A Case of Acute Myeloid Leukemia Harboring a Rare Three-Way Translocation t(5;7;7) Involving the PDGFRB Gene and Successfully Treated with Imatinib.

Platelet-derived growth factor-beta (PDGFRB) gene maps for the receptor tyrosine kinase PDGRFß. PDGFRB gene fusions have been implicated in multiple myeloid and lymphoid neoplasms and have shown exquisite sensitivity to tyrosine kinase inhibitors. We report a case of a 29-year-old male who presented with acute myeloid leukemia who was eventually found to harbor a unique three-way translocation t(5;7;7)(q33.2;q32;q11.2) involving the PDGFRB gene. The patient initially achieved a complete response after induction with daunorubicin and cytarabine, but when he returned for consolidation, his white cell count had increased, and he was found to have an underlying myeloproliferative neoplasm. He was given consolidation with high-dose cytarabine and imatinib with excellent response, and ultimately received a matched unrelated donor transplant. The patient remains in remission to this day more than eight years later.

Preparation, Culture, and Analysis of Amniotic Fluid Samples.

Amniotic fluid obtained via amniocentesis provides a source of fetal material used in prenatal diagnosis. The fluid may be used directly for biochemical analyses, fluorescence in situ hybridization (FISH), and isolation of DNA for molecular studies, including chromosomal microarray analysis (CMA). The fluid is typically cultured as a source of metaphase cells for chromosome analysis and to provide additional material for biochemical and DNA-based testing. This unit describes an in situ method for the preparation, culture, and harvest of amniotic fluid samples for metaphase chromosome analysis. Cells are grown, harvested for metaphase spreads, and analyzed on glass coverslips. The unit also describes methods to obtain cells for additional studies (such as molecular genetic analyses) by growing cells in flasks either following passaging of cells from a glass coverslip culture or by directly establishing a flask culture from the amniotic fluid specimen. When cells are grown in flasks, they must be removed from the flask with trypsin before they can be used in studies. Lastly, this unit describes a method for isolating DNA for CMA from uncultured amniotic fluid and cultured cells. © 2018 by John Wiley & Sons, Inc.

Preparation, culture, and analysis of amniotic fluid samples.

Amniotic fluid obtained via amniocentesis provides a source of fetal material used in prenatal diagnosis. The fluid is used directly for biochemical analyses and as a source of fetal cells for biochemical assays, DNA testing, and chromosome studies. This unit describes an in situ method for the preparation, culture, and harvest of amniotic fluid samples for chromosome analysis. Cells are grown, harvested for metaphase spreads, and analyzed on glass coverslips. The unit also describes methods to obtain cells for additional studies (such as molecular genetic analyses) by growing cells in flasks either following passaging of cells from a glass coverslip culture or by directly establishing a flask culture from the amniotic fluid specimen. When cells are grown in flasks, they must be removed from the flask with trypsin before they can be used in studies.

Anaplastic lymphoma kinase-positive large B-cell lymphoma with complex karyotype and novel ALK gene rearrangements.

Anaplastic lymphoma kinase-positive large B-cell lymphoma is a rare non-Hodgkin lymphoma that exhibits a characteristic immunoblastic/plasmablastic morphology and is frequently associated with t(2;17)(p23;q23) and expression of Clathrin-anaplastic lymphoma kinase fusion protein. Here, we report a refractory anaplastic lymphoma kinase-positive large B-cell lymphoma in a 49-year-old human immunodeficiency virus-positive man. The neoplastic cells expressed CD138, epithelial membrane antigen, CD45, and perforin, and showed a strong granular cytoplasmic anaplastic lymphoma kinase staining pattern. Conventional chromosome analysis revealed a clone with multiple anomalies and a chromosome count of 76 to 79. Fluorescence in situ hybridization studies showed 5 copies of the ALK gene with 2 intact signals and 3 signals resulting from 2 independent, previously unreported, rearrangements. One rearrangement, seen in 2 copies, involved translocation of ALK sequences to chromosome Xq21. The second rearrangement involved translocation of ALK sequences to chromosome 12q24.1. This case broadens the cytogenetic alterations in anaplastic lymphoma kinase-positive large B-cell lymphoma, and it also demonstrates the high genetic instability of this tumor.

Trisomy 8 in B-cell chronic lymphocytic leukemia.

Association between trisomy 8 and myeloid disorders/malignancies has been well documented. We report on two patients with a known history of B-cell chronic lymphocytic leukemia (B-CLL) with bone marrow involvement. In addition to the classic B-CLL cytogenetic abnormalities in one of the patients, both showed a trisomy 8 clone in their bone marrow specimens. Using a BioView Duet automated scanning system, which allowed us to combine histology with fluorescence in situ hybridization, we showed that the trisomy 8 clone was restricted to the myeloid lineage. We believe that this finding signifies the development of myelodysplastic syndrome (de novo or therapy related), rather than progression of B-CLL with the occurrence of a new clone, and that in general, it has implications for the finding of trisomy 8 in CLL.

Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma with a complex karyotype and cryptic 3' ALK gene insertion to chromosome 4 q22-24.

Anaplastic lymphoma kinase (ALK)-positive diffuse large B-cell lymphoma (DLBCL) is a rare tumor that is frequently associated with t(2;17)(p23;q23), a translocation fusing the ALK gene at 2p23 to the clathrin heavy chain gene (CLTC) at 17q23. Here, we report a unique case of ALK-positive DLBCL with plasmablastic morphology and focal cytoplasmic granular ALK stain in an HIV-negative 33-year-old man. By conventional karyotyping, the lymphoma cells were near-tetraploid and included 4 structurally normal copies each of chromosomes 2 and 17. Fluorescence in situ hybridization revealed an apparently normal, intact ALK gene on each of the 4 chromosome 2 homologs plus a cytogenetically cryptic ALK gene insertion into 2 of the 4 chromosome 4 homologs at band 4q22-24. The lymphoma cells expressed CD138, lambda light chain, focal and weak CD30, and exhibited aberrant T-cell antigens, including perforin. This case indicates that ALK-positive DLBCL is more heterogeneous at the cytogenetic/molecular level than previously recognized.

Single-cell DNA and FISH analysis for application to preimplantation genetic diagnosis.

The goal of preimplantation genetic diagnosis (PGD) is to avoid transfer of embryos affected with a specific genetic disease or condition. This unit describes the steps involved in amplifying DNA from a single blastomere and specific assays for detecting a variety of DNA mutations. For some assays, whole-genome amplification by primer-extention preamplification (PEP) is performed prior to analysis. Support protocols describe the biopsy of one or two blastomeres from the developing preimplantation embryo, isolation for further investigation of all blastomeres from embryos shown to have the mutant allele, and isolation of single lymphocytes or lymphoblastoid cells as models for single-cell DNA analysis. A procedure for FISH analysis on single interphase blastomeres is provided along with a support protocol for probe validation that is recommended as a preliminary step before performing any PGD FISH analysis.