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Malignant gastrointestinal neuroectodermal tumour (GNET) is a rare, aggressive neoplasm with fewer than 100 cases reported in the literature. Most cases arise in the tubular gastrointestinal tract. We reported a unique case of GNET arising in the extrahepatic bile ducts and reviewed the literature of GNETs. The patient is a female in her mid-30s who presented with painless jaundice and diarrhoea several months after cholecystectomy for biliary dyskinesia. Workup revealed a tumour arising from the peripheral 4B bile ducts involving the left hepatic duct and bifurcation. Histologic examination of the lesion showed a malignant spindled and epithelioid neoplasm which strongly expressed S100 and SOX-10. Neoplastic cells were negative for various cytokeratins and melanoma markers. FISH testing usingEWSR1break-apart probes showed rearrangement of theEWSR1gene region. The immunohistochemical and molecular findings are consistent with a diagnosis of GNET arising in the extrahepatic bile ducts.

Context.— Next-generation sequencing is a powerful clinical tool for cancer management but can produce incidental/secondary findings that require special consideration. Objective.— To discuss clinical and laboratory issues related to incidental or secondary germline findings in the clinical setting of tumor testing and inform future guidelines in this area. Design.— A College of American Pathologists workgroup including representation from the American Society of Clinical Oncology, the Association for Molecular Pathology, and the American College of Medical Genetics and Genomics created a review of items that should be considered when developing guidelines for incidental or secondary findings when performing clinical tumor testing. Results.— Testing recommendations should be cognizant of the differences among anticipated incidental, unanticipated incidental, and secondary findings, and whether normal tissue is also tested. In addition to defining which variants will be reported, robust recommendations must also take into account test design and validation, reimbursement, cost, infrastructure, impact on reflex testing, and maintenance of proficiency. Care providers need to consider the potential of a test to uncover incidental or secondary findings, the recommendation of upfront counseling, the need for consent, the timing of testing and counseling, and that the exact significance of a finding may not be clear. Conclusions.— As clinical oncology testing panels have become a mainstay of clinical cancer care, guidelines addressing the unique aspects of incidental and secondary findings in oncology testing are needed. This paper highlights clinical and laboratory considerations with regard to incidental/secondary findings and is a clarion call to create recommendations.

We report on a 12-year-old female with both a partial duplication and deletion involving chromosome 6. The duplication involves 6p25.3p24.3 (7.585 Mb) while the deletion includes 6q27q27 (6.244 Mb). This chromosomal abnormality is also described as distal trisomy 6p and distal monosomy 6q. The patient has a Chiari II malformation, hydrocephalus, agenesis of the corpus callosum, microcephaly, bilateral renal duplicated collecting system, scoliosis, and myelomeningocele associated with a neurogenic bladder and bladder reflux. Additional features have included seizures, feeding dysfunction, failure to thrive, sleep apnea, global developmental delay, intellectual disability, and absent speech. To our knowledge, our report is just the sixth case in the literature with concomitant distal 6p duplication and distal 6q deletion. Although a majority of chromosomal duplication-deletion cases have resulted from a parental pericentric inversion, the parents of our case have normal chromosomes. This is the first reported de novo case of distal 6p duplication and distal 6q deletion. Alternate explanations for the origin of the patient’s chromosome abnormalities include parental gonadal mosaicism, nonallelic homologous recombination, or potentially intrachromosomal transposition of the telomeres of chromosome 6. Nonpaternity was considered but ruled out by whole exome sequencing analysis.

We presented in this article a patient with Klinefelter syndrome (KS) (47,XXY) who had maternal nondisjunction and uniparental disomy of the X chromosome with regions of heterodisomy and isodisomy, an interstitial Xp22.31 deletion of both X chromosomes, and other problems. His mother also possesses the same Xp22.31 deletion. The patient presented with status epilepticus and stroke, followed by severe brain atrophy and developmental regression. His unusual clinical and cytogenetic findings apparently have not been reported with either KS or Xp22.31 deletions. Based on the patient's available genetic and biochemical information, we cannot satisfactorily explain his seizures, strokes, or catastrophic brain regression.

Chromoanagenesis, a phenomenon characterized by complex chromosomal rearrangement and reorganization events localized to a limited number of genomic regions, includes the subcategories chromothripsis, chromoanasynthesis, and chromoplexy. Although definitions of these terms are evolving, constitutional chromoanagenesis events have been reported in a limited number of patients with variable phenotypes. We report on 2 cases with complex genomic events characterized by multiple copy number gains and losses confined to a single chromosome region, which are suggestive of constitutional chromoanagenesis. Case 1 is a 43-year-old male with intellectual disability and recently developed generalized tonic-clonic seizures. Chromosomal microarray analysis identified a complex rearrangement involving chromosome region 14q31.1q32.2, consisting of 16 breakpoints ranging in size from 0.2 to 6.2 Mb, with 5 segments of normal copy number present between these alterations. Interestingly, this case represents the oldest known patient with a complex rearrangement indicative of constitutional chromoanagenesis. Case 2 is a 2-year-old female with developmental delay, speech delay, low muscle tone, and seizures. Chromosomal microarray analysis identified a complex rearrangement consisting of 28 breakpoints localized to 18q21.32q23. The size of the copy number alterations ranged from 0.042 to 5.1 Mb, flanked by 12 small segments of normal copy number. These cases add to a growing body of literature demonstrating complex chromosomal rearrangements as a disease mechanism for congenital anomalies.

Biospecimens acquired during routine medical practice are the primary sources of molecular information about patients and their diseases that underlies precision medicine and translational research. In cancer care, molecular analysis of biospecimens is especially common because it often determines treatment choices and may be used to monitor therapy in real time. However, patient specimens are collected, handled, and processed according to routine clinical procedures during which they are subjected to factors that may alter their molecular quality and composition. Such artefactual alteration may skew data from molecular analyses, render analysis data uninterpretable, or even preclude analysis altogether if the integrity of a specimen is severely compromised. As a result, patient care and safety may be affected, and medical research dependent on patient samples may be compromised. Despite these issues, there is currently no requirement to control or record preanalytical variables in clinical practice with the single exception of breast cancer tissue handled according to the guideline jointly developed by the American Society of Clinical Oncology and College of American Pathologists (CAP) and enforced through the CAP Laboratory Accreditation Program. Recognizing the importance of molecular data derived from patient specimens, the CAP Personalized Healthcare Committee established the Preanalytics for Precision Medicine Project Team to develop a basic set of evidence-based recommendations for key preanalytics for tissue and blood specimens. If used for biospecimens from patients, these preanalytical recommendations would ensure the fitness of those specimens for molecular analysis and help to assure the quality and reliability of the analysis data.

