Interphase Fluorescence In Situ Hybridization Analysis Research Articles

Beyond the Surface: Uncovering the PML::RARA Fusion in Cytogenetically Cryptic and FISH-Negative Acute Promyelocytic Leukemia- A Case Report and Review of the Literature

Abstract Introduction/Objective Chromosomal t(15;17)(q22;q21), resulting in a fusion between the PML gene on chromosome 15q22 and the RARA gene on chromosome 17q21, is the cytogenetic hallmark of acute promyelocytic leukemia (APL). Conventional karyotyping, along with fluorescence in situ hybridization (FISH), can detect the PML/RARA rearrangements in up to 98% of APL cases. Reverse transcriptase-polymerase chain reaction (RT-PCR) is often used to confirm the initial diagnosis. APL with PML::RARA fusion, which is negative with FISH and conventional karyotyping approaches but positive with RT-PCR, is extremely rare. Herein, we report a de novo APL case that was positive by RT-PCR but negative by cytogenetics and FISH studies. A comprehensive review of the literature was also performed. Methods/Case Report A 23-year-old male presented to the ED for left upper extremity weakness 2 days after post- dental cleaning. Upon admission, he developed intracranial hemorrhage, necessitating urgent decompressive hemicraniectomy. Blood smear revealed promyelocytes; flow cytometric analysis showed a CD13+, CD33+, MPO+, CD117+, CD34-, and HLA-DR- abnormal cell population. STAT interphase FISH analyses for PML/RARA fusion and RARA break-apart were negative. A diagnosis of APL was made based on abnormal promyelocytes and characteristic flow cytometry findings. ATRA was immediately administrated. However, the post-operative course was complicated by significant coagulopathy, pancytopenia, and multi-organ failure, and the patient expired 7 days later. RT-PCR returned positive for PML::RARA long fusion transcript; the conventional cytogenetics study was uninformative. The clinical, pathological, and genetic features of 31 cases of FISH/Cytogenetics-negative APL harboring cryptic rearrangements of PML-RARA, detected by RT-PCR in the literature, have been reviewed. Various detection methods were also discussed. Results (if a Case Study enter NA) N/A Conclusion Conventional cytogenetics, FISH and RT-PCR analysis are complementary studies for all clinical suspected APL cases. The clinicopathological characteristics also play critical roles in APL diagnosis when genetic confirmation of the PML::RARA fusion is unavailable.

Role of Cytogenetics and Fluorescence In Situ Hybridization in the Laboratory Workup of Acute Myeloid Leukemias

AbstractA new understanding of acute myeloid leukemia as a varied group of unique biologic entity has emerged, as a result of the identification of various chromosomal aberrations and their association with clinical prognosis and diagnosis. Following induction treatment, cytogenetic examination can establish the presence of any residual malignant cells, it's recurrence, clonal evolution if any, or the formation of novel abnormalities. The G-banded karyotype has been the gold standard method for detecting all of these aberrations for years. The capacity to examine the entire genome through karyotype analysis quickly enabled the detection of deletions, duplications, and structural rearrangements across every chromosome, and the more frequent ones were associated with particular aberrant clinical symptoms. Fluorescence in situ hybridization (FISH) is a sensitive technology that aids in differential diagnosis or therapeutic planning and provides rapid results. Furthermore, the combination of cytogenetic and molecular profiling enables a more precise evaluation of disease prognosis, diagnosis, classification, risk stratification, and patient treatment. Interphase FISH analysis, in conjunction with G-banded chromosomal analysis, can be used as a major testing tool for the evaluation of hematological neoplasms. For accurate and consistent descriptions of genomic changes identified by karyotyping and FISH, a specified terminology is necessary. The International System for Human Cytogenomic Nomenclature is the main source and provides instructions for documenting cytogenetic and molecular findings in laboratory reports. This review discusses the two methods, karyotyping and FISH, their advantages and limitations, sample requirements, various FISH probes that are used, nomenclature for results reporting, and the necessary quality control measures.

Mosaic tetrasomy 9p at amniocentesis in a pregnancy associated with a favorable fetal outcome, perinatal progressive decrease of the aneuploid cell line and cytogenetic discrepancy in various tissues

