Metaphase Fluorescence In Situ Hybridization Research Articles

JAK2 V617F mutation positive primary myelofibrosis with concomitant t(9;11;22)(q34;p15;q11.2) but no BCR/ABL fusion

Dear Sir, A 50-year-old woman with a strong family history of cancer (ca: father ca lung age 52, mother ca lung age 50, brother ca colon age 42) and hepatitis B virus carriage on tenofovir presented with anemia [hemoglobin (Hb), 5.6 g/ dL; white blood cell (WCC), 19.4 9 10/L; platelet (Plat), 150 9 10/L with a leukoerythroblastic peripheral blood picture (Fig. 1a). Gross splenomegaly was also observed (Fig. 1b). Serum lactate dehydrogenase (LDH) was raised at 1985 IU/L, reference range 143–280]. A marrow biopsy showed osteosclerosis with markedly thickened bony trabeculae, narrowed intertrabecular spaces (Fig. 1c), and dilated sinusoids. Focal areas showed prominent blasts and clusters of megakaryocytes abutting on vascular sinuses (Fig. 1d). Reticulin fibres are markedly coarsened. The overall features were consistent with the fibrotic stage of primary myelofibrosis (PMF). A polymerase chain reaction (PCR) test showed the presence of JAK2 V6517F mutation. Cytogenetic studies, however, showed a sub-clone with a three-way translocation generating a Ph chromosome: 47,XX, ? 8[1]/47,idem,t(9;11;22)(q34;p15;q11.2)[5]/46,XX[1] (Fig. 1e). Reverse transcription PCR and fluorescent in situ hybridization (FISH) studies showed no BCR/ABL fusion (Fig. 1e). There was no clinical response to a trial of imatinib mesylate. She was treated with oral hydroxyurea, and a splenectomy (1.5 kg) specimen showed extramedullary haemopoiesis only. One year later, she remained stable (Hb, 7.9 g/dL; WCC, 22.7 9 10/L; Plat, 226 9 10/L; LDH, 655 IU/L) on regular transfusion and chelation. She refused work up for stem-cell transplantation and remained well at 2 years post-diagnosis. In myeloproliferative disorders (MPD) JAK2 mutations and BCR/ABL fusion are regarded as hallmark events. They occur in polycythemia vera/essential thrombocytosis/PMF and chronic myelogenous leukemia (CML), respectively. Although there may be preceding genetic events, both are definitive clonal markers and are mutually exclusive. There are many reports of patients possessing both the JAK2 V617F mutation and BCR/ABL transcripts. These two lesions can occur in either sequence, resulting in a transition in disease phenotype [1, 2]. Rarely, they may occur concomitantly at diagnosis, resulting in a mixed disease phenotype [3, 4]. A review of nine cases showed a median age of 48 years (range 32–66), male predominance (8/9), and a median lapse of 48 months (0–144) between the two disorders [3]. The two clones are always distinct [1–3, 5, 6]. The response of the CML element to tyrosine kinase inhibitor (TKI) is good and overall prognosis is favorable. The emergence of the second MPD should not be mistaken for TKI failure [5]. In our patient, the three-way translocation generates a morphological Ph chromosome-looking der(22). This was a subclonal evolution from a stem trisomy 8 clone. There was no BCR/ABL fusion or any clinical response to TKI. The presentation and disease course is also typical for PMF. Hence the ‘‘Philadelphia’’ chromosome in our case appeared to be an incidental cytogenetic anomaly. On the metaphase FISH analysis, the entire ABL gene was translocated onto 11p, no residue ABL signal was found in 9q. W.-Y. Au (&) Department of Medicine, Queen Mary Hospital, UMU, 4/F, Professorial Block, Pokfulam Road, Hong Kong, China e-mail: auwing@hotmail.com

From the front line: the realities and challenges of microarray studies in cancer

Metaphase cytogenetics and fluorescence in situ hybridisation (FISH) play an integral role in diagnosis and clinical decision making algorithms for many malignancies. However, inherent methodological limitations mean it is not always possible to adequately characterise the malignant clone using these techniques. DNA microarrays [array comparative hybridisation (aCGH)/sin-gle nucleotide polymorphism arrays (SNP-A)] overcome some limitations of metaphase cytogenetics and FISH. Microarrays are not dependent on the ability to obtain metaphases and detect copy number changes across the genome at high resolution. SNP-A also detect copy neutral loss of heterozygosity (CN-LOH) which may flag the presence of homozygous oncogenic mutations in the neoplastic clone. We have performed microarray testing on samples from patients with acute lymphoblastic leukaemia (ALL), myelodysplastic syndromes (MDS) and chronic lymphocytic leukaemia (CLL) because these disorders are characterised by recurrent copy number changes and CN-LOH with demonstrated clinical significance. In our experience, microarrays provide additional information about the neoplastic clone that complements the standard testing approach. Karyotypic complexity and the presence of contaminating non-malignant cells can pose problems with QC, interpretation, nomenclature and reporting. The position of microarrays in the hierarchy of cytogenetic testing in cancer is likely to be disease specific and is yet to be clearly defined.

