2%) were presented in the context of a hematologic neoplasm, based on the provided clinical history, specimen type, and/or chromosomal abnormalities. For these 87 hematologic neoplasm challenges, 91 individual cases were provided and graded on the basis of abnormality recognition and karyotype nomenclature (ISCN, International System for Human Cytogenomic [previously Cytogenetic] Nomenclature). Of the 91 cases, 89 (97.8%) and 87 (95.6%) exceeded the required 80% consensus for grading of abnormality recognition and correct karyotype nomenclature, respectively. The 2 cases (2 of 91; 2.2%) that failed to meet the 80% consensus for abnormality recognition had complex karyotypes. The 4 cases (4 of 91; 4.4%) that failed to meet the 80% consensus for correct karyotype nomenclature were the result of incorrect abnormality recognition (2 cases), missing brackets in the karyotype (1 case), and incorrect breakpoint designation (1 case). CONCLUSIONS.­: This 20-year review demonstrates clinical cytogenetics laboratories have been and continue to be highly proficient in the detection and description of chromosomal abnormalities associated with hematologic neoplasms.

Clinicopathologic features of breast cancer reclassified as HER2-amplified by fluorescence in situ hybridization with alternative chromosome 17 probes.

OBJECTIVE: Dual probe fluorescence in situ hybridization (FISH) assays for determination of human epidermal growth factor receptor 2 (HER2) gene amplification in breast cancer provide a ratio of HER2 to chromosome 17. The ratio may be skewed by copy number alterations (CNA) in the control locus for chromosome 17 (CEP17). We analyzed the impact of alternative chromosome 17 control probes on HER2 status in a series of breast cancers with an emphasis on patients reclassified as amplified. METHODS: Breast cancer patients with equivocal HER2 immunohistochemistry (2+) and equivocal FISH with CEP17 were included. Reclassification of HER2 status was assessed with alternative chromosome 17 control probes (LIS1 and RARA). RESULTS: A total of 40 unique patients with 46 specimens reflexed to alternative chromosome 17 probe testing were identified. The majority (>80%) of patients had pT1-2, hormone receptor-positive tumors with an intermediate or high combined histologic grade. There were 34/46 (73.9%) specimens reclassified as amplified with alternative probes, corresponding to 29/40 (72.5%) patients. Of the patients reclassified as amplified with alternative probes, 34.5% (10/29) received HER2-targeted therapy. CONCLUSION: In this series, the majority of breast cancers tested with alternative chromosome 17 control probes under the 2013 ASCO/CAP Guidelines were converted to HER2-amplified. The treatment data and the clinicopathologic profile of the tumors suggest that most of these patients will neither receive nor benefit from HER2-targeted therapy. The findings support the recommendation in the 2018 ASCO/CAP HER2 Guidelines to discontinue the use of alternative chromosome 17 probes.

Co-occurring Down syndrome and SUCLA2-related mitochondrial depletion syndrome.

Mitochondrial DNA depletion syndrome 5 (MIM 612073) is a rare autosomal recessive disorder caused by homozygous or compound heterozygous pathogenic variants in the beta subunit of the succinate-CoA ligase gene located within the 13q14 band. We describe two siblings of Hispanic descent with SUCLA2-related mitochondrial depletion syndrome (encephalomyopathic form with methylmalonic aciduria); the older sibling is additionally affected with trisomy 21. SUCLA2 sequencing identified homozygous p.Arg284Cys pathogenic variants in both patients. This mutation has previously been identified in four individuals of Italian and Caucasian descent. The older sibling with concomitant disease has a more severe phenotype than what is typically described in patients with either SUCLA2-related mitochondrial depletion syndrome or Down syndrome alone. The younger sibling, who has a normal female chromosome complement, is significantly less affected compared to her brother. While the clinical and molecular findings have been reported in about 50 patients affected with a deficiency of succinate-CoA ligase caused by pathogenic variants in SUCLA2, this report describes the first known individual affected with both a mitochondrial depletion syndrome and trisomy 21.

Neuroblastoma in a pediatric patient with a microduplication of 2p involving the MYCN locus.

Neuroblastoma is the most common solid tumor of infancy, and mutations in several genes have been implicated as playing a role in tumor development. Here, we describe a pediatric patient with a constitutional microduplication of 2p24.3 who developed Stage 4 neuroblastoma at age 11 months. He represents the sixth patient described in the literature with partial trisomy 2p and neuroblastoma. All previous cases had duplication events spanning two genes implicated in neuroblastoma, MYCN and ALK. Our patient is unique because his duplicated region includes the MYCN gene only; the ALK gene is unaffected. These data, combined with the relatively high incidence of neuroblastoma reported in partial trisomy 2p patients, support the notion that MYCN duplication should be added to the growing list of genetic factors associated with an increased risk of neuroblastoma. The mechanism of increased risk is unclear, but the fact that our patient had dramatic amplification of MYCN in his tumor suggests that a germline duplication might predispose to further amplification. Additionally, our patient has several morphologic features common to patients with partial trisomy 2p including high forehead, hypertelorism, postaxial polydactyly, and developmental delay despite having a microduplication spanning approximately 1 Mb and including just three intact genes. This case may therefore help further delineate the genotype-phenotype correlations associated with partial trisomy 2p.

