Dual-color Interphase Fluorescence Research Articles

MiR-21-5p, miR-34a, and human telomerase RNA component as surrogate markers for cervical cancer progression

ObjectiveThis study aimed to demonstrate the predictive value of miR-21-5p, miR-34a, and human telomerase RNA component (hTERC) in cervical cancer (CC) development and evaluated their potential possibility for future clinical applications. MethodsSpecimens were collected from the normal cervix, cervical intraepithelial neoplasia (CIN) I, CIN II/III, cervical squamous cell carcinoma. Cytological evaluations and histopathologic examinations were conducted in all subjects, along with the assessment of human papillomavirus (HPV) DNA. The expression levels of the miR-21-5p and miR-34a were detected by RT-PCR. hTERC amplification was detected by dual-color interphase fluorescence in situ hybridization (FISH). Then miRNA, hTERC expressions were compared with the cytological and histologic examination. ResultsCompared to that in the benign samples, the expression of miR-21-5p and miR-34a in abnormal samples was significantly upregulated and downregulated, gradually corresponding to the severity of cervical lesions (P < 0.05). There was a trend toward an increasing amplification of hTERC with the increasing severity of cervical lesions. miR-21-5p and miR-34a expression, and hTERC amplification were more specific than HPV positivity in differentiating low-grade cervical disorders from high-grade ones (P < 0.05). ConclusionsMiR-21-5p upregulation, miR-34a downregulation, and hTERC amplification were associated with the aggressive progression of CC, which suggests that miR-21-5p, miR-34a and hTERC might serve as surrogate markers for CC progression and potential molecular targets for blockage of the development of CC.

Preservation of truncal genomic alterations in clear cell and papillary renal cell carcinomas with sarcomatoid features: An intra- and intertumoral, multifocal fluorescence in situ hybridization analysis reveals limited genetic heterogeneity.

Understanding tumor genomic heterogeneity may offer vital information in an age of targeted therapy for renal cell carcinoma. We sought to investigate hallmark truncal chromosomal alterations between conventional, sarcomatoid, and matched metastatic tumor foci in clear cell and papillary renal cell carcinomas. A retrospective review identified 58 cases including clear cell (CCRCC) and papillary renal cell carcinomas (PRCC). All cases contained sarcomatoid transformation. Additionally, 10 of 58 patients had matched metastatic disease available for analysis. Three separate foci of conventional and sarcomatoid morphologies were analyzed in each tumor using dual color interphase fluorescence in situ hybridization. In the CCRCC cohort, hallmark chromosome 3p deletion was identified in 71% of cases (37/52). Complete concordance of chromosomal status between intratumoral foci in sarcomatoid and conventional foci was 89% and 86%, respectively. Overall chromosome 3p status between matched conventional and sarcomatoid morphologies was identified in 98% of cases (51/52). Hallmark 3p deletion was present in 91% of CCRCC metastatic samples (10/11) and was concordant with the matched primary CCRCC tumor in 91% (10/11). In the PRCC cohort, trisomy 7 and 17 was identified in all six cases (6/6). Complete concordance between intratumoral foci of trisomy 7 and 17 was 83% (5/6). Trisomy 7 and 17 were identified in all metastatic PRCC samples with 100% concordance with the matched primary tumor. These data show the relative preservation of truncal chromosomal abnormalities between conventional and sarcomatoid morphologic as well as matched metastatic settings.

The clinical value of Thinprep cytology test combined with h-TERC and c-myc in the diagnosis of cervical cancer

Objective To explore the clinical value of Thinprep cytology test (TCT) combined with h-TERC and c-myc in the diagnosis of cervical cancer. Methods hTERC amplification was detected by dual-color interphase fluorescence in situ hybridization (FISH), and the results were compared with TCT and histological examination.Examination the positive which TCT, h-TERC and c-myc by pathological examination.The final diagnosis was determined by the pathological examination. Results TCT was abnormal in 26.4% of 500 case, 18.0% abnormal h-TERC gene, 16.0% abnormal c-myc gene.In 270 cases according to the cervical biopsy, the positive rate of chronic inflammation, cervical intraepithelial neoplasia (CIN)Ⅰ, CINⅡ, CINⅢ and cervical cancer: 44.4%, 38.2%, 36.4%, 18.2%, and 7.3% respectively.The positive rates of h-TERC were 18.1%, 45.4%, 52.5%, 65.9% and 100.0%, respectively.The positive rates of c-myc were 21.4%, 48.9%, 56.7%, 59.9% and 100.0%.With increased pathological grade, the expressions of h-TERC and c-myc were high. Conclusion TCT combined with h-TERC and c-myc can test cervical cancer more effective. Key words: Cytological examination; Gene, h-TERC; Gene, myc; Uterine neoplasm