Introduction Chromosomal abnormalities are frequent events in hematological malignancies. The degree of HLA compatibility between donor and recipient in hematopoietic stem cell transplantation is critical. Purpose of the study In this report, we describe an acute myeloid leukemia case with loss of heterozygosity (LOH) encompassing the entire HLA. Materials and methods HLA molecular typing was performed on peripheral blood (PB) and buccal swabs (BS). Chromosomal microarray analysis (CMA) was performed using a whole genome platform. Results Typing results on PB sample collected during blast crisis demonstrated homozygosity at the -A, -B, -C, -DR, and -DQ loci. A BS sample demonstrated heterozygosity at all loci. A subsequent PB sample drawn after count recovery confirmed heterozygosity. The CMA performed on PB samples collected during and after blast crisis revealed a large terminal region of copy-neutral LOH involving chromosome region 6p25.3p21.31, spanning approximately 35.9 Mb. The results of the CMA assay on sample collected after count recovery did not demonstrate LOH. Conclusions LOH at the HLA gene locus may significantly influence the donor search resulting in mistakenly choosing homozygous donors. We recommend confirming the HLA typing of recipients with hematological malignancies when homozygosity is detected at any locus by using BS samples, or alternatively from PB when remission is achieved.

Two siblings with the same male unbalanced karyotype demonstrate sex reversal. The older sib appeared phenotypically female and the younger sib demonstrated a male gender. The female had gonadal dysgenesis with bilateral ovatestes. The male had bilateral testes. The report discusses the phenotypical differences and genes associated with sex reversal.

Many aspects of genetic counseling training programs have been examined over the years. However, no study has explored professional or unprofessional behaviors genetic counseling graduate students experience during their training, and how these behaviors influence satisfaction with their training. This exploratory study examined students' experiences with program leaders, instructors, supervisors, and other trainees. Specific experiences included actions of favoritism, bias, negativity, abuse of power, and examples of positive role modeling. A survey was sent to all members of the National Society of Genetic Counselors and program directors in order to reach graduates of Accreditation Council for Genetic Counseling (ACGC)-accredited programs from 2015-2019 who were eligible to participate. Responses to questions relating to demographics, satisfaction with graduate education, behaviors experienced or seen during graduate school, and reporting of inappropriate behaviors were collected and analyzed. Results demonstrated that 95% of the genetic counseling graduates were highly satisfied with their graduate education and those who experienced inappropriate behaviors during their training were somewhat less satisfied (p = .04). Individuals who felt more prepared by their graduate education were more satisfied with their graduate education (p < .01). Being publicly embarrassed or humiliated, being made to feel like a burden in clinic, or being subjected to negative or offensive behavior based on their personal beliefs or personal characteristics (excluding areas of gender, race/ethnicity, or sexual orientation) were all negatively associated with satisfaction (all p < .04). We conclude that this survey could serve as a "Genetic Counseling Training Experiences Assessment" which could be incorporated into annual evaluations required by the ACGC. Implementation of this assessment would enhance the current evaluations of genetic counseling training programs and provide important information regarding student experiences during their training.

Introduction: Multiple myeloma (MM) is a genetically heterogeneous disease where risk stratification and outcomes are associated with translocations involving the immunoglobulin (Ig) loci and MYC, copy number abnormalities including gain(1q), del(1p), and del(17p), as well as mutations. Additionally, MM tumors may harbor rare mutations in genes that are targetable in other tumors, such as in IDH1 and IDH2. Therefore, we designed a comprehensive MM targeted sequencing panel to interrogate the common genomic abnormalities in MM and validated it against known standards. Methods: The targeted panel was designed to include the exons of 228 genes which are either frequently mutated, associated with prognosis or risk stratification, clinically actionable, or sites of important copy number abnormalities. Additional targets were added across the genome to identify hyperdiploidy. These targeted regions encompass the mutation detection part of the panel and involve approximately 990 kb. The Ig loci and region surrounding MYC were tiled to capture translocations and copy number changes. In total, this translocation part of the panel involves approximately 4.7 Mb. The mutation and translocation panels are manufactured separately and combined during the assay resulting in a 5:1 sequencing ratio, respectively, which prevents over-sequencing of the large translocation panel. 100 ng DNA extracted from CD138+ bone marrow cells (n=223) and from non-tumor tissue (peripheral blood or saliva) was processed using the HyperCap workflow (KAPA Biosystems). Of the 223, 48 samples were processed in a clinical diagnostic laboratory. Adapter ligated DNA was hybridized with a mixture of the mutation and translocation panel and purified, amplified libraries were sequenced using 75 bp paired end reads. Sequences were aligned to hg19 and mutations and translocations identified using Strelka and Manta. Copy number was determined using the ratio of non-tumor to tumor reads in each targeted region. Data were validated using clinical FISH (translocations, n=116), MLPA (copy number, n=101), known standards (mutations), ddPCR (mutations), and whole genome sequencing (WGS; translocations and copy number, n=122). Results: Canonical IgH translocations were detected in 43.2% of patients by the panel, and all agreed with WGS. FISH detected one additional "variant" t(4;14), but did not detect 4 translocations detected by both sequencing methods. In the remainder of the samples no canonical IgH translocation was detected, agreeing with FISH results. Non-canonical translocations were detected in 14.5% of samples, 43% of which were to the MYC locus. MYC translocations were detected in 37.3% of samples with copy number abnormalities occurring surrounding MYC in 32.7% of samples. Overall, MYC abnormalities were detected in 46.4% of samples. Copy number was determined by panel sequencing and MLPA for 22 regions that were directly comparable between the technologies in 101 patient samples and 13 myeloma cell lines. The copy number concordance between the technologies was 96.9% and 99.6% in patient samples and cell lines, respectively. For the important prognostic regions, the concordance was R 2=0.962 (CDKN2C), R 2=0.986 (CKS1B), and R 2=0.973 (TP53). Panel copy number data were also compared to WGS data and showed complete concordance across the three prognostic regions, which the exception of 2 samples. In these 2 samples a homozygous deletion was detected by the panel but not by WGS. The deletions were 6.2 and 8.0 kb in size, one encompassing the coding sequencing of TP53 and the other exons 1-4 of TP53. A larger homozygous deletion of 36.3 kb was detected by both sequencing methods. Mutation detection validation was performed using Horizon Discovery samples with known variant allele frequencies (VAF) for common mutations. We were able to determine the sequencing VAF for 74 mutations across 5 samples which had a concordance of R 2=0.9849 between the expected and observed frequencies. The minimum detected VAF was 1.3% at an average depth of 891x. We also performed ddPCR on 6 patient samples with the common KRAS, NRAS and BRAF mutations which resulted in a VAF concordance of R 2=0.9983. Conclusion: We have developed a targeted sequencing panel for MM patient samples that is as robust or better than both FISH and WGS. A full protocol for sample processing and analysis is available, and has been used in a clinical diagnostic laboratory. Disclosures Ahsan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Abu Zaid: Pieris: Current equity holder in publicly-traded company; Incyte: Research Funding; Pharamcyclic: Research Funding; Syndax: Consultancy, Research Funding. Ramasamy: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; Celgene (BMS): Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Conference registration, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive biotech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yong: GSK: Honoraria; Amgen: Honoraria; BMS: Research Funding; Sanofi: Honoraria, Research Funding; Takeda: Honoraria; Autolus: Research Funding; Janssen: Honoraria, Research Funding. Morgan: Takeda: Honoraria. Abonour: Celgene-BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; Jensen: Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding. Flynt: Bristol Myers Squibb: Current Employment. Ansari-Pour: Bristol Myers Squibb: Consultancy. Gooding: Bristol Myers Squibb: Research Funding. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Walker: Bristol Myers Squibb: Research Funding; Sanofi: Speakers Bureau.