ObjectiveWe present mosaic tetrasomy 9p at amniocentesis in a pregnancy associated with a favorable fetal outcome, perinatal progressive decrease of the aneuploid cell line and cytogenetic discrepancy in various tissue. Case reportA 33-year-old primigravid woman underwent elective amniocentesis at 18 weeks of gestation because of anxiety, and the karyotype of cultured amniocytes was 47,XX,+i (9) (p10)[20]/46,XX [55]. Cordocentesis was performed at 20 weeks of gestation, and the karyotype of cord blood was 47,XX,+i (9) (p10)[7]/46,XX [15]. She was referred for genetic counseling at 23 weeks of gestation, and repeat amniocentesis revealed a karyotype of 47,XX,+i (9) (p10)[1]/46,XX [16] with seven cells in one colony having tetrasomy 9p in cultured amniocytes, and in uncultured amniocytes, quantitative fluorescence polymerase chain reaction (QF-PCR) analysis excluded uniparental disomy (UPD) 9 and determined paternal origin of the extra i (9p), array comparative genomic hybridization (aCGH) analysis on the DNA extracted from uncultured amniocytes revealed arr 9p24.3p13.1 × 3.0 consistent with 50% mosaicism for tetrasomy 9p, and interphase fluorescence in situ hybridization (FISH) on uncultured amniocytes showed 22.6% (12/53 cells) mosaicism for tetrasomy 9p. A third amniocentesis at 27 weeks of gestation revealed a karyotype of 46, XX (10/10 colonies) in cultured amniocytes, and interphase FISH analysis on uncultured amniocytes revealed 20% (20/100 cells) mosaicism for tetrasomy 9p. The parental karyotypes and prenatal ultrasound were normal. At 39 weeks of gestation, a phenotypically normal 3388-g female baby was delivered. The karyotypes of cord blood, umbilical cord and placenta were 47,XX,+idic (9) (q12)[19]/46,XX [21] or 47,XX,+idic (9) (pter→q12:q12→pter)[19]/46,XX [21], 47,XX,+idic (9) (q12)[1]/46,XX [39] and 47,XX,+idic (9) (q12)[4]/46,XX [36], respectively. When follow-up at age two months, the neonate was phenotypically normal, the peripheral blood had a karyotype of 47,XX,+idic (9) (q12)[18]/46,XX [22], and interphase FISH analysis on 100 buccal mucosal cells revealed 1% (1/100 cells) mosaicism for tetrasomy 9p. When follow-up at age seven months, the neonate was phenotypically normal, and the peripheral blood had a karyotype of 47,XX,+idic(9)(q12)[14]/46,XX[26]. ConclusionMosaic tetrasomy 9p at amniocentesis can be a transient and benign condition, and can be associated with a favorable fetal outcome and perinatal progressive decrease of the aneuploid cell line and cytogenetic discrepancy in various tissue.

Prevalence of t(11;14) Multiple Myeloma in Real-World Setting: Interim Analysis from the Medici Study

Introduction: Multiple myeloma (MM) is the second most common hematologic malignancy characterized by bone marrow infiltration by monoclonal plasma cells (PC). Several well-defined genetic abnormalities have been identified as relevant with the disease. Although novel therapies have improved overall survival most patients with MM will eventually relapse or become refractory (RR). Therefore, there is an emerging need for better therapeutic regimens including personalized medicine based on specific biomarkers. Venetoclax (Ven) is a selective and potent oral B-cell lymphoma-2 (BCL-2) inhibitor that has shown significant antimyeloma activity in patients with t(11;14) RRMM (Kumar et al. Blood 2017; 130 (22): 2401-2409). In the phase 3 BELLINI (NCT02755597) study, patients with t(11;14) RRMM showed improved response rates and progression-free survival when treated with Ven in combination with bortezomib and dexamethasone compared to placebo-bortezomib-dexamethasone (Kumar et al. Lancet Oncol. 2020; 21:1630-1642). These results suggest that t(11;14)-based selection of patients with MM may represent a potential biomarker for venetoclax-based treatment regimens. An ongoing phase 3 study, CANOVA (NCT03539744), is evaluating the combination of Ven and dexamethasone in patients with t(11;14) RRMM compared to standard of care pomalidomide-dexamethasone (Mateos et al. J Clin Oncol 2020; 38: no. 15_suppl) . Understanding the prevalence of t(11;14) MM in a real-world setting will provide important insights for targeted therapy in this patient subpopulation. Therefore, the MEDICI study was designed to evaluate the real-world prevalence of t(11;14) as detected by interphase fluorescence in situ hybridization (FISH) technology in patients with MM. Methods: MEDICI (NCT04721002) is an ongoing, prospective, multicenter minimally interventional study. The primary objective is to determine the prevalence of t(11;14) in both newly diagnosed (ND) and RRMM as determined by interphase FISH analysis of monoclonal bone marrow (BM) PCs. All patients (≥18 years) included in the study signed informed consent. BM aspirates (BMA) were collected as part of standard of care procedures performed at diagnosis and disease relapse. Patients with no BMA sample at the time of diagnosis or confirmation of relapse were excluded from the trial. The primary endpoint was the t(11;14) status of the earliest bone marrow sample collected at initial diagnosis or across subsequent lines of therapies. CD138-enriched BMA samples were evaluated for t(11;14) by FISH (Abbott Molecular). Results: Sixty-two patients were included at the time of the interim analysis (data cut-off 09 March 2022). Median age was 67.5 years, 51.6% were male and 95.2% were Caucasian. Patients with ECOG performance status ≤2/>2/unknown was 83.9%/8.1%/8.1%. ISS stage across patients were stage I (30.6%), stage II (22.6%), stage III (33.9%) and unknown (12.9%) respectively. Del 17p was observed in 6.5% of patients and gain of chromosome 1q in 25.8% of patients. There were 36 and 26 patients with NDMM and RRMM respectively. One patient had longitudinal samples. The overall prevalence of patients with t(11;14) MM was 30.0%, which was similar between NDMM (25.0%) and RRMM (37.5%) patients (Table). The distribution of patients across the observed t(11;14) FISH fusion (F) categories (1F/2F/≥3F) for NDMM was 33.3%/22.2%/22.2% and 44.4%/55.6%/0.0% in RRMM pts (Table). Across lines (L) of therapy, the distribution of t(11;14)+ patients were similar between 1L (25.0%) , 2L (35.7%), and 3L+ (36.4%) (Table). The distribution of t(11;14)+ patients across disease stage was also similar between stage I (31.6%), stage II (28.6%), and stage III (25.0%) respectively. There were 8 patients with unknown stage of disease, 3 among them were t(11;14)+. Conclusion: Interim analysis of MEDICI demonstrated that prevalence of t(11;14) was consistent across ND and RRMM setting and independent of line of therapy and disease stage. This study is currently ongoing, and further updates will be provided upon presentation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Fluorescence In Situ Hybridization: A Sensitive Method for Trisomy 8 Detection in Bone Marrow Specimens