Utility of Routine Bone Marrow Fluorescent in Situ Hybridization (FISH) Panel Testing in Patients Suspected of Myelodysplastic Syndrome (MDS): An Analysis of 3,418 Patients and Comparison of Academic Versus Community Practices

AbstractAbstract 3855 Background:Metaphase cytogenetics and FISH panel testing are commonly performed on bone marrow aspirate samples from patients suspected of having MDS. We performed this study to determine the frequency of simultaneous testing in academic versus community practices and whether routine FISH panel provides additional information to metaphase cytogenetics. Methods:After approval from our institutional review boards, a list of patients who had bone marrow aspirate samples submitted to the Wisconsin State Laboratory of Hygiene between January 2000 to June 2011, with a diagnosis of anemia, thrombocytopenia, neutropenia, or pancytopenia, and suspected MDS was obtained from Wisconsin State Laboratory of Hygiene. If multiple samples were submitted for the same patient, we included only the first sample. We collected data pertaining to demographics, indication/diagnosis, requesting institution, date of request, and cytogenetics/FISH results. The FISH panel included probes for chromosomes 5q31, 7q31, 8, and 20q (Abbott Molecular, Abbott Park, IL). Results:We included 3,418 patients who met our study criteria. The median age was 57.5 years (18–97 years) and 55.2% were males. The samples came from 1 academic center (36.9%), 4 community practices (44.7%), and 3 referral laboratories (18.4%). The top clinical indications for bone marrow aspiration were anemia (48.5%), pancytopenia (26.8%), thrombocytopenia (15.2%), neutropenia (5.2%) and possible MDS (2.3%). Karyotype was normal in 2,527 (73.9%), abnormal in 617 (18.1%), and could not be determined in 274 (8.0%) due to culture failure or inadequate karyotypes.Simultaneous testing for cytogenetics and FISH was performed in 379 patients (11.1%). In this subgroup, abnormal karyotypes were identified in 64 (16.8%) and abnormal FISH results in 55 (14.5%). Among the 299 samples which had adequate metaphases for cytogenetic analysis, FISH panel detected additional cytogenetic abnormalities in 11 (3.6%) patients. The cytogenetic abnormalities detected by FISH but not by karyotyping were −20q (5 patients), −7q (4 patients), −5q (1 patient), and −5q/-20q (1 patient). However, this would have changed the MDS International Prognostic Scoring System karyotype score in only 5 of these patients (1.7%) patients. In cases with inadequate karyotypes or no growth (n = 80), abnormal FISH result was found in 13 (16.2%). The cytogenetic abnormalities detected were −20q (4 patients), −7q (4 patients), −5q (3 patients), −5q/-7q/-20q (1 patient) and −5q/-7q (1 patient).The karyotype inadequacy/failure rates were similar regardless of whether samples came from academic center or the community (8.7% vs 7.6%; P = 0.277). However, community physicians were more likely to perform simultaneous cytogenetic and FISH testing compared to academic physicians (14.0% vs 6.2%; P < 0.0001). FISH testing detected additional abnormalities at a similar rate among samples coming from the academic and community practices (2.6% vs 7.3%; P = 0.190). There was a significant variability in the rates of simultaneous cytogenetic and FISH testing among community practices ranging from 0% to 23.4% (P < 0.0001). Conclusions:In our study of a large sample of patients being evaluated for cytopenias and suspected of MDS, routine bone marrow FISH panel testing added little to cytogenetic study except when karyotype failed or was inadequate. Community physicians requested simultaneous testing for cytogenetics and FISH significantly more often than their academic counterparts even though karyotype inadequacy or failure rate was similar. There was a large variation in simultaneous testing rates among community practices. Disclosures:No relevant conflicts of interest to declare.

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from ring chromosome 2

ObjectiveTo present prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome (sSMC) derived from ring chromosome 2 [r(2)]. Methods and ResultsA 35-year-old woman underwent amniocentesis at 17 weeks of gestation, because of advanced maternal age. Amniocentesis revealed a de novo ring-shaped sSMC in 11 of 23 colonies of cultured amniocytes. Repeated amniocenteses were made. The sSMC was characterized by array comparative genomic hybridization (aCGH), interphase fluorescence in situ hybridization (FISH) and quantitative fluorescent polymerase chain reaction (QF-PCR) on uncultured amniocytes. In uncultured amniocytes, aCGH showed a 39.49-Mb genomic gain in chromosome 2 encompassing 2q11.2→q21.2, interphase FISH revealed a mosaic level of 52% (52/100 cells), and QF-PCR manifested a diallelic pattern for chromosome 2, with gene dosage increase in the paternal allele of proximal 2q-specific DNA markers. In cultured amniocytes, the sSMC was characterized by metaphase FISH, spectral karyotyping (SKY) and multicolor banding (MCB) to contain the centromere and proximal 2q, and the karyotype was 47,XX,+r(2)(p11.1q21.2)[14]/46,XX[11]. The pregnancy was terminated. The fetus postnatally manifested facial dysmorphisms. Postnatal cytogenetic analyses revealed the karyotypes of 47,XX,+r(2)[12]/46,XX[28] in cord blood, 47,XX,+r(2)[7]/46,XX[33] in umbilical cord, 47,XX,+r(2)[13]/47,XX,+idic r(2)[3]/46,XX[24] in placenta and 47,XX,+r(2)[8]/47,XX,+idic r(2)[1]/46,XX[31] in amnion. ConclusionMolecular cytogenetic techniques such as aCGH, interphase FISH and QF-PCR on uncultured amniocytes, and SKY, MCB and metaphase FISH on cultured amniocytes are useful for characterization of the nature of a prenatally detected sSMC.