Introduction/Objective PAX5 is a transcription factor critical for B-cell development and its genetic alterations are common in B lymphoblastic leukemia/lymphoma (B-ALL). We report a case of PAX5 P80R mutated acute leukemia with predominantly monocytic immunophenotype followed by a genetically-related histiocytic proliferation. Methods/Case Report A 27-year-old male presented with pancytopenia, epistaxis, and blurry vision. Bone marrow exam showed 95% blasts with nuclear indentations, occasional prominent nucleoli and basophilic cytoplasm. Blasts were positive for bright CD33, HLA-DR, CD14, bright CD64, partial CD123 and partial CD19. In addition, a minute population of B lymphoblasts positive for CD19, CD20, and dim TdT was seen. The AML FISH panel showed markedly aneuploid population with all probes demonstrating 3 to 5 signals. PAX5 P80R and KRAS G13D mutations were detected by NGS. Post induction bone marrow showed no evidence of acute leukemia with normal cytogenetics and FISH results. Subsequent two bone marrow exams performed due to progressive cytopenias demonstrated a prominent histiocytic proliferation with sheets of mature appearing histiocytes with abundant cytoplasm, oval to folded nuclei and occasional hemophagocytosis. This population was positive for CD163, CD14, CD68R, CD68, cyclin D1 with variable OCT2 expression and low proliferative activity. PAX5 P80R and KRAS G13D were detected. AML FISH panel showed aneuploidy in histiocytic appearing cells and IgH gene rearrangement studies by PCR showed prominent clonal population. The patient remained pancytopenic and died of disseminated fungal infection. Results (if a Case Study enter NA) NA Conclusion PAX5 P80R mutation has been primarily reported in B-ALL and is exceedingly rare in acute myeloid leukemias. This alteration has been linked to downregulation of B-cell lineage genes affecting B-cell maturation, and not surprisingly a proportion of PAX5 P80R mutated B-ALL cases show a switch to a monocytic lineage. The reported case demonstrates diagnostic caveats including unusual features of acute leukemia at the time of initial diagnosis and subsequent genetically-related histiocytic proliferation.

Plasma elevations of the amino acids alloisoleucine and argininosuccinic acid (ASA) are pathognomonic for maple syrup urine disease and argininosuccinate lyase deficiency, respectively. Reliable detection of these biomarkers is typically achieved using methods with tedious sample preparations or long chromatographic separations, and many published amino acid assays report poor specificity and/or sensitivity for one or both of these compounds. This report describes a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method that provides rapid quantification of alloisoleucine and ASA in human plasma. The basis of this method is a mixed-mode solid phase separation that achieves baseline resolution of alloisoleucine from isobaric interferents without the use of derivatization or ion pairing agents. The inject-to-inject time is 6 min including elution, column washing and re-equilibration. Validation studies demonstrate excellent limits of quantification (1 μmol/L), linearity (r = 0.999 from 1 to 250 μmol/L), accuracy (bias = -3.8% and -10.1%), and inter-assay imprecision (CV 8.06%) for plasma analyses. Data from long-term clinical application confirms chromatographic consistency equivalent to more traditional reversed-phase or HILIC based columns. Additional matrix studies indicate low suppression (10%) for a wide range of amino acids and compatibility with other matrixes such as blood spot analyses. Finally, analysis of our first 257 clinical specimens demonstrates high analytic specificity and sensitivity, allowing the detection of subtle but clinically relevant elevations of alloisoleucine and ASA that may be missed by other less sensitive methods. In conclusion, the novel LC-MS/MS method reported here overcomes a number of the challenges associated with alloisoleucine and ASA quantification. Combining this approach with published incomplete amino acid quantification methods allows, for the first time, a rapid and comprehensive LC-MS/MS analysis of underivatized amino acids without the use of ion pairing agents.

