In vitro propagation of XXY human Klinefelter spermatogonial stem cells: A step towards new fertility opportunities.

Klinefelter Syndrome (KS) is characterized by a masculine phenotype, supernumerary sex chromosomes (47, XXY), and impaired fertility due to loss of spermatogonial stem cells (SSCs). Early testicular cryopreservation could be an option for future fertility treatments in these patients, including SSCs transplantation or in vitro spermatogenesis. It is critically essential to adapt current in vitro SSCs propagation systems as a fertility option for KS patients. KS human testicular samples (13,15- and 17-year-old non-mosaic KS boys) were donated by patients enrolled in an experimental testicular tissue banking program. Testicular cells were isolated from cryopreserved tissue and propagated in long-term culture for 110 days. Cell-specific gene expression confirmed the presence of all four main cell types found in testes: Spermatogonia, Sertoli, Leydig, and Peritubular cells. A population of ZBTB16+ undifferentiated spermatogonia was identified throughout the culture using digital PCR. Flow cytometric analysis also detected an HLA-/CD9+/CD49f+ population, indicating maintenance of a stem cell subpopulation among the spermatogonial cells. FISH staining for chromosomes X and Y showed most cells containing an XXY karyotype with a smaller number containing either XY or XX. Both XY and XX populations were able to be enriched by magnetic sorting for CD9 as a spermatogonia marker. Molecular karyotyping demonstrated genomic stability of the cultured cells, over time. Finally, single-cell RNAseq analysis confirmed transcription of ID4, TCN2, and NANOS 3 within a population of putative SSCs population. This is the first study showing successful isolation and long-term in vitro propagation of human KS testicular cells. These findings could inform the development of therapeutic fertility options for KS patients, either through in vitro spermatogenesis or transplantation of SSC, in vivo.

Sudden Unexpected Death in a Child From Acute Myeloid Leukemia.

ABSTRACT: Acute myeloid leukemia can rarely cause sudden, unexpected death in children. Presentation may be non-specific and death may occur in children with no prior medical history. Herein we present the case of a previously healthy 2-year and 2 month-old White girl, who on autopsy, was found to have acute myeloid leukemia with KMT2A rearrangement extensively involving all major thoracic and abdominal organs. This case is presented to the forensic community to discuss the presentation and findings in sudden death caused by acute leukemia. The case highlights when acute leukemia should enter the differential as a potential cause of death, as well as potential resources available in the postmortem workup of acute leukemias.

In Vitro Propagation of XXY Undifferentiated Mouse Spermatogonia: Model for Fertility Preservation in Klinefelter Syndrome Patients.

Klinefelter syndrome (KS) is characterized by a masculine phenotype, supernumerary sex chromosomes (usually XXY), and spermatogonial stem cell (SSC) loss in their early life. Affecting 1 out of every 650 males born, KS is the most common genetic cause of male infertility, and new fertility preservation strategies are critically important for these patients. In this study, testes from 41, XXY prepubertal (3-day-old) mice were frozen-thawed. Isolated testicular cells were cultured and characterized by qPCR, digital PCR, and flow cytometry analyses. We demonstrated that SSCs survived and were able to be propagated with testicular somatic cells in culture for up to 120 days. DNA fluorescent in situ hybridization (FISH) showed the presence of XXY spermatogonia at the beginning of the culture and a variety of propagated XY, XX, and XXY spermatogonia at the end of the culture. These data provide the first evidence that an extra sex chromosome was lost during innate SSC culture, a crucial finding in treating KS patients for preserving and propagating SSCs for future sperm production, either in vitro or in vivo. This in vitro propagation system can be translated to clinical fertility preservation for KS patients.

PML-RARA Fusion Transcripts Detectable 8 Months prior to Promyelocytic Blast Crisis in Chronic Myeloid Leukemia.

