The Role of Repeat DNA Sequences in Human Evolution and Disease.

OBJECTIVES: Segmental chromosome duplications are an important evolutionary mechanism to produce new gene functions. Once an initial duplication takes place, the probability of a second event (structural change) increases. Segmental duplications (SDs) occur in many sizes and configurations. It has long been thought that SDs contribute to rapid evolution in primate genes. SDs tend to cluster around core duplicons, evolutionarily conserved sequences that are often shared between primate species. Many known SDs are associated with predisposition to human chromosome abnormalities and genetic diseases. Chromosome abnormalities in which SDs are implicated as the etiology include deletions, interstitial duplications, inversions, supernumerary marker chromosomes and translocations. Genomic abnormalities include DiGeorge and Velocardiofacial syndromes, Williams-Beuren syndrome, Prader-Willi and Angelman syndromes, cat eye syndrome, and many more. Segmental duplications also may play an important role in the etiology of some cancers. Most models for the likely mechanism of SD formation and expansion are based upon misalignment between juxtaposed non-allelic segmental duplications, followed by recombination.

A case of recurrent epilepsy-associated rosette-forming glioneuronal tumor with anaplastic transformation in the absence of therapy.

Rosette-forming glioneuronal tumor (RGNT) most commonly occurs adjacent to the fourth ventricle and therefore rarely presents with epilepsy. Recent reports describe RGNT occurrence in other anatomical locations with considerable morphologic and genetic overlap with the epilepsy-associated dysembryoplastic neuroepithelial tumor (DNET). Examples of RGNT or DNET with anaplastic change are rare, and typically occur in the setting of radiation treatment. We present the case of a 5-year-old girl with seizures, who underwent near total resection of a cystic temporal lobe lesion. Pathology showed morphologic and immunohistochemical features of RGNT, albeit with focally overlapping DNET-like patterns. Resections of residual or recurrent tumor were performed 1 year and 5 years after the initial resection, but no adjuvant radiation or chemotherapy was given. Ten years after the initial resection, surveillance imaging identified new and enhancing nodules, leading to another gross total resection. This specimen showed areas similar to the original tumor, but also high-grade foci with oligodendroglial morphology, increased cellularity, palisading necrosis, microvascular proliferation, and up to 13 mitotic figures per 10 high power fields. Ancillary studies the status by sequencing showed wild-type of the isocitrate dehydrogenase 1 (IDH1), IDH2, and human histone 3.3 (H3F3A) genes, and BRAF studies were negative for mutation or rearrangement. Fluorescence in situ hybridization (FISH) showed codeletion of 1p and 19q limited to the high-grade regions. By immunohistochemistry there was loss of nuclear alpha-thalassemia mental retardation syndrome, X-linked (ATRX) expression only in the high-grade region. Next-generation sequencing showed an fibroblast growth factor receptor receptor 1 (FGFR1) kinase domain internal tandem duplication in three resection specimens. ATRX mutation in the high-grade tumor was confirmed by sequencing which showed a frameshift mutation (p.R1427fs), while the apparent 1p/19q-codeletion by FISH was due to loss of chromosome arm 1p and only partial loss of 19q. Exceptional features of this case include the temporal lobe location, 1p/19q loss by FISH without true whole-arm codeletion, and anaplastic transformation associated with ATRX mutation without radiation or chemotherapy.

Interphase FISH for Residual Disease: Proposal for a Qualitative Determination of Rare Events.

OBJECTIVES: Interphase fluorescence in situ hybridization (FISH) cutoff values are calculated using various mathematical methods to determine whether abnormalities seen are at reportable (statistically significant) levels. However, for interphase FISH studies of samples obtained from oncology patients who have been transplanted or treated, these cutoff values may result in reporting a false negative result due to the small percentage of residual disease that falls below such a cutoff value. Failure to detect the rare abnormal cells may impact patient care and prognosis. For such situations, the two questions are: is the disease still present, and if so, how prevalent is it? The first question is qualitative and the second is quantitative. Traditionally, only the quantitative parameters have been used for determining reportability. Here we propose a method to account for both qualitative and quantitative evaluations of interphase FISH results.

MECOM (EVI1) Rearrangements: A Review and Case Report of Two MDS Patients with Complex 3q Inversion/Deletions.