A systematic analysis of small supernumerary marker chromosomes using array CGH exposes unexpected complexity

A small supernumerary marker chromosome is often seen in patients with developmental disorders. Prior to array-based comparative genomic hybridization markers were rarely genotyped end to end. In this study, a valid genotype-to-phenotype correlation was possible because the supernumerary marker chromosomes were fully characterized by array-based comparative genomic hybridization in a genome-wide analysis. Ten consecutive de novo small supernumerary marker chromosome cases were systematically genotyped using G-banding, C-banding, AgNOR staining, whole-genome array-based comparative genomic hybridization, and fluorescence in situ hybridization. Among 10 small supernumerary marker chromosome cases studied, 4 (40%) were not identified by array-based comparative genomic hybridization because of low-level mosaicism or because they lacked euchromatin. One case (10%) was a simple pericentromeric marker extending from 5p13.3 to 5q11.2. Five (50%) markers showed unexpected complexity. Two cases had markers that were derivative acrocentric (AgNOR+) chromosomes with the euchromatin from chromosomes 18p or 19p. Each of the other three cases with complex markers had unusual characteristics including a marker from noncontiguous segments of chromosome 19q, a highly complex rearrangement involving a chromosome 20 homolog as well as the small supernumerary marker chromosome, and a mosaic duplication of a proximal 8p marker. Small supernumerary marker chromosomes are frequently complex on the basis of our small sample. Whole-genome array-based comparative genomic hybridization characterization of the small supernumerary marker chromosome provided informed genetic counseling.

Recurrent Rearrangement of the PHF1 Gene in Ossifying Fibromyxoid Tumors

Ossifying fibromyxoid tumor (OFMT) is a soft tissue tumor of unknown lineage. Although most cases are histologically and clinically benign, some show malignant morphological features and local recurrences are not uncommon; a few may even metastasize. In the present study, cytogenetic analysis identified different structural rearrangements of chromosome band 6p21 in tumor cells from three cases of OFMT, including one with typical, one with atypical, and one with malignant morphological features. Mapping of the 6p21 breakpoint by fluorescence in situ hybridization (FISH) indicated that the PHF1 gene was rearranged in all three cases. Further FISH, 5'-rapid amplification of cDNA ends, and RT-PCR analyses disclosed an EP400/PHF1 fusion transcript in one of the cases. Interphase FISH on tumor sections from 13 additional cases of OFMT showed rearrangement of the PHF1 locus in four of four typical, two of three atypical, and one of six malignant lesions. Thus, the PHF1 gene, previously shown to be the 3'-partner of fusion genes in endometrial stromal tumors, is also recurrently involved in the pathogenesis of OFMTs, irrespective of whether they are diagnosed as typical, atypical, or malignant lesions. The PHF1 protein interacts with the polycomb-repressive complex 2 (PRC2), which, in turn, regulates the expression of a variety of developmental genes. Thus, the results indicate that deregulation of PRC2 target genes is crucial for OFMT development.

FOSL1 as a candidate target gene for 11q12 rearrangements in desmoplastic fibroblastoma

Desmoplastic fibroblastoma (DF) is a benign fibroblastic/myofibroblastic tumor. Cytogenetic analyses have revealed consistent rearrangement of chromosome band 11q12, strongly suggesting that this region harbors a gene of pathogenetic importance. To identify the target gene of the 11q12 rearrangements, we analyzed six cases diagnosed as DF using chromosome banding, fluorescence in situ hybridization (FISH), single-nucleotide polymorphism array and gene expression approaches. Different structural rearrangements involving 11q12 were found in five of the six cases. Metaphase FISH analyses in two of them mapped the 11q12 breakpoints to an ∼20-kb region, harboring FOSL1. Global gene expression profiling followed by quantitative real-time PCR showed that FOSL1 was expressed at higher levels in DF with 11q12 rearrangements than in desmoid-type fibromatoses. Furthermore, FOSL1 was not upregulated in the single case of DF that did not show cytogenetic involvement of 11q12; instead this tumor was found to display a hemizygous loss on 5q, including the APC (adenomatous polyposis coli) locus, raising the possibility that it actually was a misdiagnosed Gardner fibroma. 5′RACE-PCR in two 11q12-positive DF did not identify any fusion transcripts. Thus, in agreement with the finding at chromosome banding analysis that varying translocation partners are involved in the 11q12 rearrangement, the molecular data suggest that the functional outcome of the 11q12 rearrangements is deregulated expression of FOSL1.

Dual-color FISH karyotype and rDNA distribution analyses on four Cucurbitaceae species

Karyotype and ribosomal DNA distribution on four Cucurbitaceae species was analyzed through dual-color fluorescence in situ hybridization (FISH) using 5S and 45S rDNA probes. Chromosome sizes varied slightly among the species with Cucumis sativus relatively the largest (∼2.5 μm) and Momordica charantia the smallest (∼1 μm). In Cucumis sativus L. (2n = 14), the 45S rDNA hybridized on the pericentromeric area of five chromosomes (metacentric a, b, c, g, and submetacentric d) and the 5S rDNA on the region proximal to the centromere of the short arm of chromosome e. In Luffa cylindrica (L.) Roem. (2n = 26), the 45S rDNA hybridized on the distal regions of the short arms of five chromosomes (metacentric a, b, c, f, and submetacentric d) and the 5S rDNA on the region proximal to the centromere of the short arm of chromosome e. In Lagenaria siceraria (Molina) Standl. (2n = 22), the 45S rDNA hybridized on the distal regions of the short arms of two chromosomes (submetacentric b and metacentric e) and the 5S rDNA juxtaposed with the 45S rDNA signal in a region proximal to the centromere on chromosome e. In Momordica charantia L. (2n = 22), the 45S rDNA hybridized on the majority of the short arms of two metacentric chromosomes (d and k) and the 5S rDNA on the proximal region of the short arm of chromosome e. The interphase and metaphase rDNA distribution and FISH karyotype analyses of the four species showed the possible fate of rDNAs through the process that lead to the interspecific variations among the cucurbits. These results will be useful in elucidating the phylogenetic relationships among Cucurbitaceae species.