Promyelocytic blast crisis arising from chronic myeloid leukemia (CML) is rare. We present a 40-year-old male who developed promyelocytic blast crisis 17 months after CML diagnosis, confirmed by the presence of the t(15;17) and t(9;22) translocations in the leukemic cells. Preserved nucleic acids from routine BCR-ABL1 testing provided a unique opportunity to evaluate clonal progression over time. Retrospective analysis demonstrated PML-RARA fusion transcripts were first detectable 8 months prior to blast crisis presentation. A review of 21 cases of promyelocytic blasts crisis published in the literature reveals a male predominance with earlier age at onset as compared to females. Interestingly, TKI therapy during chronic phase did not impact the time interval between diagnosis and promyelocytic blast crisis. Treatment with standard acute promyelocytic leukemia regimens provides more favorable outcomes with complete molecular remission. Although rare, it is important to consider a promyelocytic blast crisis when evaluating for transformation of CML due to its effective treatment with specific therapies.

Non-Nodal CD5-Negative Mantle Cell Lymphoma with Secondary TP53 Deletion.

Mantle cell lymphoma is a non-Hodgkin lymphoproliferative neoplasm with several clinical and morphologic variants linked, primarily, through genetic derangement of the cyclin D1 locus. Aberrant phenotypes have been described, though prognostic data in such cohorts are limited due to a paucity of cases. We report a case of mantle cell lymphoma with non-nodal clinical presentation, aberrant loss of CD5 expression, and concomitant cytogenetic deletion of 17p. While non-nodal disease is often associated with an improved prognosis in mantle cell lymphoma, this 67-year-old patient experienced a more challenging clinical course with a poor initial response to chemotherapy. Therefore, this case may represent a type of non-nodal mantle cell lymphoma with a prognosis similar to that of classical cases due to the additional phenotypic and genetic alterations found in this patient.

Evaluation of Cytotoxic and Genotoxic Effects of Extremely Low-frequency Electromagnetic Field on Mesenchymal Stromal Cells.

BACKGROUND: Interest in the use of extremely low-frequency (ELF) electromagnetic field (EMF) for the treatment of pain and inflammation is increasing due to the ability of this promising therapy to compete with pharmaceuticals without the adverse effects caused by drugs. However, there continues to be concerns regarding cytotoxic and genotoxic effects that may occur as a result of exposure to EMF. OBJECTIVE: To investigate this concern, we tested the effect of our known therapeutic 5 Hz, 0.4 milliTesla (mT) EMF on a human mesenchymal stromal cell (hMSC) line to determine whether ELF-EMF exposure would cause cytotoxic or genotoxic effects. METHODS: Treated samples along with controls were exposed to 5 Hz, 0.4 mT ELF-EMF for 20 min/day, 3×/week for 2 weeks and then assayed for cell viability, proliferation rates, and chromosome breaks. RESULTS: Cytogenetic analysis of the viability and proliferation rates along with analysis of morphological genome stability showed no cytotoxicity, and no chromosome breaks per karyotype analysis-therefore no genotoxicity. CONCLUSION: Exposure to an ELF-EMF of 5 Hz, 0.4 mT for 20 min/day, 3×/week for 2 weeks does not cause cytotoxic or genotoxic effects in hMSCs.

Donor-derived myeloid sarcoma in two kidney transplant recipients from a single donor.

We report the rare occurrence of donor-derived myeloid sarcoma in two kidney transplant patients who received organs from a single deceased donor. There was no evidence of preexisting hematologic malignancy in the donor at the time of organ recovery. Both recipients developed leukemic involvement that appeared to be limited to the transplanted organ. Fluorescence in situ hybridization (FISH) and molecular genotyping analyses confirmed that the malignant cells were of donor origin in each patient. Allograft nephrectomy and immediate withdrawal of immunosuppression were performed in both cases; systemic chemotherapy was subsequently administered to one patient. Both recipients were in remission at least one year following the diagnosis of donor-derived myeloid sarcoma. These cases suggest that restoration of the immune system after withdrawal of immunosuppressive therapy and allograft nephrectomy may be sufficient to control HLA-mismatched donor-derived myeloid sarcoma without systemic involvement.

Clinicopathological analysis of near-tetraploidy/tetraploidy acute myeloid leukaemia.