Trisomy 8 is a common anomaly in bone marrow (BM) cells of patients with myeloproliferative disorders (MPD), myelodysplastic syndromes (MDS), or acute nonlymphocytic leukemia (ANLL). We studied the efficacy of fluorescence in situ hybridization (FISH) detection of trisomy 8 in patients with MPD, MDS, or ANLL using directly labeled fluorescent alpha-satellite and whole chromosome paint (WCP) DNA probes specific for chromosome 8. Using FISH, we analyzed interphase nuclei and metaphase spreads from randomized series of BM specimens from normal individuals and patients with varying proportions of trisomy 8 as determined by conventional cytogenetic analysis. The BM of all normal donors contained less than or equal to 2.0% nuclei with 3 interphase FISH signals and less than or equal to 1 metaphase with 3 WCP FISH signals. Ninety-five percent and 98% of BM specimens with at least two metaphase cells with trisomy 8 by cytogenetic analysis contained greater than 2.0% nuclei with 3 interphase FISH and greater than 2 metaphases with 3 WCP FISH signals, respectively. Thirteen patients had 1 in 20 or 1 in 30 metaphase cells with trisomy 8 by conventional cytogenetic studies. Of these patients, four had greater than 2.0% nuclei with 3 interphase FISH signals. The BM of all four patients contained positive metaphase FISH results. We then studied the usefulness of FISH analysis to detect occult trisomy 8 by analyzing BM nuclei from 144 patients who had MPD, MDS, or ANLL and either 20 normal metaphase cells or an abnormal karyotype without trisomy 8. Seven patients had greater than 2.0% nuclei with 3 interphase FISH signals (range, 2.10% to 3.40%) and six patients had 2 or more cells with trisomy 8 upon metaphase FISH or extensive conventional cytogenetic analysis. Our results show that interphase and metaphase FISH analyses are useful methods to detect trisomy 8 cells in BM specimens, especially for specimens with normal or uncertain conventional cytogenetic results.

Prenatal diagnosis and molecular cytogenetic characterization of a small supernumerary marker chromosome derived from inv dup(15)

ObjectiveWe present prenatal diagnosis and molecular cytogenetic characterization of an inverted duplication of proximal chromosome 15 [inv dup(15)] presenting as a small supernumerary marker chromosome (sSMC) at amniocentesis associated with concomitant microduplication of 8q22.1. Materials and methodsA 39-year-old woman underwent amniocentesis at 16 weeks of gestation because of advanced maternal age, and the result was 47, XY, +mar dn. The woman requested for repeat amniocentesis at 20 weeks of gestation. Array comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), quantitative fluorescent polymerase chain reaction (QF-PCR) and DNA methylation analysis were applied to determine the nature of the sSMC.Results: aCGH on the uncultured amniocytes revealed the result of arr 8q22.1 (93,918,763–96,618,539) × 3.0, arr 15q11.2q13.2 (22,765,628–30,658,876) × 4.0, arr 15q13.2q13.3 (30,653,877–32,509,926) × 3.0 [GRCh37 (hg19)]. Interphase FISH analysis using RP11-34H12 [15q13.2; Texas Red, 30,709,033–30,893,021 (hg19)] on 100 uncultured amniocytes showed that 38 cells had three signals, 45 cells had four signals and 27 cells had two signals. The parental bloods had normal aCGH results. The karyotype of cultured amniocytes was 47, XY, +inv dup(15) (pter→q13::q13→pter) which was confirmed by metaphase FISH analysis. No informative markers could be found in QF-PCR analysis. DNA methylation analysis on cord blood confirmed a maternal origin of the 15q11-q13 gene dosage increase with a result of 15q11.2 SNRPN DNA hypermethylation. Postnatal cytogenetic analysis on cord blood, umbilical cord and placenta showed the results consistent with the prenatal diagnosis. ConclusionMolecular cytogenetic techniques are useful for rapid diagnosis of an inv dup(15) chromosome presenting as an sSMC at amniocentesis.

Prenatal diagnosis of low-level mosaic trisomy 17 with maternal uniparental disomy 17 by amniocentesis in a pregnancy with a favorable outcome

ObjectiveWe present prenatal diagnosis low-level mosaic trisomy 17 with maternal uniparental disomy (UPD) 17 at amniocentesis in a pregnancy with a favorable outcome. Materials and methodsA 40-year-old, primigravid woman underwent amniocentesis at 18 weeks of gestation because of advanced maternal age. This pregnancy was conceived by in vitro fertilization and embryo transfer. Amniocentesis revealed a karyotype of 47,XX,+17 [13]/ 46, XX [23]. Repeat amniocentesis was performed at 21 weeks of gestation. Conventional cytogenetic analysis was applied on cultured amniocytes, parental bloods and cord blood. Simultaneous molecular genetic analysis such as interphase fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and quantitative fluorescent polymerase chain reaction (QF-PCR) assays were applied on uncultured amniocytes. Interphase FISH was applied on postnatal buccal cells. ResultsRepeat amniocentesis revealed a karyotype of 47,XX,+17[6]/46,XX[28]. Genetic analyses on uncultured amniocytes showed the results of mosaic trisomy 17 (12/101 cells = 11.9%) in FISH analysis, no genomic imbalance in aCGH analysis and maternal UPD 17 in QF-PCR assays. The parental karyotypes were normal. Prenatal ultrasound findings were unremarkable. The parents decided to continue the pregnancy, and a 1449-g, phenotypically normal female baby was delivered prematurely at 31 weeks of gestation. The cord blood had a karyotype of 46,XX. She had a normal psychomotor development at age 22 months at follow-up. Interphase FISH analysis on buccal cells showed trisomy 17 signals in 1/66 cells (1.5%). ConclusionsLow-level mosaicism for trisomy 17 associated with maternal UPD 17 detected by amniocentesis without ultrasound abnormality can be associated with a favorable outcome. Molecular genetic analysis of uncultured amniocytes at repeat amniocentesis is useful for confirmation and genetic counseling under such as circumstance.

