Detection of three common translocation breakpoints in non-Hodgkin's lymphomas by fluorescence in situ hybridization on routine paraffin-embedded tissue sections

The receptor for advanced glycation end-products (RAGE) is a newly recognized factor regulating cancer cell invasion and metastasis. This study investigated the expression of RAGE in gastric carcinomas and its association with invasion and metastasis. Of eight gastric cancer cell lines examined, seven constitutively expressed RAGE messenger ribonucleic acid (mRNA), MKN45 being the exception. RAGE protein expression of MKN28 cells treated with RAGE antisense S-oligodeoxynucleotide was nine times less than that of sense S-oligodeoxynucleotide-treated cells. Growth of cells under RAGE antisense S-oligodeoxynucleotide treatment was not different from that seen under sense S-oligodeoxynucleotide treatment in MKN28 (a cell line producing high levels of RAGE) and MKN45 (a non-RAGE-expressing cell line). RAGE antisense S-oligodeoxynucleotide treatment suppressed the invasive activity of RAGE-positive MKN28 cells, as estimated by in vitro invasion assay. The number of MKN28 cells invading the type IV collagen-coated membrane under RAGE antisense S-oligodeoxynucleotide treatment was significantly lower than under RAGE sense S-oligodeoxynucleotide treatment (p<0.0001). In contrast, antisense and sense S-oligodeoxynucleotide-treated RAGE-negative MKN45 cells showed no difference. A wound-healing assay showed that no RAGE antisense S-oligodeoxynucleotide-treated MKN28 cells migrated into the scraped area, whereas sense S-oligodeoxynucleotide-treated cells showed many budding nests in the scraped area. Immunohistochemistry of gastric carcinoma tissue showed that 62 (65%) of the 96 cases examined were RAGE-positive and that poorly differentiated adenocarcinomas preferentially expressed RAGE protein (38/42, 90%) (p<0.0001). Strong RAGE immunoreactivity was also correlated with depth of invasion and lymph node metastasis (p<0.0001). RAGE-positive cancer cells tended to be distributed at the invasive front of primary tumours and were detected in all metastatic foci in lymph nodes. In contrast, a major RAGE ligand, amphoterin, was expressed in 82 (85%) of the 96 cases, regardless of histological type and disease progression. RAGE expression appears to be closely associated with invasion and metastasis in gastric cancer.     

Specific metaphase and interphase detection of the breakpoint region in 8q24 of Burkitt lymphoma cells by triple-color fluorescence in situ hybridization.

Triple fluorescence in situ hybridization with a plasmid DNA library from sorted human chromosomes 8 in combination with bacteriophage clones flanking the breakpoint in 8q24 of the Burkitt lymphoma cell line J1 was used for the specific delineation of this breakpoint in individual tumor cells. With this approach, tumor-specific breakpoints in translocation chromosomes can be detected at all stages of the cell cycle with high specificity.     

Interphase fluorescence in situ hybridization assay for the detection of 3q21 rearrangements in myeloid malignancies.

In myeloid malignancies, chromosome rearrangements involving band 3q21 are associated with a particularly poor prognosis of the disease. Their sensitive and unequivocal detection is therefore of great clinical importance. In this report, we describe the establishment of an interphase fluorescence in situ hybridization (FISH) assay that complements classical cytogenetic analysis in the diagnosis of such aberrations. PACs that map centromeric and telomeric of known 3q21 breakpoints were labeled with different fluorescent dyes, and the separation of the normally colocalizing signals was used as an indicator of the presence of a 3q21 rearrangement. Two cell lines and 10 primary samples from myeloid leukemia and myelodysplastic syndrome (MDS) patients with 3q21 rearrangements were investigated using the newly established method. The rate of false positivity was determined in 27 control samples from patients with various types of myeloid malignancies. In addition to providing a sensitive and rapid test for the detection of 3q21 aberrations, the interphase FISH assay yields preliminary information about the localization of individual breakpoints. Six of the 10 breakpoints in the patient samples map to an only recently described breakpoint cluster region (BCR) 60 kb centromeric of the originally reported 3q21 BCR. These findings may contribute to the understanding of the molecular basis of the clinical features associated with 3q21 rearrangements.     