AIMS: Near-tetraploidy/tetraploidy (NT/T) is a rare cytogenetic alteration in acute myeloid leukaemia (AML). NT/T-AML is categorised as complex cytogenetics and therefore, presumed to have an unfavourable prognosis. Our aim is to further characterise the clinical, morphological, cytogenetic and prognostic features of NT/T-AML. METHODS: We searched our cytogenetic laboratory database from 1991 to 2012 to reveal 13 cases of NT/T-AML. Each case was evaluated with regard to its demographics, morphology, immunophenotype and prognosis. Specific morphological features included blast size, irregularity of nuclear contours, cytoplasmic vacuoles, and presence and lineage of dysplasia. RESULTS: Eleven men and two women had a median age of 68 years. Blasts were predominately large (11/13). Eight of 13 patients had AML with myelodysplasia-related changes. Sixty-nine per cent of patients achieved complete remission (CR). Median overall survival (OS) was 8.6 months. CR rate and median OS in cases with ≥ 5 cytogenetic abnormalities were 71% and 6 months, compared with 67% and 18.1 months in cases with <5 abnormalities. CONCLUSIONS: NT/T-AML occurs in older males, exhibits large blast size and is associated with myelodysplasia. Unlike previously reported data, our study reveals an overall better prognosis in this older population with NT/T-AML than was expected for a complex karyotype AML. Cytogenetic complexity independent of ploidy status did not greatly affect the high CR rates, but did appear to be a better estimation of prognostic risk in terms of median OS.

Pdx1 and controlled culture conditions induced differentiation of human amniotic fluid-derived stem cells to insulin-producing clusters.

This study investigated the differentiation of human amniotic fluid-derived stem cells (hAFSCs) into insulin-producing clusters in vitro. Adenovirally-delivered mouse Pdx1 (Ad-Pdx1) induced human Pdx1 expression in hAFSCs and enhanced the coordinated expression of downstream ß-cell markers. When Ad-Pdx1-transduced hAFSCs were sequentially treated with activin A, bFGF and nicotinamide and the culture plate surface coated with poly-l-ornithine, the expression of islet-associated human mRNAs for Pdx1, Pax6, Ngn3 and insulin was increased. C-peptide ELISA confirmed that Ad-Pdx1-transduced hAFSCs processed and secreted insulin in a manner consistent with that pathway in pancreatic ß-cells. To sustain the ß-cell-like phenotype and investigate the effect of three-dimensional (3D) conformation on the differentiation of hAFSCs, Pdx1-transduced cells were encapsulated in alginate and cultured long-term under serum-free conditions. Over 2 weeks, partially differentiated hAFSC clusters increased in size and increased insulin secretion. Taken together, these data demonstrate that ectopic Pdx1 expression initiates pancreatic differentiation in hAFSCs and that a ß-cell-like phenotype can be augmented by culture conditions that mimic the stromal components and 3D geometry associated with pancreatic islets.

MDM2 copy numbers in well-differentiated and dedifferentiated liposarcoma: characterizing progression to high-grade tumors.

OBJECTIVES: MDM2 gene amplification is associated with well-differentiated (WDL) and dedifferentiated liposarcomas (DDL). Using fluorescent in situ hybridization (FISH), we sought to characterize various patterns of MDM2 amplification among the morphologic spectrum of liposarcoma. METHODS: Forty-six cases of liposarcoma in various sites were examined and included 22 WDLs, 14 DLLs, and 10 negative control subjects. RESULTS: The MDM2 amplification ratio (MDM2/CEP12) was lower in WDL (10.2) compared with DDL (18.3) cases (P = .0000002). An amplification ratio of 16 showed optimal sensitivity (0.86) and specificity (0.96) as a cutoff point for progression to DDL. Borderline areas, defined as tumors with increased cellularity and atypia but with preserved lipomatous differentiation, showed a significantly higher MDM2 ratio (17.5; P = .0007) compared with WDL. Central (retroperitoneal and intra-abdominal) tumors also showed a significantly higher MDM2 ratio than peripheral ones (P = .02). CONCLUSIONS: Differences in MDM2 amplification profiles among liposarcomas could help further define and predict progression to high-grade neoplasia.

Biclonal light chain gammopathy with aberrant CD33 expression in secondary plasma cell leukemia.