Mosaic deletion-duplication syndrome of chromosome 3: Prenatal molecular cytogenetic diagnosis using cultured and uncultured amniocytes and association with fetoplacental discrepancy

Objective To present prenatal molecular cytogenetic diagnosis of mosaicism for terminal 3p deletion and distal 3q duplication using cultured and uncultured amniocytes, and the association with fetoplacental discrepancy. Materials, Methods, and Results A 35-year-old primigravid woman was referred for genetic counseling at 21 weeks of gestation because of 20% (5/25 colonies) mosaicism for add(3)(p26) detected by amniocentesis. Repeated amniocenteses were performed. Array comparative genomic hybridization (aCGH) and interphase fluorescence in situ hybridization (FISH) were applied in the uncultured amniocytes. aCGH analysis detected 0.15-Mb microdeletion of 3p26.3 with CHL1 haploinsufficiency and a 49.42-Mb duplication of 3q24-q29 in the uncultured amniocytes. Interphase FISH analysis revealed 27.3% mosaicism (12/44 cells) in the uncultured amniocytes. Metaphase FISH analysis revealed 23.3% mosaicism (7/30 cells) in the cultured amniocytes. Conventional cytogenetic analysis showed a karyotype of 46,XX,der(3)(qter → q24::p26.3 → qter)[10]/46,XX[20] (33% mosaicism). Subsequent fetal blood sampling showed a karyotype of 46,XX,der(3) (qter → q24::p26.3 → qter)[5]/46,XX[35] (12.5% mosaicism). The parents elected to terminate the pregnancy, and a malformed fetus was delivered at 24 weeks of gestation with characteristic facial dysmorphism and clinodactyly of the hands. Cytogenetic analysis of the extraembryonic tissues revealed the results of 46,XX (40 cells) in placenta, 25% mosaicism (10/40 cells) in amniotic membrane and 50% mosaicism (20/40 cells) in umbilical cord. Conclusion Our presentation highlights the utility of molecular cytogenetic technologies in prenatal diagnosis of rare mosaic chromosome rearrangements and provides evidence for fetoplacental discrepancy under such circumstances.

IAMP21 Is Associated with Inferior Outcomes in Children with Acute Lymphoblastic Leukemia (ALL) on Contemporary Children's Oncology Group (COG) Studies

Abstract 739Intrachromosomal amplification of a region of chromosome 21 (iAMP21) occurs in a 1–3% of children with ALL and can be identified by RUNX1 fluorescence in situ hybridization (FISH). We monitored the outcome of patients with iAMP21 in recent COG trials for newly diagnosed standard risk (SR; AALL0331) and high risk (HR; AALL0232) B-precursor ALL based on reports of inferior outcomes associated with this cytogenetic alteration. (Moorman et al Blood 2007)We examined the incidence, clinical characteristics and outcome for 7799 children, adolescents and young adults 1–30 years old enrolled on COG AALL0331 and AALL0232 between 2003 and 2011. All patients had testing for prognostically relevant cytogenetic alterations including ETV6-RUNX1 performed in COG central laboratories (2003-2006) or approved local cytogenetics laboratories with central review (2007-2011). Ascertainment of iAMP21 may have been incomplete prior to 2007 as ETV6-RUNX1 was primarily assessed centrally by RT-PCR. Classification as iAMP21 required >4 RUNX1 signals on a single chromosome (>5 total RUNX1 signals). If metaphase FISH was not possible, iAMP21 was identified as multiple copies of RUNX1 clumped in at least some of the nuclei. ALL cases defined as very high risk with t(9;22), hypodiploidy with <44 chromosomes or MLL rearrangements and slow early response to treatment were excluded from this analysis. Treatment on AALL0331 and AALL0232 consisted of a 3- (AALL0331) or 4-drug (AALL0232) induction, with post-induction therapy based on early response and established prognostic cytogenetic features. Therapy was not altered for patients with iAMP21.iAMP21 was identified in 158/7799 (2%) cases; 75/5060 (1.5%) SR cases and 83/2739 (3.0%) HR cases. Patients with iAMP21 were more likely to be ≥10 years old (49% vs. 33%, p<0.0001), have white blood cell counts (WBC) <50,000/μL (96% vs. 85%, p<0.0001), be female (54% vs. 34%, p=0.036) and to have ≥0.01% end Induction bone marrow minimal residual disease (MRD) (41% vs. 21%, p<0.0001) than those without iAMP21.While earlier analyses suggested that iAMP21 was not associated with inferior outcomes in COG AALL0232 and AALL0331 (Heerema et al, ASH 2009 abstract; 114: 2598), new analyses with larger patient numbers and longer follow-up show that the outcome for patients with iAMP21 is worse than for patients without iAMP21 (Table 1). These differences were statistically significant in SR, but not HR patients.Outcome comparisons were also made examining iAMP21 status and end induction MRD (<0.01% vs. ≥0.01%). Pooled SR and HR patients with iAMP21 who were MRD positive (≥0.01%) had significantly inferior outcomes with 4-year event-free survival (EFS) of 55.8±12.4% vs. 76.5±2% among non-iAMP21 MRD positive patients, p=0.037. For MRD negative patients, 4-year EFS for those with iAMP21 was significantly worse (81.4±7.8%) than for those without this feature (92.2±0.6%; p=0.016).In multivariate Cox regression analysis of AALL0331 patients, iAMP21 (hazard ratio (HR) 2.246; p=0.0021), day 29 MRD ≥0.01% (HR 2.430; p<0.0001) and favorable genetics (ETV6-RUNX1 or trisomies of chromosomes 4 and 10 (HR 0.361; p<0.0001) all had high prognostic significance, while iAMP21 was not significant in a multivariate Cox model in AALL0232 patients.In conclusion, patients with iAMP21 have inferior outcomes with contemporary chemotherapy in COG ALL trials and may benefit from more intensive or novel treatment approaches. In particular, lower intensity therapy given to SR ALL patients in COG AALL0331 led to significantly inferior outcomes for those with iAMP21.Table 1Outcomes in iAMP21 SR and HR ALLiAMP21Othersp-valueAALL0232+AALL0331N15876414-year EFS70.2 ± 7%88.1 ± 0.7%<0.00014-year OS84.7 ± 5.6%93.9 ± 0.5%0.0132AALL0331N7549854-year EFS70.4 ± 9.3%91.8 ± 0.7%<0.00014-year OS87 ± 6.9%96.5 ± 0.5%0.004AALL0232N8326564-year EFS70 ± 10.6%81 ± 1.3%0.464-year OS82.7 ± 9.2%88.9 ± 1.1%0.65 Disclosures:Borowitz:BD Biosciences: Research Funding. Mattano:Pfizer, Inc.: Employment. Wood:BD Biosciences: Research Funding.