Acute myelogeneous leukemia (AML) with inv(3)/t(3;3)(q13q25) is associated with aberrant expression of the stem-cell regulator MECOM (aka EVI1). Two bone marrow samples received in the OHSU Knight Diagnostic Laboratories (KDL) Cytogenetics Laboratory for chromosomes and FISH for a question of progression of myelodysplastic syndrome (MDS) to AML showed complex abnormalities including a deletion of chromosome 3q, one with del(3)(q13q25) and the other with del(3)(q22q25). In light of the prognostic importance of the activation of the MECOM oncogene and the concurrent inactivation of the GATA2 tumor suppressor that occurs with the classic inversion of chromosome 3q, fluorescence in situ hybridization (FISH) was performed using two different probe designs to better define the 3q deletions in the two cases. Using the Abbott Molecular Laboratories dual fusion MECOM/RPN1 probe, interphase and metaphase cells in both patients showed a variant single fusion (orange/green/fusion) signal pattern consistent with fusion and deletion. Using the three-color (red/green/aqua) Cytocell EVI1 probe, interphase cells in both cases showed a split red/green signal with the aqua signal remaining with the green signal. The distance between the split signals was generally less than is usually seen in the commonly described inverted chromosome 3. These findings are therefore consistent with a complex inversion and concurrent deletion/deletions of chromosome 3q. Thus, the deletion 3q seen in G-banded chromosomes from bone marrow from these two patients is most consistent with the activation of MECOM and the inactivation of GATA2.

A New Rhesus Macaque Karyotype Based on Human-rhesus Synteny.

Rhesus macaque (Macaca mulatta), because of their similarity to humans, are often used to study complex neurobiology and anatomy, cardiovascular disease, and in vaccine development. While the rhesus genome is studied on its own by primatologists, the grand majority of rhesus macaque research is done with the intention of extrapolating the findings to human diseases and traits. As such, it makes sense that the rhesus genome and karyotype be arranged based on homology to human chromosomes in an effort to ease the comparisons between the two, and aide in interpreting data generated using rhesus macaque model systems. Various approaches have been utilized, including linkage analyses using radiation hybrid markers and human microsatellite loci, and next generation sequencing, to create a comprehensive rhesus genome. Here, we present for the first time, the rhesus macaque karyotype adjusted and renumbered to reflect human homology, and to complement the newly completed sequencing data.

The New Equivocal: Changes to HER2 FISH Results When Applying the 2013 ASCO/CAP Guidelines.

OBJECTIVES: Human epidermal growth factor receptor 2 (HER2, ERBB2) testing is an important prognostic/predictive marker in breast cancer management, especially in selecting HER2-targeted treatment. American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines address HER2 status and were recently revised in 2013, replacing the 2007 version. For in situ hybridization interpretation, 2013 guidelines return to the prior threshold of a HER2/CEP17 ratio of 2.0 or greater for positive and eliminate 1.8 to 2.2 as the equivocal range. Also, the HER2 signal/nucleus ratio is accounted for, with 6.0 or greater for positive and 4.0 to less than 6.0 for equivocal, even in cases with a HER2/CEP17 ratio less than 2.0. METHODS: With institutional review board approval, we reviewed our 2006 to 2012 HER2 fluorescence in situ hybridization (FISH) results and classified them according to both the 2007 and 2013 guidelines as negative, positive, or equivocal. RESULTS: Of 717 HER2 FISH results, 55 (7.7%) changed category when reassessed by 2013 guidelines. Nineteen of 25 results in the 2007 equivocal category were reassigned as positive (n = 13) or negative (n = 6). Thirty-five previously negative cases became equivocal in the 2013 scheme, 12 of these with 1+ immunohistochemistry. The positive category increased from 71 to 85. CONCLUSIONS: The 2013 ASCO/CAP guidelines increased the number of HER2 FISH positive and equivocal results. The equivocal group is substantially different, posing a dilemma for clinical management.

ALK FISH Assays for Non-small Cell Lung Cancer Treatment Selection: Scoring Issues and Probe Design.

Lung cancer is becoming more treatable with new pharmaceuticals. There is a growing interest in FISH testing to determine which drugs to use on lung cancer patients for the best results, with a concurrent need for laboratories to provide ALK FISH testing for nonsmall-cell lung cancer. However, the test has some unique difficulties and requires a great deal of experience to interpret in some cases. A three-color probe may help make the ALK FISH test more reliable.

FISH testing for deletions of chromosome 6q21 and 6q23 in hematologic neoplastic disorders.

Deletions of chromosome 6q are found in many types of cancer, including melanoma, prostate cancer, fibroadenomas, and carcinoma of breast and other sites. Chromosome 6q deletions are also commonly found in lymphoid malignancies such as acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkins lymphoma (NHL), multiple myeloma (MM), mantle zone lymphoma (MZL), and Waldenström's macroglobulinemia (WM). These deletions may appear to be terminal or interstitial. In childhood B- and T-cell ALL a deletion of 6q is the hallmark of a neutral prognosis; however, it may be cytogenetically obscure or cryptic, requiring interphase FISH analysis. In adult ALL it indicates a favorable prognosis, but in CLL, B-cell small lymphocytic lymphoma (SLL), WM, and MM it has a poor prognosis. Since the deletion of 6q has prognostic implications and may be used to monitor residual disease, FISH analysis remains a valuable tool in treatment of these disorders. Using our three-color FISH probe cocktail for 6 centromere, 6q21 and 6q23, we began a validation series. We report the results of the first three patients in our laboratory with deletions of 6q in lymphoid malignancies using this cocktail.