The purpose of this study is to evaluate the incidence of maternal cell contamination (MCC) in the first few milliliters of amniotic fluid withdrawn during amniocentesis.A prospective observational study was performed. The initial 2-3 ml of amniotic fluid withdrawn during amniocentesis was divided into direct analysis (uncultured) and cultured samples. A matching maternal buccal swab was obtained for MCC testing. MCC was determined by short-tandem repeat analysis. The primary outcome was measurement of clinically significant contamination (MCC 5%). Secondary outcomes included the determination of risk factors associated with MCC 5%. Outcomes were assessed by fisher's exact, independent t-test, binary logistic regression, and ANOVA.Direct analysis measured clinically significant contamination (MCC 5%) in 26% of specimens, while any amount of MCC was present in 68% of specimens. Cultured specimens had MCC 5% in 2%, and any amount of MCC in 24%. Only blood-tinged fluid was associated with an increased risk for MCC 5%. Larger volumes of the discard sample were not associated with increased incidence of MCC greater than 5%.A significant amount of MCC is present with direct analysis of the initial few milliliters of amniotic fluid withdrawn and is not influenced by the volume of the discard sample. Our results suggest that the first few milliliters of amniotic fluid be removed and discarded when direct analysis is utilized for prenatal genetic testing.

Background. We investigated the reliability of combined DOG1 and mammaglobin immunohistochemistry compared with ETV6 fluorescence in situ hybridization (FISH) in the assessment of salivary tumors previously diagnosed as acinic cell carcinoma (ACC). Ultrastructural features of cases reclassified as mammary analogue secretory carcinoma (MASC) were assessed by transmission electron microscopy (TEM). Methods. Immunohistochemical (IHC) reactivity to DOG1 and mammaglobin was validated against FISH targeting the ETV6 gene in all 14 cases. Results. Three cases with papillary cystic histomorphology previously diagnosed as ACC were revised to MASC. TEM features of the ETV6 rearrangement-positive MASC cases showed large numbers of secretory granules with extrusion into the intercellular spaces, well-developed endoplasmic reticulum, lipid-laden vacuoles, well-formed microvilli, and large lining cystic spaces. Conclusions. Combined DOG1 and mammaglobin immunohistochemistry is comparable to ETV6 -breakapart analysis for differentiating between papillary cystic variants of ACC and MASC.

Purpose To update key recommendations of the American Society of Clinical Oncology/College of American Pathologists human epidermal growth factor receptor 2 (HER2) testing in breast cancer guideline. Methods Based on the signals approach, an Expert Panel reviewed published literature and research survey results on the observed frequency of less common in situ hybridization (ISH) patterns to update the recommendations. Recommendations Two recommendations addressed via correspondence in 2015 are included. First, immunohistochemistry (IHC) 2+ is defined as invasive breast cancer with weak to moderate complete membrane staining observed in > 10% of tumor cells. Second, if the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test may (not “must”) be ordered on the excision specimen based on specific clinical criteria. The HER2 testing algorithm for breast cancer is updated to address the recommended work-up for less common clinical scenarios (approximately 5% of cases) observed when using a dual-probe ISH assay. These scenarios are described as ISH group 2 ( HER2/chromosome enumeration probe 17 [CEP17] ratio ≥ 2.0; average HER2 copy number < 4.0 signals per cell), ISH group 3 ( HER2/CEP17 ratio < 2.0; average HER2 copy number ≥ 6.0 signals per cell), and ISH group 4 ( HER2/CEP17 ratio < 2.0; average HER2 copy number ≥ 4.0 and < 6.0 signals per cell). The diagnostic approach includes more rigorous interpretation criteria for ISH and requires concomitant IHC review for dual-probe ISH groups 2 to 4 to arrive at the most accurate HER2 status designation (positive or negative) based on combined interpretation of the ISH and IHC assays. The Expert Panel recommends that laboratories using single-probe ISH assays include concomitant IHC review as part of the interpretation of all single-probe ISH assay results. Find additional information at www.asco.org/breast-cancer-guidelines .

Background Supernumerary marker chromosomes derived from chromosome 5 (SMC5) and 5p13 duplication syndrome are rare disorders, and phenotypic descriptions of patients are necessary to better define genotype-phenotype correlations for accurate, comprehensive genetic counseling. The purpose of this study is to highlight the unique findings of a patient with a 5p13.3-q11.2 duplication arising from a SMC5 and compare and contrast the phenotype with cases in the literature. Case presentation We report on an adult male with a 22 Mb duplication of chromosome 5p13.3-q11.2 resulting from a small SMC5. The patient has a history of prenatal polyhydramnios, dysmorphic features, respiratory issues, talipes equinovarus, hypotonia, developmental delay, and autistic features. The patient also has novel features of aortic dilation, pectus excavatum, kyphoscoliosis, and skin striae, suggestive of a connective tissue disorder. Despite these features he did not meet clinical diagnostic criteria for a well-characterized connective tissue disorder. Additional molecular genetic testing for syndromic and non-syndromic aortic aneurysms was negative. Conclusions Many of the patient’s features are consistent with individuals reported with 5p13 duplication syndrome and similar cases of SMC5, including polyhydramnios, macrocephaly, dolichocephaly, pre-auricular pits, arachnodactyly, respiratory problems, and developmental delays. It is unclear if the patient’s unique features of aortic dilation, pectus excavatum, kyphoscoliosis, and skin striae could be novel features of the SMC5 given its rarity and the few well-phenotyped adults in the literature. This report reviews the literature and provides additional phenotypic information to define the genotype-phenotype correlation of SMC5 and 5p13 duplication syndrome.

Purpose: Taxane-induced peripheral neuropathy (TIPN) is an important survivorship issue for many cancer patients. Currently, there are no clinically implemented biomarkers to predict which patients might be at increased risk for TIPN. We present a comprehensive approach to identification of genetic variants to predict TIPN. Experimental Design: We performed a genome-wide association study (GWAS) in 3,431 patients from the phase III adjuvant breast cancer trial, ECOG-5103 to compare genotypes with TIPN. We performed candidate validation of top SNPs for TIPN in another phase III adjuvant breast cancer trial, ECOG-1199. Results: When evaluating for grade 3–4 TIPN, 120 SNPs had a P value of Conclusions: rs3125923 represents a validated SNP to predict grade 3-4 TIPN. Genetically determined AA race represents the most significant predictor of TIPN. Clin Cancer Res; 21(22); 5082–91. ©2015 AACR.