Application of Fluorescence in situ Hybridization (FISH) Technique to Discern Complete/Partial Monosomy 21

The present study pertains to a 7 month old female infant who showed dysmorphic features and developmental delay. Conventional cytogenetic analysis by GTG banding technique was carried out and this revealed monosomy 21. Interphase FISH and metaphase FISH were employed for better delineation of the observed results. FISH analysis confirmed unbalanced X;21 translocation. This study concludes that FISH technique along with conventional cytogenetic analysis serves for better understanding and interpretation of complete/ partial autosomal monosomies.

Diagnostic Yield of Bone Marrow and Peripheral Blood FISH Panel Testing in Clinically Suspected Myelodysplastic Syndromes and/or Acute Myeloid Leukemia

It is unclear how often and in what setting fluorescence in situ hybridization (FISH) panels for myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) provide additional information over metaphase cytogenetics alone. Furthermore, the usefulness of peripheral blood vs bone marrow FISH has also not been directly compared. We prospectively compared metaphase cytogenetics and FISH for -5/5q-, -7/7q-, +8, and 20q- in 433 cases of suspected MDS/AML. FISH testing was abnormal in 6 (14%) of 43 and 10 (19%) of 54 cases with fewer than 20 normal metaphases or no growth, respectively. FISH was only rarely abnormal in cases with 20 normal metaphases obtained (6/222 [2.7%]). Comparison of peripheral blood and bone marrow results in 48 cases showed abnormal peripheral blood FISH results in 18 (69%) of 26 cases with abnormal bone marrow FISH results and in 5 (23%) of 22 cases with normal bone marrow FISH results. These findings, the largest published comparison of FISH vs metaphase cytogenetics in MDS/AML, provide a rational strategy for FISH testing in peripheral blood and bone marrow.

Unknown partner for USP6 and unusual SS18 rearrangement detected by fluorescence in situ hybridization in a solid aneurysmal bone cyst

USP6 rearrangement is the most common genetic abnormality in primary aneurysmal bone cyst, and SS18 rearrangement has not been previously described in any type of tumor where synovial sarcoma was excluded from the differential diagnosis. We report a case of solid aneurysmal bone cyst in which fluorescence in situ hybridization (FISH) analysis indicated rearrangements of both USP6 and SS18, but histologic features were consistent with aneurysmal bone cyst throughout the lesion. Reverse-transcription polymerase chain reaction (RT-PCR) for the SS18-SSX1 and SS18-SSX2 translocations, identity testing, and SS18 FISH were performed on cytogenetic monolayer cultures and formalin-fixed paraffin-embedded (FFPE) tissue. Genomic microarray, FISH, and immunohistochemistry were performed on follow-up studies of the FFPE specimen. The karyotype was 45,X,add(X)(p11.2),add(4)(q13),add(8)(p21),-13,add(17)(p11.2),add(18)(q11.2) in all 20 cells analyzed from monolayer cultures. The karyotype showed no cytogenetically visible alterations of chromosomal regions harboring known partners for USP6. Metaphase FISH with a commercial SS18 break-apart probe showed translocation of the 5' portion of the SS18 probe to the short arm of the derivative X, as is observed in synovial sarcoma. RT-PCR showed no evidence of a SS18-SSX fusion, and immunohistochemistry was negative for TLE1, EMA, and cytokeratin AE1/3 expression. FISH on FFPE sections with a custom break-apart probe flanking USP6 showed evidence for a USP6 rearrangement throughout the tumor (25-50%). FISH on FFPE sections with a commercial SS18 break-apart FISH probe showed more variable results (0-50% split signals). There was no evidence of a SS18-USP6 fusion by FISH or RT-PCR. A molecular inversion probe array revealed a deletion encompassing the entire SS18 gene and its promoter, as well as portions of the region targeted by the commercial SS18 FISH probe. In conclusion, results obtained from commercially available FISH probes may occasionally yield misleading results. In this case, the SS18 rearrangement by FISH resulted from a complex rearrangement of 18q11.2 with a deletion of the SS18 gene. The translocation partner for USP6 remains unknown in this case.