Plasma cell leukemia is a rare neoplastic proliferation of circulating plasma cells. Clonal proliferations of plasma cells, such as in plasma cell leukemia or plasma cell myeloma, are typically characterized by production of a monoclonal heavy and/or light chain immunoglobulin. We present a case of a secondary plasma cell leukemia arising from plasma cell myeloma with dual expression of lambda and kappa light chains along with aberrant expression of CD33, CD20, and dim CD56. This case emphasizes the importance of recognizing aberrant immunophenotypes in plasma cell leukemias and represents the first reported case of biclonal light chain expression in a secondary plasma cell leukemia.

Burkitt's Lymphoma Presenting as Late-Onset Posttransplant Lymphoproliferative Disorder following Kidney and Pancreas Transplantation: Case Report and Review of the Literature.

Posttransplant lymphoproliferative disorders (PTLD) are a rare, but serious complication following transplantation. Late-onset PTLD are often associated with more monoclonal lesions and consequently have a worse prognosis. There are only isolated case reports of Burkitt's lymphoma presenting as PTLD. We present an extremely rare, aggressive Burkitt's lymphoma years after kidney and pancreas transplantation which was successfully treated with combination chemotherapy along with withdrawal of immunosuppression. The patient remains in complete remission more than 2 years after his diagnosis. We also provide a succinct review of treatment of various PTLD and discuss the role of Epstein-Barr virus infection in the pathogenesis of PTLD.

New recurrent balanced translocations in acute myeloid leukemia and myelodysplastic syndromes: cancer and leukemia group B 8461.

Acquired chromosome abnormalities in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are among the most valuable determinants of diagnosis and prognosis. In search of new recurrent balanced translocations, we reviewed the Cancer and Leukemia Group B (CALGB) cytogenetics database containing pretreatment and relapse karyotypes of 4,701 adults with AML and 565 with MDS who were treated on CALGB trials. We identified all cases with balanced structural rearrangements occurring as a sole abnormality or in addition to one other abnormality, excluded abnormalities known to be recurrent, and then reviewed the literature to determine whether any of what we considered unique, previously unknown abnormalities had been reported. As a result, we identified seven new recurrent balanced translocations in AML or MDS: t(7;11)(q22;p15.5), t(10;11)(q23;p15), t(2;12)(p13;p13), t(12;17)(p13;q12), t(2;3)(p21;p21), t(5;21)(q31;q22), and t(8;14)(q24.1;q32.2), and additionally, t(10;12)(p11;q15), a new translocation in AML previously reported in a case of acute lymphoblastic leukemia. Herein, we report hematologic and clinical characteristics and treatment outcomes of patients with these newly recognized recurrent translocations. We also report 52 unique balanced translocations, together with the clinical data of patients harboring them, which to our knowledge have not been previously published. We hope that once the awareness of their existence is increased, some of these translocations may become recognized as novel recurring abnormalities. Identification of additional cases with both the new recurrent and the unique balanced translocations will enable determination of their prognostic significance and help to provide insights into the mechanisms of disease pathogenesis in patients with these rare abnormalities.

inv(16)/t(16;16) acute myeloid leukemia with non-type A CBFB-MYH11 fusions associate with distinct clinical and genetic features and lack KIT mutations.

The inv(16)(p13q22)/t(16;16)(p13;q22) in acute myeloid leukemia results in multiple CBFB-MYH11 fusion transcripts, with type A being most frequent. The biologic and prognostic implications of different fusions are unclear. We analyzed CBFB-MYH11 fusion types in 208 inv(16)/t(16;16) patients with de novo disease, and compared clinical and cytogenetic features and the KIT mutation status between type A (n = 182; 87%) and non-type A (n = 26; 13%) patients. At diagnosis, non-type A patients had lower white blood counts (P = .007), and more often trisomies of chromosomes 8 (P = .01) and 21 (P < .001) and less often trisomy 22 (P = .02). No patient with non-type A fusion carried a KIT mutation, whereas 27% of type A patients did (P = .002). Among the latter, KIT mutations conferred adverse prognosis; clinical outcomes of non-type A and type A patients with wild-type KIT were similar. We also derived a fusion-type-associated global gene-expression profile. Gene Ontology analysis of the differentially expressed genes revealed-among others-an enrichment of up-regulated genes involved in activation of caspase activity, cell differentiation and cell cycle control in non-type A patients. We conclude that non-type A fusions associate with distinct clinical and genetic features, including lack of KIT mutations, and a unique gene-expression profile.