The ATP-binding cassette, subfamily C [CFTR/MRP], member 2 (ABCC2) gene is a member of the ATP-binding cassette transporters and is involved in the transport of molecules across cellular membranes. Substrates transported by ABCC2 include antiepileptics, statins, tenofovir, cisplatin, irinotecan, and carbamazepine. Because of the pharmacogenomics implications, we developed a clinical laboratory–developed assay to test for seven variants in the ABCC2 gene: c.3563T>A (p.V1188E, rs17222723), c.1249G>A (p.V417I, rs2273697), c.3972C>T (p.I1324I, rs3740066), c.2302C>T (p.R768W, rs56199535), c.2366C>T (p.S789F, rs56220353), c.-24C>T (5′UTR, rs717620), and c.4544G>A (p.C1515Y, rs8187710). During the validation process, we noted several DNA samples, obtained from the Coriell Cell Repository, that contained both c.3563T>A, c.4544G>A, and a third variant, suggesting that c.3563T>A and c.4544G>A are in cis on the chromosome in some individuals. We obtained DNA samples from a trio (father, mother, and child), tested their ABCC2 variants, and confirmed that c.3563T>A and c.4544G>A were in cis on the same chromosome. Here, we report a new haplotype in ABCC2.

Background Hydranencephaly is a relatively rare but severe structural brain abnormality that often results in perinatal death. Although several factors including infection and multiple births have been reported to be associated with this birth defect, the underlying etiology is not well understood. Recently, FLVCR2 gene mutations have been implicated in a subset of hydranencephaly cases, following an autosomal recessive pattern of inheritance. Case We report a male infant with hydranencephaly found to have a previously unreported six amino acid deletion in one copy of the FLVCR2 gene following a pregnancy complicated by poor prenatal care and maternal cocaine use. Although our patient currently presents with developmental delays, he is showing progress and gaining some skills. Conclusion We discuss the possibility of a synergistic effect between the FLVCR2 genetic mutation and environmental cocaine exposure, creating a susceptible brain, as an explanation for this infant's phenotype. This case demonstrates the potential clinical utility of testing for mutations in FLVCR2 for patients with hydranencephaly after other possible etiologies, such as congenital infection, have been reasonably eliminated. Current literature on FLVCR2 is relatively sparse; identifying additional patients with similar mutations will aid in defining the clinical significance of a gene mutation and the contribution to the etiology of hydranencephaly. Birth Defects Research (Part A) 103:45–50, 2015. © 2014 Wiley Periodicals, Inc.

10032 Background: Burkitt Lymphoma (BL) is a common pediatric cancer in sub-Saharan Africa. Despite advances in care, prognosis is poor. The BL treatment protocol at our center in Kenya used since 2010, had a one-year survival of 29% (Martijn et al. BMJ Paed Open 2017). We hypothesized that financial burdens and delays in start of therapy impact outcomes. Methods: Our trial tested interventions aimed to: 1) Shorten time from presentation to start of treatment to improve survival; and 2) Support families during therapy to reduce abandonment. Initial eligibility included clinical suspicion of BL in a child 0-13 years of age. Patients with confirmed diagnosis of a mature B-cell lymphoma received support to complete therapy. Children with prior treatment of cancer were excluded. The trial was approved by human protection boards in Kenya and Indiana and consent was obtained prior to study entry. We enrolled 96 children with possible BL. Study personnel expedited routing of tissue samples to laboratories. Touch preps or smears were stained with Giemsa and reviewed by pathologists. Fresh tissue from all sources was used for flow cytometry. Tissue from core needle or incisional biopsies was processed for routine and immunohistochemical staining. Of 96 patients, 43 had BL confirmed by pathologic studies and were treated with combination chemotherapy given over 25 weeks. We paid families for transport costs for care, called after missed appointments, and provided direct monetary support (~$200 USD) for the 200 days children were on treatment. Results: Facilitating the biopsy shortened, by 3 days, the median time from admission to initiation of therapy for BL, because families did not need to raise funds for testing. Monetary support reduced abandonment of treatment by patients. Only 2 of 43 (5%) patients abandoned therapy, compared to abandonment by 22 of a cohort of 63 (35%) patients treated previously (p < 0.001). The combination of rapid diagnosis, early therapy, and financial support and communication with families improved event free survival of children with BL to 50% at 1 year (p = 0.045). Conclusions: Simple interventions to improve efficiency of diagnosis and reduce abandonment leads to improved outcomes for children with BL in Kenya.

We compared survival outcomes following myeloablative allotransplant (MAT) or cyclophosphamide/fludarabine (Cy/Flu) nonmyeloablative allotransplant (NMAT) for 165 patients with acute myelogenous leukemia (AML) in remission or without frank relapse. Patients who received NMAT were more likely to be older and have secondary AML and lower performance status. At a median follow-up of 61 months, median event-free survival and overall survival survival were not different between NMAT and MAT in univariate as well as multivariate analyses. Cy/Flu NMAT may provide similar disease control and survival when compared with MAT in patients with AML in remission or without frank relapse.

We herein describe 2 cases of adult multivisceral transplant patients who developed graft-versus-host disease manifesting predominantly as lichenoid skin papules and plaques. The diagnosis was supported by histopathology but ultimately corroborated by the utilization of the fluorescence in situ hybridization (FISH) technique using X and Y chromosome probes on unstained biopsy slides. In both cases, FISH revealed a high percentage of donor-derived cells as part of the inflammatory infiltrate in the skin biopsy. This report adds to the previous publications showing the utility of FISH in corroborating the diagnosis of graft-versus-host disease in transplant patients with unmatched sex donor.