Variant Philadelphia Chromosome Identified by Interphase Fluorescence In Situ Hybridization (FISH) without Evidence on G-banded Karyotyping and Metaphase FISH

A variant Philadelphia chromosome (Ph) is generated from translocation of one or more partner chromosomes in addition to chromosomes 9 and 22. We have described the cases of 2 patients bearing variant Ph detected by interphase FISH but not detected by G-banded karyotyping and metaphase FISH. FISH was performed using BCR/ABL dual color dual fusion translocation probes (Abbott Molecular, USA). A 52-year-old man was diagnosed with acute leukemia of mixed phenotype. G-banded karyotyping showed 46,XY,t(9;22)(q34;q11.2)[12]/47,idem,+der(22)t(9;22)[5]/46,XY[3]. Interphase FISH revealed nuc ish(ABL1,BCR) × 3(ABL1 con BCR × 2)[329/450]/(ABL1,BCR) × 4(ABL1 con BCR × 3)[5/450]/(AL1,BCR) × 3(ABL1 con BCR × 1)[44/450]. Metaphase FISH showed ish (9;22)(ABL1+,BCR1+;BCR+,ABL+)[22]/der(22)(BCR+,ABL1+)[3]. The other case was that of a 31-yr-old male patient diagnosed with CML in the blastic phase. G-banded karyotyping of all 20 metaphase cells showed 47,XYYc,dup(1)(q21q32),del(7)(p11.2),t(9;22)(q34;q11.2). Interphase FISH revealed nuc ish(ABL1,BCR) × 3(ABL1 con BCR × 2)[254/600]/(ABL1,BCR) × 3(ABL1 con BCR × 1)[191/600]. Metaphase FISH showed ish t(9;22)(ABL1+,BCR+;BCR+,ABL1+)[16]. These results suggest that typical t(9;22) and variant Ph may coexist in the same patient, and interphase FISH may facilitate the detection of the variant Ph that cannot be detected by G-banded karyotyping alone.

Responses and Survival Are Not Affected by Cytogenetics In Patients with Relapsed and Refractory Multiple Myeloma (R/R MM) Treated with Single-Agent Carfilzomib

AbstractAbstract 1942 Background:The impact of cytogenetic abnormalities on the response to therapies and survival in patients (pts) with multiple myeloma (MM) has been well established in the literature. Bortezomib (BTZ), either alone or in combination with other agents, has been shown to overcome the adverse impact of several common unfavorable cytogenetic features. In addition, responses with lenalidomide (LEN)/dexamethasone and pomalidomide have also been reported in patients with unfavorable cytogenetic profiles. Carfilzomib (CFZ) is a novel and highly selective epoxyketone proteasome inhibitor which produces durable single-agent activity in pts with R/R MM. The objective of this analysis was to evaluate the influence of cytogenetics in a large Ph 2b study (PX-171-003-A1), with single-agent CFZ in pts with R/R MM. Methods:Of the 266 pts enrolled in the 003-A1 study, 229 pts (86%) were response evaluable and had available metaphase cytogenetics and/or fluorescence in situ hybridization (FISH) analyses for hypodiploidy, chromosome 13 deletions, del 17p13, t(4:14), and t(14;16) chromosomal abnormalities. Metaphase cytogenetic analyses were available for 200 pts (75%) and FISH results were available for 205 pts (77%). All pts received CFZ at 20 mg/m2 IV on days 1, 2, 8, 9, 15, and 16 in a 28-day cycle (C) in C1 followed by 27 mg/m2 in each C thereafter for up to 12 C. Pts who completed 12 C of therapy were eligible to continue CFZ on an extension study. For the analysis, overall response rate was defined as ≥PR by International Myeloma Working Group (IMWG) criteria. Clinical benefit response (CBR; ORR + MR as defined by EBMT criteria) was also assessed. All responses were assessed and confirmed by an Independent Response Review Committee (IRC). Results:The median age in this subpopulation was 64 yrs. Pts must have had disease that was relapsed after ≥2 regimens including BTZ and either thalidomide (THAL) or LEN, and must have been refractory to their most recent regimen. In this heavily pre-treated pt population, pts had received median of 13 prior anti-myeloma drugs and a median of 5 prior regimens. 99.6% of pts had previously received treatment with BTZ, 74% received prior thalidomide, 94% prior lenalidomide, and 74% prior stem cell transplantation. The ORR (≥PR) for the pts with available metaphase cytogenetics and/or FISH analyses was 25% and the CBR (≥MR) was 38%. 71 of 229 pts (31%) had at least one cytogenetic abnormality. Of these, 27/71 (38%) pts had abnormalities detected via metaphase cytogenetics, 24 (34%) pts were detected by FISH and 20 (28%) pts had abnormalities by both methods. The presence of deletion 13 or hypodiploidy by cytogenetics or del17p13, t(4;14), or t(14;16) by FISH did not significantly impact the ORR. Specifically, 30% of pts with ≥1 abnormality responded compared to 23% with none. The CBR was also unaffected at 34% vs. 39% for pts with ≥1 and no abnormalities, respectively. [Display omitted] For all pts in this analysis, the median duration of response (DOR) was 8 months (95% CI 6–10). For pts with ≥1 abnormality, the DOR was 7 months (95% CI 4–10) and did not differ significantly from the DOR of 8 months (95% CI 6–not reached) in pts with no abnormalities. A complete breakdown of response categories and time-to-progression (TTP) will also be presented. Conclusions:CFZ demonstrated durable and comparable activity in pts with R/R MM in both the absence or presence of one or more cytogenetic abnormalities including hypodiploidy, chromosome 13 deletions, del 17p13, t(4:14), or t(14;16). Notably, these results are consistent with data obtained in another Ph2 study with CFZ in pts with relapsed MM following 1–3 prior therapies and who had abnormal cytogenetics (PX-171-004). The results of the present study suggest that durable responses to CFZ can be achieved in heavily pretreated patients and responses are not impacted by poor prognostic cytogenetic features. Disclosures:Jakubowiak:Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Centocor Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Martin:Celgene: Honoraria; Onyx: Consultancy. Singhal:Celgene: Speakers Bureau; Takeda/Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx : Research Funding. Wang:Celgene: Research Funding; Onyx: Research Funding; Millenium: Research Funding; Novartis: Research Funding. Vij:Onyx: Honoraria. Jagannath:Millenium, OrthoBiotec, Celgene, Merck, Onyx: Honoraria; Imedex, Medicom World Wide, Optum Health Education, PER Group: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lonial:Millennium: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. Kukreti:Celgene: Honoraria; Roche: Honoraria; Ortho Biotech: Honoraria. Bray:Onyx Pharmaceuticals: Employment. Vallone:Onyx Pharmaceuticals: Employment. Kauffman:Onyx Pharmaceuticals: Employment. Siegel:Millenium: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.