Molecular subtyping of metastatic renal cell carcinoma: implications for targeted therapy

BackgroundRenal cell carcinoma (RCC) is known for its ability to metastasize synchronously or metachronously to various anatomic sites. Distinguishing histologic subtypes of metastatic RCC has become increasingly important, as prognosis and therapy can differ dramatically between subtypes. We propose a combination of immunohistochemistry (IHC) and molecular cytogenetics for subtyping metastatic RCC in light of these potential therapeutic implications.ResultsSpecimens from 103 cases of metastatic RCC were retrieved, including 32 cases originally diagnosed as metastatic clear cell renal cell carcinoma (CCRCC), 8 as metastatic papillary renal cell carcinoma (PRCC), and 63 metastatic RCC without a specific subtype. Immunohistochemistry was performed with antibodies against cytokeratin 7 (CK7) and alpha-methylacyl-CoA racemase (AMACR). Dual color interphase fluorescence in situ hybridization was utilized to assess for deletion of chromosome 3p and trisomy of chromosomes 7 and 17 in all tumors. Chromosome 3p deletion was detected in 41% of all metastatic RCC specimens, and trisomy of chromosomes 7 and/or 17 was detected in 16%. Of metastatic CCRCC, chromosome 3p deletion was detected in 63%. Of metastatic PRCC, 75% showed trisomy of chromosomes 7 and/or 17. Of the tumors not previously classified, 6% were positive for CK7, and 64% were positive for AMACR; 35% showed chromosome 3p deletion, and 16% showed trisomy of chromosomes 7 and/or 17. Combined analysis of immunohistochemistry and cytogenetics enabled reclassification of 52% of these metastatic tumors not previously classified.ConclusionOur findings support the utility of immunohistochemistry and cytogenetics for subtyping metastatic RCC.

The detection of hTERC amplification using fluorescence in situ hybridization in the diagnosis and prognosis of cervical intraepithelial neoplasia: a case control study

BackgroundCurrently the routine non-invasive screening methods for cervical intraepithelial neoplasia (CIN) and cervical cancer are Thinprep cytology test (TCT) and human papillomavirus testing. However, both methods are limited by the high false positive and false negative rates and lack of association with patients’ prognosis, especially for the early detection of pro-malignant CIN. The aim of the study was to investigate the role of genomic amplification of human telomerase gene (hTERC) in the diagnosis and prognosis of CIN.MethodsThe study group consisted of specimens of exfoliated cervical cells from 151 patients, including 27 with CIN I, 54 with CIN II/III, 17 with carcinoma in situ, and 28 with invasive squamous carcinoma, as well as 25 patients who were at 2-year follow-up after either Loop Electrosurgical Excision treatment (n = 11) or radical surgery (n = 14). hTERC amplification was detected by dual-color interphase fluorescence in situ hybridization (FISH), and the results were compared with TCT and histologic examination. The final diagnosis was determined by the pathological examination. The control group consisted of specimens of exfoliated cervical cells from 40 normal women.ResultsThe percentage of cervical exfoliated cells with positive hTERC amplification and incidence rates of hTERC amplification were 9.2% ± 4.6% and 44.4% (12/27) respectively in patients with CIN I; 16.0% ± 14.4% and 85.1% (46/54) in patients with CIN II/III; 19.7% ± 13.3% and 88.3% (15 /17) in patients with carcinoma in situ; 47.0% ± 25.2% and 100% (28/28)in patients with invasive squamous carcinoma. There was statistically significant difference between the control and study group (P <0.01), and between the patients with various diseases within the study group (P <0.05).ConclusionThe detection of genomic amplification of hTERC using FISH is a non-invasive and effective approach for CIN.