Hemizygous deletions of chromosome band 16q24 in Wilms tumor: detection by fluorescence in situ hybridization

Loss of heterozygosity (LOH) for markers on chromosome arm 16q in Wilms tumor has been linked to an increased risk of treatment failure. We therefore postulated that fluorescence in situ hybridization (FISH) with probes from this region might enhance current strategies for identifying high-risk patients at diagnosis. In a blinded comparative pilot study of 19 Wilms tumor samples from 18 patients with favorable histology, FISH and DNA polymorphism analysis yielded concordant results in 14 cases, either retention (n = 6) or loss (n = 8) of chromosome arm 16q markers. Discordant findings in 4 of the 5 remaining cases resulted from detection of LOH, but no loss by FISH. Two of these cases, directly comparable at marker D16S422, appeared to have tumor-specific uniparental disomy, in that 2 copies of D16S422 and the 16 centromere were evident, despite LOH. In 2 other cases, the discrepancies could be explained by LOH confined to loci distal to the D16S422 locus. In the fifth case, FISH detected 2 distinct populations of tumor cells, one characterized by normal diploidy and the other by monosomy 16, whereas DNA polymorphism analysis failed to indicate LOH altogether. Thus, FISH confirmed the presence of allelic loss (hence, the possible location of biologically important tumor suppressor genes) on the distal long arm of chromosome 16 in cases of favorable-histology Wilms tumor, with the advantages of technical simplicity, successful analysis of samples that were otherwise uninformative by analysis of DNA polymorphisms, and the addition of internal controls for chromosomal aneusomy. We suggest that combined analysis of the chromosome 16q region in Wilms tumor by FISH and DNA polymorphism analysis would improve evaluations to identify high-risk patients who might benefit from alternative therapy.     

Frequent deletions of 6q23–24 in B-cell non-Hodgkin's lymphomas detected by fluorescence in situ hybridization

Deletions of the long arm of chromosome 6 (6q) are among the most frequent chromosome aberrations in malignant lymphomas and often occur as secondary changes in addition to typical translocations, such as t(14; 18). Using fluorescence in situ hybridization (FISH) with two YAC probes hybridizing to 6q23–24 and with the centromeric probe D6Z1 as internal control, we studied 31 cases of low-grade and eight cases of high-grade B-cell lymphoma. Deletions of 6q23–24 were detected in 21 patients (56.8%) by FISH, compared to 13 patients (33.3%) by chromosome analysis. Deletions of 6q23–24 were found by FISH in 5 of 13 cases of small lymphocytic lymphoma, in 2 of 3 cases of mantle cell lymphoma, in 10 of 14 cases of t(14; 18) positive low-grade follicular lymphoma, and in 4 of 8 cases of high-grade follicular lymphoma. This study shows that deletions of 6q23–24 are more frequent in B-cell lymphomas than previously suggested and that they can be detected more sensitively by FISH than by chromosome analysis. Contrary to previous reports indicating that the region 6q23–24 is preferentially deleted in low-grade lymphomas without t(14; 18), our results indicate that deletions of 6q23–24 appear to be common in other pathological subsets of B-cell lymphoma as well, especially in follicular lymphomas with t(14; 18). Genes Chromosom. Cancer 18:310–313, 1997. © 1997 Wiley-Liss, Inc.     

Interphase fluorescence in situ hybridization studies for the detection of 9q34 deletions in chronic myelogenous leukemia: a practical approach to clinical diagnosis

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome (Ph) in more than 90% of cases. Recent studies using fluorescence in situ hybridization (FISH) have shown that in a subset of patients with CML, deletions of 9q34 involving the argininosuccinate synthetase region occur at the time of the Philadelphia translocation and are associated with a poor prognosis. We performed interphase FISH studies in 152 cases of CML using a dual-color, dual-fusion probe system with a third probe directed at 9q34. Cytogenetic studies showed a simple (typical) Ph in 124/152 (82%), a cryptic Ph in 11/152 (7%), and a variant Ph chromosome with a complex translocation in 17/152 (11%) of cases. Interphase FISH studies showed single BCR/ABL fusion patterns in 48/152 (32%) of cases. Deletions of 9q34 were observed in 14% of all the cases and were present in 46% of cases with single BCR/ABL fusion pattern. All the 9q34 deletions occurred in cases with single BCR/ABL fusion signal. However, a single-fusion pattern is not specific for 9q34 deletions, and cases should be routinely screened for the presence of this prognostically significant abnormality by using a third probe directed specifically at 9q34.     

Interphase FISH analysis on histologic sections of fixed tissues for t(11;14) (q13;q32) detection in mantle cell lymphoma and t(8;14)(q24;q32) in Burkitt's lymphoma

We describe an interphase FISH analysis for formol-fixed, paraffin-embedded tissue sections with commercial probes detecting the t(11;14)(q13;q32) in mantle cell lymphoma and the t(8;14)(q24;q32) in Burkitt's lymphoma. Staining of an adjacent section allowed identification of tumoral areas. The cut-off value was evaluated in reactive lymph nodes at 5% for both probes. An incomplete hybridization pattern was found in 18 to 26% of the nuclei but was always lower than the percentages of translocated cells in tumoral regions. A t(11;14) was detected in 4/4 mantle cell lymphomas and a t(8;14) in 2/3 Burkitt's lymphomas. Interphase FISH analysis of fixed-tissue sections is a reliable technique for the direct detection of lymphoma-associated translocations on routine histological material.     