Variant Philadelphia Translocation with BCR/ABL Fusion Gene Site In 10q24 In a Case of Chronic Myeloid Leukemia

AbstractAbstract 4839 Introduction.Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph) observed in more than 90% of patients with CML as a result of t(9;22)(q34;q11), leading to the formation of chimeric gene BCR/ABL encoding for proteins with abnormal tyrosine kinase activity. Cytogenetic variants of Ph chromosome can be identifed in 5 to 10% of CML patients, involving additional chromosomes other than 9 and 22. To explain the formation of variant translocations one-step, two-step and multi-step mechanisms have been proposed. Rarely, the variant Ph chromosome results from a BCR insertion on the ABL region and form a BCR/ABL fusion gene, generally mapping to 9q34, instead of the usual location at 22q11. In very few variant Ph cases, the insertion of the BCR/ABL product in a third chromosome was demonstrated. Case Report 28 year-old man, with bilateral central scotoma and gingivorragia. Physical examination: Grade 4 splenomegaly. Peripheral blood count showed hemoglobin concentration 11.5 g/dl, platelet count: 300.000/mm3, and white blood cell count 590.000/mm3. Blood smear: myelemia exhibiting 30% of myeloid blasts. Bone marrow biopsy: panmyelosis showing 20% of myeloid blasts. Cytogenetic analysis by G-banding performed in peripheral blood verified the following karyotype: 46, XY, t(9;22;10)(q34;q11;q24)[20] The analysis of the BCR-ABL fusion gene according to standard protocols detected the presence of the b3a2 isoform. Fluorescence in situ hybridization (FISH) studies using dual color dual fusion probes in metaphases showed a signal pattern 1F2G1R. The fusion signal mapped to 10q24, the red signal to 9q34, and the normal green signal to chromosome 22, while a second low intensity green signal mapped to the Ph chromosome. No signal was observed in der(9). Interphase FISH analysis in nuclei (n=200) presented the same signal pattern. Instead of using whole chromosome probes for 9 and 22, we hybridised probes used to detect DiGiorge syndrome. These probes detect gene control ARSA (spectrum green) localized at 22q13 and Tuple1 at 22q11 (spectrum orange). Two signals, green and orange were identified in normal chromosome 22. Ph chromosome showed the orange signal, whereas the green signal mapped to der(10). Discussion. The localization of the hybrid BCR/ABL gene on chromosomes other than 22q is a rare event wich can only be detected by FISH techniques. When these unusual translocation occurs, the hypothesis most often put forward is that several consecutive chromosome rearrangements have taken place. In the present case the interpretation of karyotypes, FISH data and molecular evidence lead to the following hypothesis: Insertion of the BCR sequence from chromosome 22 to chromosome 9 may have ocurred, producing a BCR/ABL fusion in der(9). The Ph chromosome detected by G-banding showed a different green fluorescence intensity in the metaphase FISH signal pattern with BCR/ABL dual color dual fusion probes, as a result of an insertion on chromosome 9. This first event was followed by the translocation between the derivative 9 and chromosome 10, being the final localization of the BCR/ABL gene in 10q24. FISH analysis using a DiGeorge syndrome probe, supports the hypothesis of a multistep mechanism underlying insertion and translocations events in the present case. The relocation of BCR/ABL fusion sequence on sites other than chromosme 22q11 represent a rare type of variant Ph translocation. At least 21 cases described in the literature, showed fusion gene BCR/ABL located at 9q24. Only 12 patients with variant Ph were reported bearing BCR/ABL on a third chromosome. All of them involved a masked Ph chromosome. To our best knowledge this is the first report showing a variant Ph chromosome detected by G-banding in a CML patient due to a BCR insertion on ABL sequences and exhibiting the fusion signal in a third chromosome. Disclosures:No relevant conflicts of interest to declare.