Fluorescence In situ hybridization signal patterns and intrachromosomal breakpoint cluster region-abelson murine leukemia viral oncogene homolog 1 amplification analysis in imatinib-resistant chronic myelogenous leukemia patients using tricolor dual fusion probe

BACKGROUND: Cytogenetic evaluation is required till a complete cytogenetic remission is achieved in chronic myelogenous leukemia (CML) patients on tyrosine kinase inhibitor (TKI) therapy. The routine dual colour fluorescence in situ hybridization (FISH) probes are less sensitive in identifying der(9) abnormalities. BCR/ABL/ASS1 tri-colour dual fusion (TCDF) probe is highly sensitive and specific in identifying der(9) deletions and random signal overlaps. METHODS: Peripheral blood interphase FISH analysis was performed on imatinib-resistant CML patients using TCDF probe. RESULTS: On analyzing 37 adult patients, all had residual Philadelphia (Ph) chromosome. Classic Ph fusion pattern was seen in 33 (89%), derivative chromosome 9 [der(9)] abnormalities in 25 (67.5%) and supernumerary Ph chromosomes in 11 (30%) patients. Coexistence of classical fusion and der(9) abnormalities was seen in 21 patients (57%); and classical fusion, der(9) abnormalities and supernumerary Ph chromosome in 8 patients (22%). None of the patients had BCR-ABL1 gene amplification. There was significant difference in the der(9) abnormal cell percentages between patients with e13a2 and e14a2 transcripts (P = 0.008) and patients with disease transformation (P = 0.007). CONCLUSION: A high frequency of der(9) abnormalities and absence of BCR-ABL1 gene amplification was seen in imatinib-resistant CML patients analyzed. The use of TCDF probe for cytogenetic follow-up in CML patients was found to be useful in identifying BCR-ABL1 related aberrations. The identified patterns in this study, can serve as a reference material for I-FISH signal interpretation using TCDF probe.

Evaluation of multiprobe fluoresence in-situ hybridization panel in the detection of common chromosomal abnormalities of acute myeloid leukemia

Background Numerous recurrent chromosomal aberrations have been identified in acute myeloid leukemia (AML), and their detection has become essential for accurate diagnosis, classification, and prognosis of the disease. Fluorescence in-situ hybridization (FISH) provides a powerful technique complementary and even alternative to chromosome banding studies for the identification of selected chromosome aberrations. Aim Evaluation of the multiprobe FISH panel in the detection of common cytogenetic abnormalities in AML and to investigate their association with clinical diagnosis, chemotherapy, and prognosis. Patients and methods This study was conducted on 20 newly diagnosed AML patients. All patients were subjected to full history taking, clinical examination, laboratory investigations including complete blood count, bone marrow aspiration, immunophenotyping, and interphase FISH using cytocell multiprobe AML/myelodysplastic syndrome panel designed to detect AML/Eight-twenty-one (ETO), Promyelocytic leukaemia- Retinoic acid receptor alpha (PML-RARA), and core-binding factor beta/Myosin, heavy chain 11, smooth muscle (CBFβ/MYH11) translocations, mixed-lineage leukaemia (MLL) break apart, P53, 5q, 7q, and 20q deletions. Results Interphase FISH analysis showed 5q deletion in 8/20 (40%) cases, positive PML/RARA in 3/20 (15%) cases p53 deletion in 15/20 (75%) cases, positive AML1/ETO in 1/20 (5%) cases, no MLL break apart cases (0%), 7q deletion in 4/20 (20%) cases, positive CBFβ/MYH11 fusion gene in 4/20 (20%) cases, 20q deletion in 9/20 (45%) cases, and trisomy 8 in 7/20 (35%) cases. There was a statistically significant relationship between 5q deletion and prognosis (P=0.028). Conclusion Multiprobe FISH is more cost effective and time effective compared with traditional FISH. It is an efficient technique for the detection of cytogenetic aberrations AML, providing critical information for diagnosis and prognosis, and for monitoring the course of the disease.