Timeless functions independently of the Tim-Tipin complex to promote sister chromatid cohesion in normal human fibroblasts.

The Timeless-Tipin complex and Claspin are mediators of the ATR-dependent activation of Chk1 in the intra-S checkpoint response to stalled DNA replication forks. Tim-Tipin and Claspin also contribute to sister chromatid cohesion (SCC) in various organisms, likely through a replication-coupled process. Some models of the establishment of SCC posit that interactions between cohesin rings and replisomes could result in physiological replication stress requiring fork stabilization. The contributions of Timeless, Tipin, Claspin, Chk1 and ATR to SCC were investigated in genetically stable, human diploid fibroblast cell lines. Whereas Timeless, Tipin and Claspin showed similar contributions to UVC-induced activation of Chk1, siRNA-mediated knockdown of Timeless induced a 100-fold increase in sister chromatid discohesion, whereas the inductive effects of knocking down Tipin, Claspin and ATR were 4-20-fold. Knockdown of Chk1 did not significantly affect SCC. Consistent findings were obtained in two independently derived human diploid fibroblast lines and support a conclusion that SCC in human cells is strongly dependent on Timeless but independent of Chk1. Furthermore, the 10-fold difference in discohesion observed when depleting Timeless versus Tipin indicates that Timeless has a function in SCC that is independent of the Tim-Tipin complex, even though the abundance of Timeless is reduced when Tipin is targeted for depletion. A better understanding of how Timeless, Tipin and Claspin promote SCC will elucidate non-checkpoint functions of these proteins at DNA replication forks and inform models of the establishment of SCC.

Deletion of hepatocyte nuclear factor-1-beta in an infant with prune belly syndrome.

Prune belly syndrome is a rare congenital disorder characterized by deficiency of abdominal wall muscles, cryptorchidism, and urinary tract anomalies. We have had the opportunity to study a baby with prune belly syndrome associated with an apparently de novo 1.3-megabase interstitial 17q12 microdeletion that includes the hepatocyte nuclear factor-1-beta gene at 17q12. One previous patient, an adult, has been reported with prune belly syndrome and a hepatocyte nuclear factor-1-beta microdeletion. Hepatocyte nuclear factor-1-beta is a widely expressed transcription factor that regulates tissue-specific gene expression and is expressed in numerous tissues including mesonephric duct derivatives, the renal tubule of the metanephros, and the developing prostate of the mouse. Mutations in hepatocyte nuclear factor-1-beta cause the "renal cysts and diabetes syndrome," isolated renal cystic dysplasia, and a variety of other malformations. Based on its expression pattern and the observation of two affected cases, we propose that haploinsufficiency of hepatocyte nuclear factor-1-beta may be causally related to the production of the prune belly syndrome phenotype through a mechanism of prostatic and ureteral hypoplasia that results in severe obstructive uropathy with urinary tract and abdominal distension.

A rational approach to genetic testing for sarcoma.

Diagnosis of sarcoma increasingly relies on identifying genetic defects using modern molecular technologies. Each analytic method has unique advantages and specimen requirements that should be considered when allocating tissue for downstream testing. Karyotype on fresh tissue represents a genome-wide screen of gross chromosomal alterations, whereas fluorescence in situ hybridization and polymerase chain reaction detect specific defects that are characteristic of a given tumor type such as t(11;22) EWSR1-FLI1 in Ewing family tumors, t(X;18) SS18-SSX1 in synovial sarcoma, t(2;13) PAX3-FOXO1A in alveolar rhabdomyosarcoma, and MYCN gene amplification in neuroblastoma. Identifying a clonal genetic defect also provides a tumor marker that could help stage the extent of spread of the neoplasm or monitor the efficacy of therapy. In research laboratories, array-based methods identify genes and biochemical pathways contributing to tumor growth and maintenance, opening avenues for pharmacogenetic tests that predict which therapy is likely to overcome the biochemical defects with minimal toxicity. Array-based discoveries are also spurring validation of smaller test panels that rely on conventional technologies such as immunohistochemistry and reverse transcription polymerase chain reaction. The pathologist's expertise is critical in: (1) consulting with clinicians about specimen collection and handling; (2) preserving tissue for immediate testing and for any downstream testing that is indicated once morphology and immunophenotype are known; (3) performing tests that maximize outcome on the basis of the strengths and limitations of each assay in each available specimen type; and (4) conveying results to the rest of the healthcare team using proper gene nomenclature and interpreting the findings in a way that facilitates optimal clinical management.