Expression of human telomerase gene in esophageal cancer

Objective To evaluate the dual-color interphase fluorescence in situ hybridization (FISH) of human telomerase gene (hTERC) in the esophageal cancer and preneoplastic lesions. Methods The fluorescence signals of hTERC in the esophageal cancer and preneoplastic lesions were detected by using interphase FISH. According to histology, biopsy and pathology certification, the samples include 45 cases of esophageal normal tissue, 27 cases of atypical hyperplasia Ⅰ , 22 cases of atypical hyperplasia Ⅱ / Ⅲ and 55 cases of squamous cell carcinomas (SCC) of the esophagus. Results None normal samples revealed copy number increases of 3q, while 25.9% of atypical hyperplasia Ⅰ , 54. 5% of atypical hyperplasia Ⅱ/Ⅲ, 90. 9% of the squamous esophageal cancer showed extra copies of 3q. The comparison between groups showed statistically significant difference ( P ＜ 0. 05 ). The mean copy number was 2. 19 ±0. 11,2. 35 ± 0. 30, 2. 64 ± 0. 27 respectively. The positive amplification of the hTERC gene was not correlated with histological stages, lymph nodes metastasis and the grades of carcinoma differentiation in SCC (P ＞ 0. 05 ). Conclusion The presence of hTERC gene may play an important role in the development of SCC. Key words: Fluorescence in situ hybridization; hTERC; Esophageal cancer

Multilocular cystic renal cell carcinoma is a subtype of clear cell renal cell carcinoma

Multilocular cystic renal cell carcinoma is an uncommon low grade renal cell carcinoma with unique morphologic features. Its cytogenetic characteristics have not been fully investigated. Its relationship to typical clear cell renal cell carcinoma is uncertain. We evaluated 19 cases of multilocular cystic renal cell carcinoma diagnosed by strict morphologic criteria using the 2004 WHO classification system. The control group consisted of 19 low grade (Fuhrman grades 1 or 2) clear cell renal cell carcinomas. Chromosome 3p deletion status was determined by dual color interphase fluorescence in situ hybridization analysis. Chromosome 3p deletion was identified in 17 out of 19 (89%) of the clear cell renal cell carcinoma cases and 14 out of 19 (74%) of the multilocular cystic renal cell carcinoma cases, respectively. There was no difference in the status of chromosome 3p deletion between clear cell renal cell carcinoma and multilocular cystic renal cell carcinoma (P=0.40). These results support the concept that multilocular cystic renal cell carcinoma as a subtype of clear cell renal cell carcinoma.

Evidence for Polyclonal Origin of Multifocal Clear Cell Renal Cell Carcinoma

Renal cell carcinomas are often multifocal. We investigated the genomic signatures of multifocal clear cell renal cell carcinoma to determine whether multiple tumors in the same kidney bear a clonal relationship to one another. A total of 62 tumors from 26 patients who underwent radical nephrectomy were examined. All patients had multiple separate clear cell renal carcinomas. Loss of heterozygosity analyses were done using five microsatellite polymorphic markers that represent putative tumor suppressor genes on chromosome 3p14 (D3S1300), 7q31 (D7S522), 8p22 (D8S261), 9p21 (D9S171), and 17p13 (TP53). X chromosome inactivation analyses were also done on the renal tumors from the 10 female patients. Chromosome 3p deletion status was determined by dual color interphase fluorescence in situ hybridization analysis in all tumors. Nineteen of the 26 (73%) patients with multifocal clear cell renal cell carcinoma showed allelic loss in at least 1 of 5 microsatellite loci in separate tumors analyzed. A disconcordant pattern of allelic loss between coexisting kidney tumors was observed in 7 cases. Six cases showed discordant 3p deletion patterns by dual color interphase fluorescence in situ hybridization analysis. Of the eight informative female cases studied by X chromosome inactivation, one showed a discordant nonrandom pattern of X chromosome inactivation. Overall, evidence of independent origin of the multifocal renal tumors was observed in 12 of 26 cases (46%). Our data suggest that in a significant number of cases of multifocal clear cell renal cell carcinoma, the spatially separate tumors are of different clonal origin and arise independently.