Cytogenetic analysis of a hemosiderotic fibrolipomatous tumor.

Hemosiderotic fibrolipomatous tumor is an extremely rare, nonencapsulated, fatty lesion with a consistent histologic appearance that was originally considered reactive in nature. To our knowledge, there are no previous reports on the cytogenetics of this lesion. Reported here is a case of hemosiderotic fibrolipomatous tumor arising within the subcutaneous tissue of the right foot, dorsal aspect, of an otherwise healthy 35-year-old woman. Subsequent cytogenetic analysis revealed a clonal reciprocal translocation between chromosomes 1 and 10, with a further rearrangement involving this derivative chromosome 1 and chromosome 3. This, in addition to its characteristic morphology and immunophenotype, supports the neoplastic nature of this tumor and may aid in its diagnosis.

Evaluation of Her-2/neu status in carcinomas with amplified chromosome 17 centromere locus.

Accurate assessment of Her-2/neu (erb-b2) status in breast carcinoma is essential for therapy planning. Clinical assays are targeted at protein overexpression (immunohistochemical analysis) or gene amplification (fluorescence in situ hybridization [FISH]). Cases with aberrant FISH signal patterns are problematic and may lead to underreporting of Her-2/neu amplification. We performed FISH with additional chromosome 17 probes, SMS (Smith-Magenis syndrome critical region) and RARA (retinoic acid receptor), on 7 cases with unusual Her-2/CEP17 (chromosome 17 centromere control probe) results to assess whether different measurements of chromosome 17 copy number might clarify the Her-2/neu amplicon status. Although the Her-2/CEP17 ratio scores were within normal range (<2.0), the Her-2/SMS or Her-2/RARA ratio revealed amplification of Her-2/neu in 5 of 7 cases. Immunohistochemical analysis demonstrated Her-2/neu protein overexpression in the same 5 cases only. We describe novel application of SMS/RARA FISH probes for assessing cases with complex Her-2/CEP17 FISH patterns. Such additional data, correlated with immunohistochemical analysis, may help guide therapy in patients with breast carcinoma.

Split centromeric alpha-satellite FISH signals in a whole arm translocation 5p;10p: consequences and implications for interphase FISH studies.

This is a case report of an apparently balanced whole arm translocation between the short arms of chromosomes 5 and 10 in which the centromeric alpha-satellite DNA is split between both derivative chromosomes for both probes, leading to abnormal signal patterns. The patient requested preimplantation genetic testing for the unbalanced products of the translocation. However, using centromeric alpha-satellite DNA probes as controls for the subtelomeric-specific probes in interphase was not informative because of the split signals. The ramifications of such variation in the alpha-satellite regions of chromosomes for other interphase FISH tests are discussed.

Meiotic exchange event within the stalk region of an inverted chromosome 22 results in a recombinant chromosome with duplication of the distal long arm.

Meiotic recombination occurs between homologous euchromatic regions of human chromosomes in early meiosis. However, such exchanges have been thought not to occur in the stalk regions of acrocentric chromosomes. We describe a child whose chromosome analysis suggests that crossovers do occur in homologous stalk regions. The proband, initially seen as a term female infant, was born to a 28-year-old mother. Dysmorphic features included wide metopic sutures, low anterior hairline, hypertelorism, external ear malformations, and cleft lip and palate. Blood chromosomes of the proband and parents were studied by G-banding, Q-banding, R-banding, and silver staining. The infant karyotype showed a sub-metacentric chromosome 22; that of the mother showed a pericentric inversion of chromosome 22. Chromosomes of the father were normal. In the infant, the abnormal chromosome 22 long arm appeared normal, but with additional long arm material attached to the distal short arm. In the mother, the distal long arm of the abnormal chromosome 22 was translocated to the distal short arm. The abnormal chromosome stalk in the child was intermediate in size to the stalk size of the abnormal and normal chromosomes 22 in the mother. Fluorescent in situ hybridization (FISH) analysis using chromosome 22 paint and ARSA gene probe confirmed that the duplicated material in the proband was of chromosome 22 origin; the karyotype interpretation is: 46,XX,rec(22)dup(22q)inv(22)(p13q13.1)mat. This abnormal karyotype is most likely due to a crossover event within the inversion loop during meiosis. The stalk length discrepancy suggests that the crossover site occurred in the stalk region.