Purpose: Clinically validated biomarkers for anti-angiogenesis agents are not available. We have previously reported associations between candidate VEGFA single-nucleotide polymorphisms (SNP) and overall survival (OS) in E2100. The associations between tumor VEGFA amplification and outcome are evaluated here. Experimental Design: E2100 was a phase III trial comparing paclitaxel with or without bevacizumab for patients with metastatic breast cancer. FISH to assess gene amplification status for VEGFA was conducted on paraffin-embedded tumors from 363 patients in E2100. Evaluation for association between amplification status and outcomes was conducted. Results: Estrogen receptor (ER)+ or progesterone receptor (PR)+ tumors were less likely to have VEGFA amplification than ER/PR− tumors (P = 0.020). VEGFA amplification was associated with worse OS (20.2 vs. 25.3 months; P = 0.013) in univariate analysis with a trend for worse OS in multivariate analysis (P = 0.08). There was a significant interaction between VEGFA amplification, hormone receptor status, and study arm. Patients with VEGFA amplification and triple-negative breast cancers (TNBC) or HER2 amplification had inferior OS (P = 0.047); amplification did not affect OS for those who were ER+ or PR+ and HER2−. Those who received bevacizumab with VEGFA amplification had inferior progression-free survival (PFS; P = 0.010) and OS (P = 0.042); no association was seen in the control arm. Test for interaction between study arm and VEGFA amplification with OS was not significant. Conclusion: VEGFA amplification in univariate analysis was associated with poor outcomes; this was particularly prominent in HER2+ or TNBCs. Additional studies are necessary to confirm the trend for poor OS seen on multivariate analysis for patients treated with bevacizumab. Clin Cancer Res; 19(5); 1281–9. ©2012 AACR.

Context Signet-ring cell lymphoma (SRCL) is a rare morphologic variant of non–Hodgkin lymphoma. Although it was initially reported as a rare morphologic variant of follicular lymphoma (FL), SRCL has to date been described in most types of non–Hodgkin lymphoma, mostly as single-case reports. Objective To study SRCL systematically by immunohistochemical stains and fluorescent in situ hybridization analyses. Design Seven SRCL cases were stained for CD3, CD5, CD20, PAX-5, CD10, CD21, CD23, cyclin D1, BCL2, BCL6, Ki-67, and MUM-1, and were analyzed by fluorescent in situ hybridization for BCL2, BCL6, MYC , and MALT1 rearrangements. Clinical information and patient outcome were reviewed in all patients. Results The patients were 3 women and 3 men, ranging in age from 31 to 75 years (average 60.3 years). The lesions involved lymph nodes, tonsil, parotid gland, soft tissue, and breast. There were 4 FLs, 1 diffuse large B-cell lymphoma (DLBCL), 1 DLBCL with FL, and 1 DLBCL with marginal zone lymphoma. All cases had typical signet-ring cell morphology. They were positive for CD20 and BCL-2, and had low-to-intermediate Ki-67 proliferation index (10%-40%) except in the parotid DLBCL with FL (70%). BCL-6 was detected in all but 1 FL (6/7). Fluorescent in situ hybridization detected IGH/BCL2 translocation in 1 FL, increased BCL6 copy number in another FL, BCL6 rearrangement, and increased copy number of MYC and MALT1 in the DLBCL with marginal zone lymphoma. Conclusions The FL with signet-ring cell morphology (1/5) tends to lack IGH/BCL2 translocation, and an extended immunohistochemical study is recommended for correct diagnosis and classification of SRCL.

Background Fanconi anaemia (FA) is a heterogeneous inherited disorder clinically characterised by progressive bone marrow failure, congenital anomalies and a predisposition to malignancies. Objective Determine, based on correction of cellular phenotypes, whether XRCC2 is a FA gene. Methods Cells (900677A) from a previously identified patient with biallelic mutation of XRCC2 , among other mutations, were genetically complemented with wild-type XRCC2. Results Wild-type XRCC2 corrects each of three phenotypes characteristic of FA cells, all related to the repair of DNA interstrand crosslinks, including increased sensitivity to mitomycin C (MMC), chromosome breakage and G2–M accumulation in the cell cycle. Further, the p.R215X mutant of XRCC2, which is harboured by the patient, is unstable. This provides an explanation for the pathogenesis of this mutant, as does the fact that 900677A cells have reduced levels of other proteins in the XRCC2–RAD51B-C-D complex. Also, FANCD2 monoubiquitination and foci formation, but not assembly of RAD51 foci, are normal in 900677A cells. Thus, XRCC2 acts late in the FA–BRCA pathway as also suggested by hypersensitivity of 900677A cells to ionising radiation. These cells also share milder sensitivities towards olaparib and formaldehyde with certain other FA cells. Conclusions XRCC2/FANCU is a FA gene, as is another RAD51 paralog gene, RAD51C/FANCO . Notably, similar to a subset of FA genes that act downstream of FANCD2, biallelic mutation of XRCC2/FANCU has not been associated with bone marrow failure. Taken together, our results yield important insights into phenotypes related to FA and its genetic origins.

Acute myeloid leukemia (AML) is a heterogeneous disease with respect to presentation and clinical outcome. The prognostic value of recently identified somatic mutations has not been systematically evaluated in a phase 3 trial of treatment for AML.We performed a mutational analysis of 18 genes in 398 patients younger than 60 years of age who had AML and who were randomly assigned to receive induction therapy with high-dose or standard-dose daunorubicin. We validated our prognostic findings in an independent set of 104 patients.We identified at least one somatic alteration in 97.3% of the patients. We found that internal tandem duplication in FLT3 (FLT3-ITD), partial tandem duplication in MLL (MLL-PTD), and mutations in ASXL1 and PHF6 were associated with reduced overall survival (P=0.001 for FLT3-ITD, P=0.009 for MLL-PTD, P=0.05 for ASXL1, and P=0.006 for PHF6); CEBPA and IDH2 mutations were associated with improved overall survival (P=0.05 for CEBPA and P=0.01 for IDH2). The favorable effect of NPM1 mutations was restricted to patients with co-occurring NPM1 and IDH1 or IDH2 mutations. We identified genetic predictors of outcome that improved risk stratification among patients with AML, independently of age, white-cell count, induction dose, and post-remission therapy, and validated the significance of these predictors in an independent cohort. High-dose daunorubicin, as compared with standard-dose daunorubicin, improved the rate of survival among patients with DNMT3A or NPM1 mutations or MLL translocations (P=0.001) but not among patients with wild-type DNMT3A, NPM1, and MLL (P=0.67).We found that DNMT3A and NPM1 mutations and MLL translocations predicted an improved outcome with high-dose induction chemotherapy in patients with AML. These findings suggest that mutational profiling could potentially be used for risk stratification and to inform prognostic and therapeutic decisions regarding patients with AML. (Funded by the National Cancer Institute and others.).