Molecular Characterization of TMPRSS2-ERG Gene Fusion in the NCI-H660 Prostate Cancer Cell Line: A New Perspective for an Old Model

Recent studies have established that a significant fraction of prostate cancers harbor a signature gene fusion between the 5' region of androgen-regulated TMPRSS2 and an ETS family transcription factor, most commonly ERG. Studies on the molecular mechanisms and functional consequences of this important chromosomal rearrangement are currently limited to the VCaP cell line derived from a vertebral bone metastasis of a hormone-refractory prostate tumor. Here we report on the NCI-H660 cell line, derived from a metastasic site of an extrapulmonary small cell carcinoma arising from the prostate. NCI-H660 harbors TMPRSS2-ERG fusion with a homozygous intronic deletion between TMPRSS2 and ERG. We demonstrate this by real-time quantitative polymerase chain reaction, a two-stage dual-color interphase fluorescence in situ hybridization (FISH) assay testing for TMPRSS2 and ERG break-aparts, and single-nucleotide polymorphism oligonucleotide arrays. The deletion is consistent with the common intronic deletion found on chromosome 21q22.2-3 in human prostate cancer samples. We demonstrate the physical juxtaposition of TMPRSS2 and ERG on the DNA level by fiber FISH. The androgen receptor-negative NCI-H660 cell line expresses ERG in an androgen-independent fashion. This in vitro model system has the potential to provide important pathobiologic insights into TMPRSS2-ERG fusion prostate cancer.

Trisomy 22 as the Sole Abnormality Is an Important Marker for Inversion 16 in Acute Myeloid Leukemia.

Inv(16) has been reported in 10%~12% of acute myeloid leukemia (AML), mostly being associated with the M4Eo subtype, and is associated with a relatively favorable outcome. However, it is a cryptic rearrangement and often difficult to recognize in conventional cytogenetics (CC). Trisomy 22 is an uncommon karyotypic aberration in AML and is often associated with inv(16)(p13q22). In order to explore the value of trisomy 22 in the diagnosis of AML with inv(16), dual-color interphase fluorescence in situ hybridization (FISH) was performed in 19 AML cases with trisomy 22 abnormality. The probe was two-color break apart probe for CBFb with SpectrumRed on the centromeric side and SpectrumGreen on the telomeric side. And the results were compared with that of R-banding CC. CC did not reveal inv(16) in any of the 19 AML with trisomy 22, but FISH analysis showed inv(16) in 11 cases and del(16)(q22) in one case. Among 11 cases with inv(16), 9 were trisomy 22 as the sole abnormality, one was complicated with trisomy 8, and one was del(16)(q22). Four AML patients with trisomy 22 and inv(16) were analyzed by multiplex FISH (M-FISH) which revealed trisomy 22 only. This study further confirmed that trisomy 22 as the sole abnormality can be regarded as an important marker for the inv(16) in AML.

Mechanisms of resistance to imatinib mesylate in gastrointestinal stromal tumors and activity of the PKC412 inhibitor against imatinib-resistant mutants

Resistance is a major challenge in the treatment of patients with gastrointestinal stromal tumors (GISTs). We investigated the mechanisms of resistance in patients with progressive GISTs with primary KIT mutations and the efficacy of the kinase inhibitor PKC412 for the inhibition of imatinib-resistant mutants. We performed a cytogenetic analysis and screened for mutations of the KIT and PDGFRA kinase domains in 26 resistant GISTs. KIT autophosphorylation status was assessed by Western immunoblotting. Imatinib-resistant GIST cells and Ba/F3 cells expressing these mutant proteins were tested for sensitivity to imatinib and PKC412. Six distinct secondary mutations in KIT were detected in 12 progressive tumors, with V654A and T670I found to be recurrent. One progressive tumor showed acquired PDGFRA -D842V mutation. Amplification of KIT or KIT / PDGFRA was found in 2 patients. Eight of 10 progressive tumors available for analysis showed phosphorylated KIT. Two remaining progressive tumors lost KIT protein expression. GIST cells carrying KIT -del557-558/T670I or KIT -InsAY502-503/V654A mutations were resistant to imatinib, while PKC412 significantly inhibited autophosporylation of these mutants. Resistance to imatinib and sensitivity to PKC412 of KIT -T670I and PDGFRA -D842V mutants was confirmed using Ba/F3 cells. This study shows the high frequency of KIT/PDGFRA kinase domain mutations in patients with secondary resistance and defines genomic amplification of KIT / PDGFRA as an alternative cause of resistance to the drug. In a subset of patients, cancer cells lost their dependence on the targeted tyrosine kinase. Our findings show the sensitivity of the imatinib-resistant KIT -T670I and KIT -V654A and of PDGFRA -D842V mutants to PKC412.