Trisomy 8 as the sole cytogenetic abnormality in a case of extraskeletal mesenchymal chondrosarcoma.

Mesenchymal chondrosarcoma is a rare malignant tumor that comprises about 3-10% of all sarcomas. Reports of cytogenetic studies of mesenchymal chondrosarcoma are limited and no consistent cytogenetic abnormality has surfaced. Some mesenchymal chondrosarcomas have a t(11;22) translocation suggesting a relationship with the PNET/Ewing tumor family. We report what to our knowledge is the first case of trisomy 8 as the sole cytogenetic abnormality in a mesenchymal chondrosarcoma.

Y chromosome heterochromatin of differing lengths in two cell populations of the same individual.

OBJECTIVE: To present a prenatal diagnosis report on a case where G-banding analysis of fetal metaphase chromosomes showed populations of cells with two different Y chromosomes; one with a short block of heterochromatin (Yqh-) and one with a longer block of heterochromatin (Yqh+). METHODS: These two populations of the Y chromosome were studied using fluorescent quinacrine banding and fluorescent in situ hybridization (FISH). A chromosome paint corresponding to the euchromatic region of the Y chromosome, and probes corresponding to the SRY, DYZ1, and DYZ3 regions were used for this study. RESULTS: Both Y chromosomes appeared to be structurally normal by these analyses. Subsequent ultrasound examination at 20 weeks' gestation revealed normal male genitalia. Follow-up with a neonatal blood sample analysis confirmed the above findings. CONCLUSIONS: This study reports a direct prenatal diagnosis case of two populations of the Y chromosome in the same individual. This apparent mosaicism may be explained by a postzygotic simple deletion or unequal crossover event between sister chromatids in the DYZ region.

The role for preimplantation genetic diagnosis in balanced translocation carriers.

OBJECTIVE: Preimplantation genetic diagnosis is an established technique that provides an alternative to prenatal diagnosis for patients who are at risk of transmitting a serious genetic disorder to their offspring. Preimplantation genetic diagnosis has been used for couples who have been at risk for having offspring with single gene or X-linked disorders and for screening for common age-related aneuploidy and in couples who themselves carry balanced chromosomal rearrangements. The aim of this study was to summarize our experience using preimplantation genetic diagnosis after the identification of a parental balanced translocation, specifically as it relates to the number of embryos that are suitable for transfer after preimplantation genetic diagnosis for a known translocation and aneuploidy screening. STUDY DESIGN: This is a retrospective review of data from a single center that involved 6 couples that initiated the process of preimplantation genetic diagnosis for translocation and aneuploidy screening by fluorescent in situ hybridization. RESULTS: A total of 65 embryos were obtained, of which 56 embryos (86%) were suitable for fluorescent in situ hybridization analysis. After fluorescent in situ hybridization, 1 embryo was diagnosed as normal or balanced (1.7%). Forty-three embryos (76.8%) were unbalanced for the translocation; 8 embryos (14.3%) were aneuploid, and 4 embryos (7.1%) were uninformative. There were no clinical pregnancies. CONCLUSION: In our experience, there are very few embryos that are available for transfer from these patients after translocation and aneuploidy screening because of multiple unbalanced segregation products and a high rate of aneuploidy. Factors that contributed to this may be related to which parent carries the translocation, methods that were used for in vitro fertilization, and advanced maternal age. Although preimplantation genetic diagnosis for translocation carriers theoretically can enhance the pregnancy rate for a couple, there are limitations. This information should be shared with couples who are contemplating preimplantation genetic diagnosis for translocation, and the options of sperm or egg donor should be considered.

Myxoinflammatory fibroblastic sarcoma with complex supernumerary ring chromosomes composed of chromosome 3 segments.

Myxoinflammatory fibroblastic sarcoma is a rare, recently described, and distinctive low-grade tumor of soft tissue. To our knowledge, there is only one previous report on the cytogenetics of this tumor. That case showed complex structural abnormalities, including a reciprocal translocation between chromosomes 1 and 10 [t(1;10)(p22;q24)] with loss of chromosomes 3 and 13. We describe here a second case showing supernumerary ring chromosomes, and a derivative chromosome 13, with additional material on the short arm. We conclude that the presence of chromosomal abnormalities supports the neoplastic nature of this tumor and aids in its diagnosis. Furthermore, we also postulate that the finding of ring chromosomes, which have been identified in other low-grade soft-tissue tumors, may have important prognostic implications regarding the aggressiveness of this neoplasm.