Prenatal diagnosis of low-level mosaicism for trisomy 13 at amniocentesis associated with a favorable outcome

ObjectiveWe present prenatal diagnosis of low-level mosaicism for trisomy 13 at amniocentesis associated with a favorable outcome. Case reportA 35-year-old woman underwent amniocentesis at 18 weeks of gestation because of advanced maternal age. Amniocentesis revealed a karyotype of 47,XY,+13[5]/46,XY[20]. Oligonucleotide array comparative genomic hybridization (aCGH) analysis on uncultured amniocytes revealed arr [GRCh37] (13)×3 [0.10], (X,Y)×1 compatible with trisomy 13 mosaicism. Prenatal ultrasound was unremarkable. Repeat amniocentesis was performed at 21 weeks of gestation. Interphase fluorescence in situ hybridization (FISH) analysis on uncultured amniocytes revealed a mosaic trisomy 13 level of 10% (10/100 cells). aCGH analysis on uncultured amniocytes revealed a result of arr 13q12.11q34 (20,407,323–115,092,619)×2.1 with a log2 ratio of 0.06 compatible with a 10% level of mosaicism. Polymorphic DNA marker analysis excluded uniparental disomy 13. The parental karyotypes were normal. Conventional cytogenetic analysis using cultured amniocytes at repeat amniocentesis revealed a karyotype of 46,XY in 23/23 colonies. The pregnancy was carried to 37 weeks of gestation, and a 3600-g phenotypically normal male baby was delivered. When examined at 8 months of age, the infant was doing well and was normal in psychomotor and growth development. The peripheral blood had a karyotype of 46,XY, and interphase FISH analysis on uncultured urinary cells revealed a mosaic trisomy 13 level of 4.4% (2/45 cells). ConclusionLow-level true mosaicism for trisomy 13 at amniocentesis without ultrasound abnormalities can be associated with a favorable fetal outcome.

Prenatal diagnosis and molecular cytogenetic characterization of low-level mosaicism for tetrasomy 18p at amniocentesis in a pregnancy with a favorable outcome

ObjectiveWe present prenatal diagnosis of low-level mosaicism for tetrasomy 18p at amniocentesis in a pregnancy with a favorable outcome. Case ReportA 40-year-old woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed a de novo supernumerary isochromosome 18p in eight of 39 colonies of cultured amniocytes. The karyotype was 47,XX,+i(18)(p10)[8]/46,XX[31]. Array comparative genomic hybridization (aCGH) analysis using uncultured amniocytes revealed arr 18p11.32p11.21 [hg 19] (148,963–14,081,887) × 2–3. Repeat amniocentesis was performed at 20 weeks of gestation. Interphase fluorescence in situ hybridization (FISH) analysis showed four 18p11.22-specific probe (RP11-918F20) signals in 11.7% (12/103 cells) of uncultured amniocytes. aCGH analysis on uncultured amniocytes did not detect genomic imbalance in chromosome 18. The parental karyotypes were normal. Polymorphic DNA marker analysis excluded uniparental disomy 18. Cytogenetic analysis of cultured amniocytes at repeat amniocentesis revealed a karyotype of 47,XX,+i(18)(p10)[2]/46,XX[12]. Prenatal ultrasound was unremarkable. The pregnancy was carried to 38 weeks of gestation, and a 2742-g phenotypically normal female baby was delivered with a cord blood karyotype of 46,XX. When examined at 8 months of age, the infant was normal in growth and psychomotor development. Interphase FISH analysis on 21 uncultured urinary cells revealed normal signals in all cells and no mosaic tetrasomy 18p. ConclusionLow-level mosaic tetrasomy 18p at amniocentesis without ultrasound abnormalities can be associated with a favorable outcome.

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 16

ObjectiveWe present prenatal diagnosis and molecular cytogenetic characterization of a small supernumerary marker chromosome (sSMC) derived from chromosome 16. Case reportA 28-year-old woman underwent amniocentesis at 17 weeks of gestation because of abnormal maternal serum screening for Down syndrome. Amniocentesis revealed a karyotype of 47,XY,+mar[5]/46,XY[9]. Parental karyotypes were normal. Prenatal ultrasound findings were unremarkable. Array comparative genomic hybridization (aCGH) analysis of cultured amniocytes revealed a de novo 16% gene dosage increase of 16q11.2-q22.1. Repeat amniocentesis at 21 weeks of gestation revealed a karyotype of 47,XY,+mar[10]/46,XY[31]. aCGH analysis of uncultured amniocytes revealed a result of arr 16q11.2q22.1 (46,492,626–68,867,969) × 2.20 with a log2 ratio of 0.15 encompassing RPGRIP1L, FTO, SLC6A2, BBS2 and CDH1. Interphase fluorescence in situ hybridization (FISH) analysis on uncultured amniocytes detected partial trisomy 16q in 36/137 (26.3%) of uncultured amniocytes. Polymorphic DNA marker analysis on amniocytes and parental bloods excluded uniparental disomy 16. Premature labor occurred at 25 weeks of gestation, and a 585-g male baby without craniofacial dysmorphism was delivered and survived. At age 1½ years, pediatric follow-ups revealed normal psychomotor development, normal body weight, short stature, congenital hypothyroidism, hearing impairment and hypospadias in the neonate, and the peripheral blood had a karyotype of 46,XY in 40 cultured lymphocytes. ConclusionaCGH, interphase FISH and polymorphic DNA marker analyses of uncultured amniocytes are useful for confirmation of prenatally detected mosaic sSMCs at amniocentesis.