Frequent occurrence of BCL6 rearrangements in nodular lymphocyte predominance Hodgkin lymphoma but not in classical Hodgkin lymphoma

We studied the genomic status of BCL6 in 23 cases of nodular lymphocyte predominance Hodgkin lymphoma (NLPHL) and 40 cases of classical Hodgkin lymphoma (cHL), using dual-color interphase fluorescence in situ hybridization (FISH). The BCL6 rearrangement was identified in 48% of NLPHL cases and was not detected in cHL cases. As a confirmation, sequential or simultaneous immunohistochemistry (IHC) and FISH using CD20 or BCL6 antibodies and BCL6 DNA probes was performed in 8 NLPHL cases. The BCL6-associated translocations, t(3;22)(q27;q11), t(3;7)(q27;p12), and the most probable t(3;9)(q27;p13), were identified in 3 cases. A consistent expression of BCL6 protein in popcorn cells with the highest number of intensely stained cells in cases with a genomic BCL6 rearrangement was shown by IHC. These findings support the hypothesis of a germinal center B cell-derived origin of NLPHL, indicate a significant role of BCL6 in the pathogenesis of NLPHL, and provide further evidence of the genetic diversity underlying the pathogenesis of NLPHL and cHL.

Human mesenchymal stem cells are not of donor origin in patients with severe aplastic anemia who underwent sex-mismatched allogeneic bone marrow transplant.

Stromal defects are part of the etiology of severe aplastic anemia (SAA), and hematopoietic engraftment is poor in unrelated and mismatched transplant. Therefore, we wanted to find out whether human mesenchymal stem cells (MSC) are partly of donor origin in patients with SAA years after successful bone marrow transplant (BMT). Three SAA patients 3, 5, and 8 years after BMT (cyclophosphamide, ATG) with bone marrow from an HLA-identical sibling donor of the opposite sex were investigated. MSC were grown from patients' bone marrow aspirates according to Caplan et al. The number of MSC that were isolated from SAA bone marrow post transplant was about 10 times lower than in normal controls. Primary cultures of adherent MSC and passage-one cells were analyzed by dual-color interphase fluorescence in situ hybridization (FISH) analysis using centromere-specific DNA probes for X and Y chromosome. FISH did not show any clear evidence of donor cells in the adherent MSC: In all cases, less than 0.5% of nuclei showed a donor-type signal pattern that is well within assay limits. In a female patient, the absence of male donor cells was confirmed by sensitive and quantitative, Y chromosome-specific TaqMan PCR (QYCS-PCR). In contrast, Ficoll-separated hematopoietic cells from the same aspirates were greater than 90% of donor origin, as expected. In SAA, as previously found in patients with lysosomal and peroxisomal storage disease, bone marrow MSC remain host-derived despite successful hematopoietic engraftment years after allogeneic BMT.

Delineation of the minimal region of loss at 13q14 in multiple myeloma.

Previous studies have focused on the incidence and prognostic implications of 13q14 deletions in multiple myeloma (MM), but none has sought to delineate the minimal common deleted region (CDR). In an effort to do so, dual-color interphase fluorescence in situ hybridization (FISH) was applied on 82 myeloma cases, initially by use of three probes for 13q14 (RB1, D13S319, and D13S25). Deletions were detected in 29/82 (35.4%) cases, and all except one were monoallelic. Subsequently, contiguous YACs, PACs, and a BAC spanning the 13q14-q21 region were employed for deletion mapping in addition to a 13q telomere probe. Large deletions extending to the 13q34 region were found in 55% of the deleted cases, whereas an additional 13.8% showed loss of both 13q34 and 13q14 regions with retention of 13q21. A CDR of approximately 350 kb was identified at 13q14 with the proximal border approximately 120 kb centromeric from D13S319, encompassing an area rich in expressed sequence tagged sites and containing DLEU1, DLEU2, and RFP2 genes. Direct sequencing of the RFP2 gene revealed no mutations in six patients and four MM cell lines harboring deletions of the CDR. However, a role for RFP2 in the pathogenesis of MM cannot yet be excluded, given that alternative mechanisms such as haploinsufficiency remain possible.