Prognostic significance of the European LeukemiaNet standardized system for reporting cytogenetic and molecular alterations in adults with acute myeloid leukemia.

PURPOSE: To evaluate the prognostic significance of the international European LeukemiaNet (ELN) guidelines for reporting genetic alterations in acute myeloid leukemia (AML). PATIENTS AND METHODS: We analyzed 1,550 adults with primary AML, treated on Cancer and Leukemia Group B first-line trials, who had pretreatment cytogenetics and, for cytogenetically normal patients, mutational status of NPM1, CEBPA, and FLT3 available. We compared complete remission (CR) rates, disease-free survival (DFS), and overall survival (OS) among patients classified into the four ELN genetic groups (favorable, intermediate-I, intermediate-II, adverse) separately for 818 younger (age < 60 years) and 732 older (age ≥ 60 years) patients. RESULTS: The percentages of younger versus older patients in the favorable (41% v 20%; P < .001), intermediate-II (19% v 30%; P < .001), and adverse (22% v 31%; P < .001) genetic groups differed. The favorable group had the best and the adverse group the worst CR rates, DFS, and OS in both age groups. Both intermediate groups had significantly worse outcomes than the favorable but better than the adverse group. Intermediate-I and intermediate-II groups in older patients had similar outcomes, whereas the intermediate-II group in younger patients had better OS but not better CR rates or DFS than the intermediate-I group. The prognostic significance of ELN classification was confirmed by multivariable analyses. For each ELN group, older patients had worse outcomes than younger patients. CONCLUSION: The ELN classification clearly separates the genetic groups by outcome, supporting its use for risk stratification in clinical trials. Because they have different proportions of genetic alterations and outcomes, younger and older patients should be reported separately when using the ELN classification.

The first case described: monozygotic twin sisters with the fragile X premutation but with a different phenotype for premature ovarian failure.

OBJECTIVE: To describe the first case of monozygotic twin sisters with fragile X premutation and discordance for premature ovarian failure (POF). DESIGN: A descriptive case study. SETTING: Academic center. PATIENT(S): Monozygotic twin sisters with fragile X premutation and discordance for POF. INTERVENTION(S): Serum laboratory testing, fragile X premutation screening, zygosity testing, X-inactivation ratio and Southern blot studies. MAIN OUTCOME MEASURE(S): Incidence of POF in this twin cohort. RESULT(S): Zygosity analysis using polymerase chain reaction of 15 polymorphic markers via capillary gel electrophoresis in these patients confirmed their monozygosity. X-inactivation studies were performed using the human androgen receptor (HUMARA) gene and revealed similar X-inactivation ratios for both the patient and her sister (11:89 and 12:88, respectively) from peripheral serum samples. Southern blot evaluation of the proband and her sister revealed a similar methylation pattern in which the premutation allele was unmethylated much more than the normal allele. The contribution of the premutation on the active allele as determined by Southern blot analysis was consistent between sisters. CONCLUSION(S): The inactivation ratio studies and subsequent Southern blot analysis do not show differences between the patients; therefore, we are unable to identify a causative mechanism for the identical sisters' discordant phenotypes. It is possible that the inactivation ratios observed from the peripheral blood specimens obtained from the sisters do not represent the allele expression and skewing present at the level of the ovary.

Possible role of engraftment syndrome and autologous graft-versus-host disease in myelodysplastic syndrome after autologous stem cell transplantations: retrospective analysis and review of the literature.