Mosaic trisomy 17 at amniocentesis: Prenatal diagnosis, molecular genetic analysis, and literature review

ObjectiveWe present prenatal diagnosis and molecular genetic analysis of mosaic trisomy 17 and a review of the literature of mosaic trisomy 17 at amniocentesis. Materials and MethodsA 42-year-old woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age, which revealed a karyotype of 47,XX,+17[4]/46,XX[17]. Prenatal ultrasound findings were unremarkable. She underwent repeat amniocentesis at 20 weeks of gestation. Interphase fluorescence in situ hybridization (FISH), array comparative genomic hybridization, and quantitative fluorescent polymerase chain reaction assays were applied to uncultured amniocytes. Conventional cytogenetic analysis was applied to cultured amniocytes and cord blood. Interphase FISH was applied to uncultured urinary cells postnatally. ResultsAt repeat amniocentesis, molecular genetic analysis of uncultured amniocytes revealed no genomic imbalance in array comparative genomic hybridization, no uniparental disomy 17 in quantitative fluorescent polymerase chain reaction, and 4.7% (5/105 cells) mosaic trisomy 17 in interphase FISH analysis. Conventional cytogenetic analysis of cultured amniocytes revealed a karyotype of 46,XX (17/17 colonies). A phenotypically normal baby was delivered at 38 weeks of gestation. The cord blood had a karyotype of 46,XX. Interphase FISH analysis of uncultured urinary cells revealed 5.6% (5/90 cells) mosaic trisomy 17. The neonate manifested normal growth and psychomotor development during follow-ups. ConclusionLow-level mosaicism for trisomy 17 detected by amniocentesis without ultrasound abnormality can be associated with a favorable outcome. Molecular genetic analysis of uncultured amniocytes at repeat amniocentesis is useful for genetic counseling. A review of the literature shows a correlation between an adverse fetal outcome and a higher trisomy 17 mosaicism level at amniocentesis associated with ultrasound abnormality.

Prenatal diagnosis and molecular cytogenetic characterization of low-level true mosaicism for trisomy 21 using uncultured amniocytes

ObjectiveWe present a prenatal diagnosis and molecular cytogenetic characterization of low-level true mosaicism for trisomy 21 using uncultured amniocytes. Case reportA 35-year-old woman presented with a borderline-positive result of noninvasive prenatal testing for trisomy 21. She underwent amniocentesis at 18 weeks of gestation, which revealed a karyotype of 47,XY,+21(5)/46,XY(53). Repeat amniocentesis at 22 weeks of gestation revealed a karyotype of 47,XY,+21(6)/46,XY(26). Array comparative genomic hybridization (aCGH) analysis on uncultured amniocytes revealed mosaic levels of 10% to 15% for trisomy 21. Interphase fluorescence in situ hybridization (FISH) analysis on uncultured amniocytes revealed a mosaic level of 21.7% (28/129 cells) for trisomy 21. Following genetic counseling and detailed ultrasound examination, the parents decided to continue the pregnancy. The pregnancy was carried to term, and a normal 3664-g male baby was delivered. The cord blood lymphocytes had a karyotype of 47,XY,+21(2)/46,XY(38). Postnatal interphase FISH analysis of urine detected no trisomy 21 in all 39/39 urinary cells. The neonate was phenotypically normal at age 7 months. ConclusionLow-level true mosaicism for trisomy 21 can be associated with a favorable fetal outcome. aCGH and interphase FISH analyses on uncultured amniocytes are useful for rapid confirmation of low-level true mosaicism for trisomy 21 at repeated amniocentesis.

Preparation of Amniocytes for Interphase Fluorescence In Situ Hybridization (FISH).

FISH has been used to detect and clarify deletions and/or other structural rearrangements, and also has applications in interphase analysis. This unit describes preparation of uncultured amniotic fluid cells for FISH analysis. Cells are swollen, and then slides are prepared by standard methods. The cells are then fixed and permeabilized for subsequent FISH. An alternate protocol describes attachment of amniocytes to a glass or plastic surface followed by hypotonic swelling, fixation, and permeabilization for subsequent FISH. Interphase FISH analysis of amniotic fluid cells is also described.

Detection of no isochromosome 20q by interphase fluorescent in situ hybridization on uncultured amniocytes in a pregnancy with mosaic isochromosome 20q in cultured amniocytes at amniocentesis

ObjectiveTo present prenatal diagnosis and molecular cytogenetic characterization of mosaic isochromosome 20q at amniocentesis. Materials and methodsA 36-year-old woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age, and conventional cytogenetic analysis revealed a karyotype of 46,XY,i(20)(q10)[12]/46,XY[7]. Repeated amniocentesis was performed at 20 weeks of gestation. During repeated amniocentesis, array comparative genomic hybridization (aCGH), interphase fluorescence in situ hybridization (FISH), and quantitative fluorescent polymerase chain reaction (QF-PCR) were performed on uncultured amniocytes, and conventional cytogenetic analysis and interphase FISH were performed on cultured amniocytes. ResultsConventional cytogenetic analysis of cultured amniocytes revealed a karyotype of 46,XY,i(20)(q10)[4]/46,XY[16]. Interphase FISH analysis on 217 uncultured amniocytes did not detect isochromosome 20q, aCGH on the DNA extracted from uncultured amniocytes showed no genomic imbalance, and QF-PCR analysis on the DNA extracted from uncultured amniocytes excluded uniparental disomy 20 (UPD 20). Interphase FISH analysis on 115 cultured untouched amniocytes revealed 13% (15/115 cells) mosaicism for isochromosome 20q. ConclusionMosaic isochromosome 20q detected at amniocentesis can be a cell culture artifact. Detailed ultrasound examination, performing interphase FISH and/or aCGH on uncultured amniocytes for confirmation of true mosaicism, and performing QF-PCR to exclude UPD 20 may be useful under such a circumstance.