ETV6/AML1 fusion by FISH in adult acute lymphoblastic leukemia.

Dual-color interphase fluorescence in situ hybridization (FISH) with ETV6 and AML1 probes was used for the first time on a series of 159 adult patients with acute lymphoblastic leukemia (ALL), for detection of the t(12;21)(p13;q22) translocation. Seven patients (4.4%) were found, with 50-100% of positive cells, of whom one of two tested, proved negative for the fusion product by RT-PCR. Two of them, aged 43 and 50 years, are the oldest patients so far confirmed to have the translocation. Three who relapsed at 10, 11 and 24 months, suggest that adults may not enjoy the good short-term prognosis reported for t(12;21)-positive children. Thirty-one-negative cases had signal numbers differing from the two expected for each gene. In 15 cases these results were consistent with the karyotype. In nine cases with uninformative cytogenetics, the numbers were consistent with those for centromeres and indicated a hidden aneuploidy. Loss of ETV6 genes in two cases and AML1 amplification in three others were not suspected from the cytogenetics. In conclusion, FISH proved to be reliable in defining ETV6/AML1 positivity in this group of patients as well as providing valuable insights into negative cases.

Identification of numerical chromosomal changes detected by interphase fluorescence in situ hybridization in high-grade prostate intraepithelial neoplasia as a predictor of carcinoma.

High-grade prostate intraepithelial neoplasia (HPIN) is the most likely precursor of prostate cancer. The condition of many patients with a diagnosis of HPIN in prostate needle core biopsy could, if left untreated, progress to invasive cancer. Currently there is no available clinical, immunohistochemical, or morphologic criteria that are predictive of this progression. To determine whether chromosomal instability in these precursor lesions could increase their predictive value for cancer detection. Dual-color interphase fluorescence in situ hybridization analysis was performed on archived prostate needle core biopsies from 54 patients with initial diagnosis of isolated HPIN and follow-up of 3 years or more. We used commercially available centromere probes for chromosomes 4, 7, 8, and 10. We had interpretable results in 44 patients as follows: (1) group A: 24 HPIN patients with persistent HPIN and/or benign lesions in the follow-up biopsies, and (2) group B: 20 HPIN patients with progression to prostate carcinoma. Twenty-five percent of the patients in group B displayed numeric chromosomal aberrations. Only 8.3% of the patients from group A had chromosomal abnormalities (P =.1). The observed overall chromosomal changes in HPIN were higher than those in normal or hyperplastic epithelium, with a statistically significant difference (P <.05). All aberrations were detected in the form of chromosomal gain. Overall, the commonest aberration was gain of chromosome 8, followed by gains of chromosomes 7 and 10. These results indicated that although no single numeric chromosomal abnormality could be assigned as a predictor of HPIN progression to carcinoma, the overall level of numeric chromosomal abnormalities shows a trend of elevation in HPIN patients whose condition subsequently progressed to carcinoma.

Detection of t(I 1; 18) in MALT-type lymphoma with dual-color fluorescence in situ hybridization and reverse transcriptase-polymerase chain reaction analysis.

t(11:8) is a recurrent chromosomal abnormality observed in mucosa-associated lymphoid tissue (MALT)-type lymphoma. API2 and MLT genes have been implicated. The authors devised a dual-color interphase fluorescence in situ hybridization (FISH) system to detect splitting of 11q22 and its fusion with 18q21. Subjects were 44 cases of extranodal lymphoma and cases of primary macroglobulinemia. Whenever RNA was available, reverse transcriptase-polymerase chain reaction followed by sequence analysis was performed. Positive cases by dual-color FISH analysis were restricted to MALT-type lymphoma and one case of primary macroglobulinemia. Among 24 cases of MALT-type lymphoma, 14 (58%) (4 gastric, 5 pulmonary, 3 orbital, 1 salivary, and 1 thyroid lymphomas) had splitting of the 11q22 region probes and fusion of signals suggesting the translocation of chromosome 11 and 18. Reverse transcriptase-polymerase chain reaction analysis showed the API2/MLT gene fusion in 9 of 10 cases. Sequence analyses showed three different modes of involvement of the MLT gene, whereas the breakpoint at API2 was the same. Monoclonal component of serum immunoglobulin M was observed in 3 of 14 positive cases for the translocation. Direct visualization using dual-color FISH on samples serves as a molecular tool for management of MALT-type lymphoma with API2/MLT gene fusion.