We describe a case of a 14-year-old boy who developed a cerebellar and brainstem glioblastoma 5 years after treatment for a medulloblastoma. The patient first presented in 2003 with 9 months of vomiting and a 9-kg weight loss. A head MRI showed a heterogeneously enhancing posterior fossa mass with hydrocephalus. Gross total resection was performed and the tumor was consistent with a classic medulloblastoma. Postoperative chemotherapy and craniospinal radiation was administered. The patient remained tumor-free until 2008, at which time he presented with right-sided weakness and numbness, left eye pain, vomiting and weight loss. Imaging showed abnormalities within the posterior pons, medulla, inferior cerebellar peduncles, cerebellar hemispheres and cervicomedullary junction with expansion of the medulla and cervical spinal cord. Due to the location of the lesion, biopsy was felt to be too risky and was avoided. Despite receiving chemotherapy, his symptoms continued to worsen and he died 4 months later. Post mortem examination limited to the brain and spinal cord confirmed the radiographic extent of the tumor. Microscopic examination showed a highly cellular infiltrative glial neoplasm with extensive palisading necrosis. A diagnosis of glioblastoma was rendered. The question of whether the first and second tumors were related is of potential clinical and academic interest. The first tumor was synaptophysin-positive and GFAP-negative, consistent with medulloblastoma. The second tumor was synaptophysin-negative and focally GFAP-positive, consistent with glioblastoma. The glioblastoma displayed EGF receptor amplification, and interestingly, it also displayed MYCN amplification; both tumors showed low level PTEN deletion. The medulloblastoma displayed a signal pattern consistent with an isochromosome 17q, while the glioblastoma showed some cells with an isochromosome 17q signal pattern amid a background of cells with abundant chromosomal instability. The relationship between these two tumors, particularly with regard to various molecular events, is discussed.

Many tumors exhibit elevated chromosome mis-segregation termed chromosome instability (CIN), which is likely to be a potent driver of tumor progression and drug resistance. Causes of CIN are poorly understood but probably include prior genome tetraploidization, centrosome amplification and mitotic checkpoint defects. This study identifies epigenetic alteration of the centromere as a potential contributor to the CIN phenotype. The centromere controls chromosome segregation and consists of higher-order repeat (HOR) alpha-satellite DNA packaged into two chromatin domains: the kinetochore, harboring the centromere-specific H3 variant centromere protein A (CENP-A), and the pericentromeric heterochromatin, considered important for cohesion. Perturbation of centromeric chromatin in model systems causes CIN. As cancer cells exhibit widespread chromatin changes, we hypothesized that pericentromeric chromatin structure could also be affected, contributing to CIN. Cytological and chromatin immunoprecipitation and PCR (ChIP-PCR)-based analyses of HT1080 cancer cells showed that only one of the two HORs on chromosomes 5 and 7 incorporate CENP-A, an organization conserved in all normal and cancer-derived cells examined. Contrastingly, the heterochromatin marker H3K9me3 (trimethylation of H3 lysine 9) mapped to all four HORs and ChIP-PCR showed an altered pattern of H3K9me3 in cancer cell lines and breast tumors, consistent with a reduction on the kinetochore-forming HORs. The JMJD2B demethylase is overexpressed in breast tumors with a CIN phenotype, and overexpression of exogenous JMJD2B in cultured breast epithelial cells caused loss of centromere-associated H3K9me3 and increased CIN. These findings suggest that impaired maintenance of pericentromeric heterochromatin may contribute to CIN in cancer and be a novel therapeutic target.

Fanconi anemia (FA) is an inherited chromosomal instability syndrome characterized by bone marrow failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). Eight FA proteins associate in a nuclear core complex to monoubiquitinate FANCD2/FANCI in response to DNA damage. Additional functions have been described for some of the core complex proteins; however, in vivo genetic proof has been lacking. Here we show that double-mutant Fancc−/−;Fancg−/− mice develop spontaneous hematologic sequelae including bone marrow failure, AML, MDS and complex random chromosomal abnormalities that the single-mutant mice do not. This genetic model provides evidence for unique core complex protein function independent of their ability to monoubiquitinate FANCD2/FANCI. Importantly, this model closely recapitulates the phenotypes found in FA patients and may be useful as a preclinical platform to evaluate the molecular pathogenesis of spontaneous bone marrow failure, MDS and AML in FA.

Primary central nervous system lymphomas are rare neoplasms characterized by a dismal prognosis relative to other extranodal lymphomas. Approximately 98% of primary central nervous system lymphomas are of B-cell origin, and most belong to the diffuse large B-cell type. Recently, diffuse large B-cell lymphomas have been subcategorized into germinal center and nongerminal center types based on gene expression profiles and immunohistochemical expression of CD10, Bcl-6, and MUM1. Studies have shown that the overall survival rate of the germinal center group is better than that of the nongerminal center lymphomas. In this study, 31 cases of primary central nervous system lymphomas of the diffuse large B-cell type were retrieved, reviewed, and immunostained for CD10, Bcl-6, MUM1, and Ki-67. Subclassification was carried out as described earlier, where CD10 and/or Bcl-6 positivity and negativity for MUM1 were considered characteristic of germinal center subtype and the opposite expression of nongerminal center subtype. Furthermore, the proliferative activity was semiquantitatively assessed using percent positive cells staining with Ki-67. Of the 31 cases examined, 26 (84%) were found to belong to the nongerminal center type. The Ki-67 index in these 26 cases ranged from 30 to 90% (mean, 69%). Five cases were categorized as the germinal center subtype. They had an Ki-67 index between 70 and 90% (mean, 78%). Interestingly, none of our patients were known to be HIV positive. One patient had a 10-year history of orthotopic liver transplant. We also performed fluorescence in situ hybridization analysis on formalin-fixed material and found that 38% of the cases where tissue was available had abnormalities of MYC/IGH and/or IGH/BCL2. We conclude that most primary central nervous system diffuse large B-cell lymphomas are of the nongerminal center origin. Regardless of the germinal center status, all cases showed a high proliferative rate. A statistically significant difference in the overall survival between the two groups was not seen.