Interphase fluorescence in situ hybridization characterization of mosaicism using uncultured amniocytes and cultured stimulated cord blood lymphocytes in prenatally detected Pallister–Killian syndrome

ObjectiveThis study aims to present molecular cytogenetic characterization of Pallister–Killian syndrome (PKS). Materials and methodsA 37-year-old woman underwent amniocentesis at 18 weeks of gestation. Amniocentesis revealed a karyotype of 47,XY,+i(12)(p10)[6]/48,XY,+i(12)(p10)×2[1]/46,XY[6]. Repeated amniocentesis was performed at 20 weeks of gestation. Array comparative genomic hybridization (aCGH) was performed using uncultured amniocytes, cord blood, and skin. Quantitative fluorescent polymerase chain reaction (QF-PCR) was performed using uncultured amniocytes and parental bloods. Interphase fluorescence in situ hybridization (FISH) analysis was performed using uncultured amniocytes and cultured stimulated cord blood lymphocytes. Conventional cytogenetic analysis was performed using cultured cells from amniotic fluid, skin, placenta, umbilical cord, and cord blood. ResultsRepeated amniocentesis revealed a mosaic tetrasomy 12p level of 25% (10/40), cultured cord blood lymphocytes had no mosaicism, cultured skin fibroblasts had a mosaic tetrasomy 12p level of 52.5% (21/40), umbilical cord fibroblasts had a mosaic tetrasomy 12p level of 72.5% (29/40), and the placental cells had a mosaic tetrasomy 12p level of 2.5% (1/40) on conventional cytogenetics. An aCGH analysis revealed that the increases in gene dosage in 12p for uncultured amniocytes, skin, and cord blood were the log2 ratios of 0.9, 0.7, and 0.7, respectively. Interphase FISH on uncultured amniocytes revealed a mosaic level of 73.1% (49/67) (tetrasomy 12p: 33; hexasomy 12p: 16). Interphase FISH analysis of stimulated cultured cord blood lymphocytes revealed a mosaic level of 58.3% (60/103) (tetrasomy 12p: 51; hexasomy 12p: 9). ConclusionIn the diagnosis of PKS by conventional culture cytogenetics, cord blood samplings and placental samplings are prone to a negative result when compared with amniocentesis. Whenever cord blood sampling is applied for prenatal diagnosis of PKS, aCGH on uncultured cord blood or interphase FISH on cultured cord blood can be used for the diagnosis, in addition to conventional cytogenetics.

Validation Of a Multi-Diagnostic Approach Including Flow Cytometry To Identify Specific Risk Categories Within Low and Intermediate-1 Risk Myelodysplastic Syndromes Within a Prospective Clinical Trial: A Study On Behalf Of The HOVON89 Study Group

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of hematopoietic stem cell disorders, characterized by ineffective hematopoiesis resulting in cytopenias and variable risk of acute myeloid leukemia (AML). To make an accurate distinction between specific risk categories in MDS, especially in low and intermediate risk MDS, a multi-diagnostic approach is recommended. To verify the efficacy of multiple diagnostic tools in MDS we used the HOVON89 study-cohort (a prospective phase II randomized multicenter study to assess the efficacy of lenalidomide with or without erythropoietin and granulocyte-colony stimulating factor in patients with low-intermediate-1 risk MDS; trial registered at www.trialregister.nl as NTR1825; EudraCT nr.: 2008-002195-10.Inclusion target of the study is 200 low/intermediate-1 risk MDS patients (134 enrolled, inclusion ongoing). We collect data on cytomorphology (CM), conventional cytogenetics (CCG), fluorescence in situ hybridization (FISH) and microarray-based genomic profiling. In addition, we performed flow cytometric (FC) analysis according to European LeukemiaNet guidelines (Van de Loosdrecht et al., Haematologica 2009 and Leukemia 2012). Current CM results (N=98) identified: 8 refractory anemia (RA); 16 refractory anemia with ringed sideroblasts (RARS); 43 refractory cytopenia with multilineage dysplasia with/without ringed sideroblasts (RCMD/RCMD-RS); 16 refractory anemia with excess blast-1 (RAEB-1), 5 chronic myelomonocytic leukemia-1 (CMML-1) and 10 patients with isolated del(5q). CCG analysis (N=101) indicated 2 very good risk, 84 good risk, 13 intermediate risk and 2 poor risk patients according to the IPSS-R risk categories (Greenberg et al., Blood 2012). In addition, interphase FISH analysis (N=72) was normal in 15 patients, in 6 patients the del(5q) was confirmed. From 68 MDS patients data from both CCG and microarray were available. Microarray-based genomic profiling identified genomic abnormalities such as copy neutral loss of heterozygosity and small (<5 Mb) copy number alterations coinciding with a cancer gene in 13 patients with normal CCG. As expected in one patient the balanced translocation t(3;3)(q21;q26) was not identified by microarray-based genomic profiling.FC analysis (N=82) evaluated aberrancies with regard to count, marker expression level and lineage infidelity marker expression on myeloid progenitors, B cell progenitors, maturing myeloid/monocytic and erythroid cells. Current, validated FC-scoring systems identified 60/81 (Della Porta et al., Haematologica 2012) and 61/81 (Wells et al., Blood 2003) patients with MDS. Both scoring systems do not evaluate dyserytropoiesis and dysmegakaropoiesis, thereby possibly not recognizing RA, RARS or RCMD patients with only dyserytropoiesis and dysmegakaropoiesis. We integrated both scoring systems into one score (Van de Loosdrecht et al., Leukemia 2012, and JNCCN 2013). Patients were divided into 3 categories: not likely MDS (A), signs of dysmyelopoiesis (B) and fitting MDS (C). This score diagnosed 69/78 patients as probably or likely MDS by FC (B or C, figure 1). Remarkably, patients scored as ‘category A' only displayed dyserytropoiesis and/or dysmegakaryopoiesis by CM. FC identified dyserytropoiesis in these patients, however, a consensus erytroid flow score is not yet validated. In addition, FC identified different risk categories within the patient group with no genetic abnormalities (based on CCG, FISH and microarray-based genomic profiling; data not shown). [Display omitted] In conclusion, this is the first prospective study in low/int-1 risk MDS that validates FC as a valuable diagnostic tool in MDS (sensitivity of FC in this cohort: 88%). FC only failed to recognize some patients with only dyserytropoiesis and dysmegakaryopoiesis by CM, not evaluated by the current scoring system. Thirteen patients with unilineage dysplasia by CM had multilineage dysplasia by FC. We postulate that RA/RARS patients with multi-lineage dysplasia by FC may have clinical features of RCMD patients and therefore a higher risk on transformation to AML. Clinical follow-up data are expected within 1.5 year.In near future, a multi-diagnostic approach may i) identify risk categories within well defined IPSS-R subgroups, ii) predict risk on transformation and iii) select patients who might benefit from new emerging drugs for low-int-1 risk MDS. Disclosures:No relevant conflicts of interest to declare.