Metaphase FISH on a Chip: Miniaturized Microfluidic Device for Fluorescence in situ Hybridization

Fluorescence in situ Hybridization (FISH) is a major cytogenetic technique for clinical genetic diagnosis of both inherited and acquired chromosomal abnormalities. Although FISH techniques have evolved and are often used together with other cytogenetic methods like CGH, PRINS and PNA-FISH, the process continues to be a manual, labour intensive, expensive and time consuming technique, often taking over 3 5 days, even in dedicated labs. We have developed a novel microFISH device to perform metaphase FISH on a chip which overcomes many shortcomings of the current laboratory protocols. This work also introduces a novel splashing device for preparing metaphase spreads on a microscope glass slide, followed by a rapid adhesive tape-based bonding protocol leading to rapid fabrication of the microFISH device. The microFISH device allows for an optimized metaphase FISH protocol on a chip with over a 20-fold reduction in the reagent volume. This is the first demonstration of metaphase FISH on a microfluidic device and offers a possibility of automation and significant cost reduction of many routine diagnostic tests of genetic anomalies.

Amplification of IGH/CCND1 fusion gene in a primary plasma cell leukemia case

The IGH/CCND1 fusion gene has been reported in many hematologic tumors such as mantle cell lymphoma, chronic lymphocytic leukemia, prolymphocytic leukemia, multiple myeloma, and plasma cell leukemia. We report a case of plasma cell leukemia showing five IGH/CCND1 fusion signals by interphase fluorescence in situ hybridization (FISH). Conventional cytogenetic analysis and multicolor spectral karyotyping showed a complex karyotype that did not include t(11;14). Metaphase FISH studies revealed three IGH/CCND1 fusion signals, two of which were amplified signals. These findings indicate that IGH/CCND1 fusion gene was amplified and interspersed in several chromosomes. The patient was treated with intensive chemotherapy. However, the clinical course was very aggressive. The amplification of the IGH/CCND1 fusion gene may contribute to the aggressive course of the disease. To our knowledge, this is the first case showing amplification of the IGH/CCND1 gene in plasma cell neoplasms.

Microdeletion Syndromes Detected by FISH – 73 Positive from 374 Cases

KEYWORDS Prader-Willi. Angelman. Williams. DiGeorge. Cytogenetics. Fluorescence. Autism ABSTRACT Fluorescence in situ hybridization (FISH) has facilitated the detection of microdeletions seen in Prader- Willi/Angelman (PW/AS), Williams and DiGeorge syndromes. Out of 374 suspected cases tested at Jaslok Hospital in the past 5 years, 73 were positive, including 29 cases of Angelman, 16 of Prader-Willi, 24 of Williams and 4 of DiGeorge syndrome. Male preponderance was seen, mainly in Williams syndrome. The mechanisms causing Prader- Willi and Angelman syndrome include microdeletions, intragenic mutations, uniparental disomy and imprinting defects, though FISH can only detect microdeletions. Metaphase FISH helped to detect 1 case each with deletion of the control (PML) signal and duplication of the critical PW/AS region, which are associated with autism. One suspected case of Prader-Willi syndrome had a Robertsonian translocation t(14;15)(q10;q10) which led to a deletion of a major part of the SNRPN region in 10% cells, resulting in low-grade mosaicism. Another FISH-positive case was due to a reciprocal translocation t(2;15)(q37;q11), where loss of critical genes at the breakpoint on chromosome 15 caused the Prader-Willi phenotype. FISH in a child with an Angelman phenotype showed no microdeletion, though Trisomy 15 was seen in 1 metaphase suggesting uniparental disomy due to trisomy rescue. A known polymorphism in the form of an additional tiny green signal on chromosome 14 was observed in 17 of 284 (6%) cases studied for Prader-Willi/Angelman syndrome. Another inherited polymorphism was seen in 5 cases, where one control signal was very small. Prenatal diagnosis was carried out with normal results, in 12 women with a previously affected child.

Microdeletion Syndromes Detected by FISH – 73 Positive from 374 Cases

KEYWORDS Prader-Willi. Angelman. Williams. DiGeorge. Cytogenetics. Fluorescence. Autism ABSTRACT Fluorescence in situ hybridization (FISH) has facilitated the detection of microdeletions seen in Prader- Willi/Angelman (PW/AS), Williams and DiGeorge syndromes. Out of 374 suspected cases tested at Jaslok Hospital in the past 5 years, 73 were positive, including 29 cases of Angelman, 16 of Prader-Willi, 24 of Williams and 4 of DiGeorge syndrome. Male preponderance was seen, mainly in Williams syndrome. The mechanisms causing Prader- Willi and Angelman syndrome include microdeletions, intragenic mutations, uniparental disomy and imprinting defects, though FISH can only detect microdeletions. Metaphase FISH helped to detect 1 case each with deletion of the control (PML) signal and duplication of the critical PW/AS region, which are associated with autism. One suspected case of Prader-Willi syndrome had a Robertsonian translocation t(14;15)(q10;q10) which led to a deletion of a major part of the SNRPN region in 10% cells, resulting in low-grade mosaicism. Another FISH-positive case was due to a reciprocal translocation t(2;15)(q37;q11), where loss of critical genes at the breakpoint on chromosome 15 caused the Prader-Willi phenotype. FISH in a child with an Angelman phenotype showed no microdeletion, though Trisomy 15 was seen in 1 metaphase suggesting uniparental disomy due to trisomy rescue. A known polymorphism in the form of an additional tiny green signal on chromosome 14 was observed in 17 of 284 (6%) cases studied for Prader-Willi/Angelman syndrome. Another inherited polymorphism was seen in 5 cases, where one control signal was very small. Prenatal diagnosis was carried out with normal results, in 12 women with a previously affected child.