Analysis of the P53, RB/D13S25, and P16 Tumor Suppressor Genes in Marginal Zone B-cell Lymphoma : An Interphase Fluorescence In Situ Hybridization Study

The genetic mechanisms underlying the genesis, disease progression, and high-grade transformation of marginal zone B-cell lymphoma (MZBCL) are poorly understood. We analyzed 33 cases of histologically and immunophenotypically well-characterized MZBCL (12 extranodal, 11 nodal, and 10 splenic MZBCL; 27 at primary diagnosis and six during the course of disease) by dual-color interphase fluorescence in situ hybridization (FISH) for deletions of tumor suppressor genes. We investigated loci known to play a role in the genesis or disease progression of other subtypes of lymphoid malignancies, namely the P53 gene (17p13), the retinoblastoma gene (RB, 13q14), the D13S25 locus (13q14), and the P16 INK4A gene (9p21). Heterozygous deletions of P53 were detected in three out of the 33 cases, including two splenic and one extranodal MZBCL. One of these patients was analyzed at primary diagnosis and two during the course of disease. Heterozygous deletions of the RB gene (nodal MZBCL) and D13S25 (splenic MZBCL) were found in one case each. P16 deletions were not detected in any of our cases. We conclude that deletions of the analyzed tumor suppressor genes are relatively rare in MZBCL, which contrasts with the findings in some other subtypes of NHL.

The value of interphase fluorescence in situ hybridization for the detection of translocation t(12;21) in childhood acute lymphoblastic leukemia.

Translocation t(12;21)(p13;q22) is the most frequent cytogenetic abnormality in childhood acute lymphoblastic leukemia (ALL) and is generally associated with favorable prognosis. In this report, we assessed the value of dual-color interphase fluorescence in situ hybridization (FISH) for the detection of t(12;21). Fifty-three patients were screened for ETV6/CBFA2 fusion by means of FISH, using two cosmid probes mapped on ETV6 and on CBFA2, respectively. The cut-off value (mean + three standard deviations) for positivity established on control patients was 9.3%. A comparison between FISH and molecular methods [reverse-transcriptase polymerase chain reaction/Southern blot (RT-PCR/SB)] was possible in 52 patients: 34 of 52 (65.4%) showed negative results with both approaches, and 13 of 52 (25%) were positive; 5 of 52 (9.6%) showed discrepancies: four patients who were positive using RT-PCR/SB were negative using FISH. Conversely, one patient negative when using RT-PCR/SB was positive with FISH. Further investigations on this patients, cytogenetically characterized by add(12p), showed an atypical breakpoint on ETV6, located 5' to the common breakpoint. Compared with RT-PCR and SB, dual-color interphase FISH with the cosmid probe set proved to be highly specific but showed limited sensitivity.

The Value of Interphase Fluorescence In Situ Hybridization in the Study of Patients with Lymphoproliferative Disorders: Further Evidence for a Higher Sensitivity of Detecting Chromosomes 7 and 8 Aneuploidy

Dual-color interphase fluorescence in situ hybridization (I-FISH) for chromosomes 7 and 8 was studied retrospectively on 32 patients with suspected lymphoid disorders, and the results were compared with standard cytogenetics. One of 29 (3.4%) patients with lymphoid malignancy showed cytogenetically detectable aneuploidy for chromosomes 7 and 8. In an additional 5 patients (17.2%), I-FISH unmasked chromosomal loss and gain that were not detected by standard metaphase analysis. This represents 19% of the 21 studied patients with acute lymphoblastic leukemia (ALL). These findings indicate that aneuploidies for chromosomes 7 and 8 are underreported in ALL and further demonstrate higher sensitivity of I-FISH for detecting numerical chromosomal rearrangements in leukemic cells.