A Comparative Study Of The Outcome Of Isolated Chromosome 13q and Clonal Progression Detected By I-FISH Do Additional Cytogenetic Abnormalities Impact Survival In Multiple Myeloma

BackgroundChromosomal abnormalities detected by interphase fluorescence in situ hybridization (I-FISH) are an important prognostic marker in patients with multiple myeloma (MM). Isolated chromosome 13q has been considered standard risk when identified by I-FISH and high risk by conventional cytogenetics. The impact of additional cytogenetic abnormalities with chromosome 13q identified by I-FISH in regards to prognosis has not been fully defined. In this report, we describe the outcome of patient's with multiple myeloma with isolated chromosome 13q and 13q+ (additional cytogenetic abnormalities) identified by I-FISH at our institution between January 2003 and January 2013 and had I-FISH analysis prior to treatment. MethodsThe primary objective was to compare patient's outcomes in regards to response, time to progression, and overall survival between patients who had an isolated 13q and 13q+ identified by I-FISH in the bone marrow plasma cells. Kaplan & Meier curves were generated to calculate overall survival (OS) between the two groups. ResultsBetween January 2003 and January 2013, we identified 76 patients by I-FISH who had either an isolated 13q or 13q+ in patients with multiple myeloma (Patient characteristics Table 1). Of the patients with an isolated 13q abnormality 33% received a bortezomib-based regimen and 38% in the 13q+ group. Of the patient's with a isolated 13q 38% went onto receive high dose chemotherapy followed by autologous stem cell transplant (ASCT) while 20% with a 13q+ received ASCT. African American patients with 13q consisted of 65% and 60% with 13q+ in our patient population. For the 13q or 13q+ who underwent high dose chemotherapy followed by autologous stem cell transplant OS was 85% compared to the non-transplant group 45% (p=0.01) (Figure 2). On follow up at a median of 2.5 years mortality occurred in 31% of the 13q patients compared to 62% in the 13q+ group. The overall survival at 5 years was 25% in the 13q+ group compared to 65% in the patient's with an isolated 13q, With the 13q+ group having an overall poor OS (p=0.03)Table 1Patient Characteristics and Outcome13q (n=42)13q+ (n=34)Age (years)65 (88-44)13q+ (n=34)GenderMale19 (45%)15Female23 (55%)19 (55%)RaceWhite13 (30%)11 (32%)African American27 (65%)20 (60%)Other23International Staging System at diagnosisI5(15%)II3(7%)4(12%)III26(62%)30(88%)Plamacytoma9 (21%)12(35%)Bortezomib based InductionRegimen14(33%)13(38%)Conditioning regimen Melphlan16(38%)7(20%)IgG21(51%)19(56%)IgA8(20%)9(26%)IgM4(9%)Mortality13(31%)21(62%)Overall survival65%25% [Display omitted] [Display omitted] ConclusionPatients who harbor the 13q and additional cytogenetic abnormalities identified by I-FISH have a significant worse outcome compared to patients with an isolated 13q. These patients should be considered high risk and consideration for treatment with novel agents and autologous stem cell transplant followed by post-transplant maintenance therapy should be considered. Disclosures:No relevant conflicts of interest to declare.

Differences in the cytogenetic alteration profiles of diffuse large B-cell lymphoma among Chinese and American patients

To study the similarities and differences of cytogenetic alterations in diffuse large B-cell lymphoma (DLBCL) between Asian and Caucasian patients, we compared the cytogenetic profiles of Chinese and American DLBCL cases by analyzing conventional karyotypes and select fluorescence in situ hybridization (FISH) findings. We used interphase FISH analyses to determine the incidence of the t(14;18) and BCL6 and MYC rearrangements. Immunohistochemical analysis was used to categorize the lymphomas into the germinal center B-cell-like (GCB) or non-GCB-DLBCL subtypes, according to the Hans algorithm. Our data suggested that Chinese patients had cytogenetic profiles for GCB-DLBCL that differed from those of their American counterparts; specifically, the Chinese GCB patients exhibited greater frequencies of BCL6 rearrangements and gains of 1q and 11q but lower incidence of the t(14;18). Non-GCB-DLBCL in both the Chinese and American patients was characterized by recurrent gains of 3/3q and 18/18q. The incidences of both BCL6 rearrangement and t(14;18) were similar in Chinese and American non-GCB-DLBCL cases.