BACKGROUND: We report a retrospective study of 452 patients with lymphoma from 1991 to 2006, with 274 men and 178 women, median age of 50 years (range, 16-76 years). PATIENTS AND METHODS: There were 85 patients with Hodgkin lymphoma (HL) and 367 with non-Hodgkin lymphoma (NHL). Eleven patients received a second autologous transplantation for progressive lymphoma, and another 4 received a second allogeneic transplantation for myelodysplastic syndrome (MDS). Twenty-seven patients had skin biopsies, and 2 patients had gastrointestinal biopsies consistent with graft-versus-host disease (GVHD), and 11 patients developed severe engraftment syndrome (ES), as defined by noninfectious fever and skin rash with or without pulmonary infiltrates requiring systemic steroids. RESULTS: The median follow-up of the patients was 6.2 years, and median overall survival was 5.3 years. Twenty-four patients (5.3%) developed MDS with median time of onset of 4.2 years (range, 8 months to 7.5 years). An additional 5 patients developed clonal karyotypic abnormalities in the bone marrow without clinical MDS. Actuarial probabilities of developing MDS at 5 and 8 years after transplantation were 5% and 15%, respectively. CONCLUSION: The incidences of MDS are similar in HL and NHL. Multivariate analysis revealed older age, occurrence of ES/GVHD, and longer intervals between the initial diagnoses to transplantation as independent factors. It is conceivable that perturbation to the host immunity caused by either previous chemotherapy or conditioning regimens in the elderly might play a role in the development of MDS after autologous transplantation.

The genetic architecture of Down syndrome phenotypes revealed by high-resolution analysis of human segmental trisomies.

Down syndrome (DS), or trisomy 21, is a common disorder associated with several complex clinical phenotypes. Although several hypotheses have been put forward, it is unclear as to whether particular gene loci on chromosome 21 (HSA21) are sufficient to cause DS and its associated features. Here we present a high-resolution genetic map of DS phenotypes based on an analysis of 30 subjects carrying rare segmental trisomies of various regions of HSA21. By using state-of-the-art genomics technologies we mapped segmental trisomies at exon-level resolution and identified discrete regions of 1.8-16.3 Mb likely to be involved in the development of 8 DS phenotypes, 4 of which are congenital malformations, including acute megakaryocytic leukemia, transient myeloproliferative disorder, Hirschsprung disease, duodenal stenosis, imperforate anus, severe mental retardation, DS-Alzheimer Disease, and DS-specific congenital heart disease (DSCHD). Our DS-phenotypic maps located DSCHD to a <2-Mb interval. Furthermore, the map enabled us to present evidence against the necessary involvement of other loci as well as specific hypotheses that have been put forward in relation to the etiology of DS-i.e., the presence of a single DS consensus region and the sufficiency of DSCR1 and DYRK1A, or APP, in causing several severe DS phenotypes. Our study demonstrates the value of combining advanced genomics with cohorts of rare patients for studying DS, a prototype for the role of copy-number variation in complex disease.

Central review of cytogenetics is necessary for cooperative group correlative and clinical studies of adult acute leukemia: the Cancer and Leukemia Group B experience.

The Cancer and Leukemia Group B has performed central review of karyotypes submitted by institutional cytogenetics laboratories from patients with acute myeloid (AML) and acute lymphoblastic (ALL) leukemia since 1986. We assessed the role of central karyotype review in maintaining accurate, high quality cytogenetic data for clinical and translational studies using two criteria: the proportion of karyotypes rejected (i.e. inadequate), and, among accepted (i.e. adequate) cases, the proportion of karyotypes whose interpretation was changed on central karyotype review. We compared the first four years during which central karyotype review was performed with a recent 4-year period and found that the proportion of rejected samples decreased significantly for both AML and ALL. However, during the latter period, central karyotype reviews still found 8% of AML and 16% of ALL karyotypes inadequate. Among adequate cases, the karyotype was revised in 26% of both AML and ALL samples. Some revisions resulted in changing the patients' assignment to particular World Health Organization diagnostic categories and/or moving patients from one prognostic group to another. Overall, when both data on rejection rates and data on karyotype revisions made in accepted cases were considered together, 32% of AML and 38% of ALL samples submitted were either rejected or revised on central karyotype review during the recent 4-year period. These data underscore the necessity of continued central karyotype review in multi-institutional cooperative group studies.