Simpson–Golabi–Behmel syndrome is an X-linked recessive overgrowth disorder characterized by prenatal onset of overgrowth, characteristic facies, and frequently mild to severe mental retardation. In addition, a number of other characteristics including supernumerary nipples, a grooved tongue or chin, chest wall malformations, and mild genital anomalies are frequently seen as well. Here we present three brothers with Simpson–Golabi–Behmel syndrome, all of which had cryptorchidism with one also having chordee of the penis, hypospadius, and penoscrotal transposition. While severe genital anomalies have been reported rarely in patients with Simpson–Golabi–Behmel syndrome, no individuals with such anomalies prior to this report had survived beyond the neonatal period. © 2009 Wiley-Liss, Inc.

Trisomy 13 mosaicism occurs when two cell lines, one with a normal complement of chromosomes and the other with an additional chromosome 13, are present in the same individual. We present two children with trisomy 13 mosaicism and summarize the literature in 47 published cases. Our first patient is a 4-year-old male with normal development and dysmorphic features that include right microtia, tall forehead, bulbous nose, and high arched palate. The second patient died at 1 day of life secondary to laryngeal stenosis, a previously undocumented finding in trisomy 13. His other dysmorphic features included micrognathia, cleft soft palate, and an axillary pterygium. Several published case reports of patients with trisomy 13 mosaicism have had the typical phenotype of complete trisomy 13 with death in the neonatal period, while others have had few dysmorphic features and prolonged survival. The most common malformations in all 49 cases included ear anomalies, cleft lip and palate, and various congenital heart defects. Intelligence varied from normal in six patients to significant delays and mental retardation in the remainder of cases. There is no clear correlation between the percentage of trisomic cells and the level of intellectual function. The dysmorphic features seen vary considerably from one patient to the next, often making the clinical diagnosis of trisomy 13 mosaicism difficult. In counseling families, medical professionals should state that trisomy 13 mosaicism may lead to physical abnormalities and poor intellectual outcomes, but that the condition does not do so universally with normal development occurring in some individuals.

Human adipose stromal cells (ASCs) reside within the stromal-vascular fraction (SVF) in fat tissue, can be readily isolated, and include stem-like cells that may be useful for therapy. An important consideration for clinical application and functional studies of stem/progenitor cells is their capacity to maintain chromosome stability in culture. In this study, cultured ASC populations and ASC clones were evaluated at intervals for maintenance of chromosome stability. Uncultured SVF (uSVF) cells were included for comparison. G-banded chromosome analysis demonstrated that ASCs are diploid and have a normal karyotype. However since only approximately 20 cells are examined, low levels of chromosome instability would not be detected. To increase detection sensitivity, fluorescence in situ hybridization was employed, to permit chromosome enumeration in larger numbers of interphase cells. Seven cultured ASC populations, two ASC clones and four uSVF samples were examined. Chromosome X and 17 probes identified diploid, tetraploid, and aneuploid interphase cells. Both cultured ASC populations [up to approximately 35 Population Doublings (PDs)] and uSVF cells exhibited a similar level of diploidy (97.8% n = 6,355 and 98.83% n = 1,197, respectively) and numerical abnormalities, suggesting that cultured ASCs are genomically stable and supporting their suitability for transplantation applications. In comparison, cultured primary human chorionic villus cells exhibited marked genomic instability resulting in an 11.6% tetraploidy rate after 8-10 PD. Thus effects of culture on genomic stability may be cell type dependent and should be tested by appropriately scaled interphase fluorescence in situ hybridization analysis in any ex vivo expanded cell population destined for transplantation.

SV at age 2 years presented with multiple congenital anomalies including an absent left kidney, anal stenosis, vertebral abnormalities, partial sacral agenesis, microcephaly, dysmorphic facial features, growth deficiency, and developmental delay. She was found to have a complex chromosomal rearrangement derived from balanced translocations in each parent.

We evaluated the efficacy of FISH to detect chromosome anomalies in the evaluation of young (

Context.—The diagnosis of B-cell lymphoid malignancy can frequently be substantiated by detecting clonal immunoglobulin heavy chain (IGH) gene rearrangements, which is typically done by polymerase chain reaction (PCR) amplification and/or Southern blot analysis. Objective.—To characterize current laboratory practice for the assessment of IGH rearrangements and to identify opportunities for improvement. Design.—The data from the Molecular Oncology Proficiency Survey distributed to participating laboratories by the Molecular Pathology Committee of the College of American Pathologists from 1998 through 2003 were analyzed. Results.—Thirty-nine proficiency survey specimens (29 positive and 10 negative for clonal IGH rearrangements) were distributed. For Southern blot analysis, 944 results were reported, with a successful response rate of 95%. For PCR detection, 2349 results were reported, with a successful response rate of 72%. A higher rate of successful responses by PCR was achieved using framework 3 primers in combination with other frameworks (82%) compared with framework 3 primers only (76%) and when fresh/ frozen (72%) compared with paraffin-embedded (65%) tissues were analyzed. Conclusions.—The performance of the participating laboratories was very good, by both Southern blot and PCR analysis. As expected, Southern blot analysis consistently detects a higher proportion of IGH rearrangements than PCR analysis. Further improvement and standardization of the IGH PCR assay is important if it is to replace Southern blot analysis as the standard method. Participation in this survey is a valuable tool for assessing laboratory performance and it directs our attention to areas where we may improve laboratory practice.