Submicroscopic genomic imbalances in burkitt lymphomas/leukemias: Association with age and further evidence that 8q24/MYC translocations are not sufficient for leukemogenesis

Chromosome banding analyses reveal secondary chromosome abnormalities in addition to the MYC translocations t(8;14)(q24;q32), t(8;22)(q24;q11), and t(2;8)(p11;q24) in 60%–80% of Burkitt lymphomas/leukemias (BL). The high incidence of such aberrations indicates that additional changes are important, perhaps necessary, for malignant transformation, i.e., the 8q24/MYC rearrangements may not be sufficient. To investigate this possibility, we performed single nucleotide polymorphism (SNP) array analysis on 20 cases of 8q24/MYC‐positive BL. Nineteen (95%) harbored genomic imbalances; the only case without such aberrations displayed secondary changes by chromosome banding analysis. Thus, all BL cases had abnormalities in addition to the 8q24 translocation. The adult cases harbored more changes (median 3; range 1–21) than did the childhood cases (median 1.5; range 0–5) (P = 0.034). Several recurrent aberrations were detected by SNP array analysis, in particular losses of 6q14.1‐q22.33, 9p21.3, and 13q14.2‐q14.3, gains of 1q23.3‐q31.3, chromosome 7, 13q31.3, and partial uniparental isodisomies for 6p12.2‐pter, 9p23‐pter, and 17p11.2‐pter. The molecular genetic consequences of these changes include deletions of the CDKN2A and TP53 genes, and gains/losses of several genes, such as MIR17HG and E2F2K, involved in the MYC pathway. Thus, deregulation of the MYC pathway, both directly through the 8q24/MYC translocation and indirectly through secondary genomic imbalances, may be essential not only for the initiation but also for the progression of BL. © 2012 Wiley Periodicals, Inc.     

Comparison of fluorescence in situ hybridization analysis of isolated nuclei and routine histological sections from paraffin-embedded prostatic adenocarcinoma specimens.

Fluorescence in situ hybridization (FISH) is a powerful tool for quantitative analysis of chromosomes and genes and can be applied in a variety of specimens, including cell cultures, isolated nuclei from fresh and fixed tissues, and histological tissue sections. However, the results of FISH analysis of isolated nuclei in prostate cancer have not been previously compared with those from histological sections from the paraffin-embedded tissue blocks. To compare these methods, we studied isolated nuclei derived from 50-microns sections and adjacent 5-microns tissue sections from 10 cases of benign nodular hyperplasia of the prostate and 16 cases of prostatic carcinoma. FISH analysis employed centromere-specific probes for chromosomes 7, 8, 11, and 12. In benign tissue, the percentage of nuclei with three or more signals for chromosomes 7, 8, 11, and 12 was less than 3% for both isolated nuclei and tissue sections. However, the percentage of nuclei with no and one signals was less than 8% for isolated nuclei and more than 24% for tissue sections. In prostatic carcinoma, numeric chromosomal anomalies were found in 75% of cases by both FISH methods. However, isolated nuclei had more chromosomal tetrasomy than tissue sections (mean, 9.2 to 11.0% versus 5.1 to 5.6%, respectively). Conversely, intratumor heterogeneity of chromosomal anomalies was identified in 5 cases by FISH analysis of tissue sections but not in isolated nuclei. Cancer ploidy analysis by FISH correlated well with ploidy analysis by flow cytometry, although FISH was more sensitive for aneuploidy. We conclude that FISH analysis of isolated nuclei and histological tissue sections from paraffin blocks are reliable methods for detection of chromosomal anomalies in archival tissue of prostate cancer, although each method has advantages and disadvantages.     

Detection of translocations involving the HOX11/TCL3-locus in 10q24 by interphase fluorescence in situ hybridization

The t(10;14)(q24;q11) and its variant t(7;10)(q35;q24), which are recurrent in acute T-cell leukemia, lead to activation of the HOX11/TCL3-gene in chromosomal region 10q24 by juxtaposing this gene to one of the T-cell receptor loci. In the present study, we established a diagnostic assay for detecting these translocations by interphase fluorescence in situ hybridization (FISH). BAC clones flanking the HOX11/TCL3-locus were obtained from a fingerprinted BAC-contig of chromosomal region 10q24. BAC clones located proximal and distal of the HOX11/TCL3-locus were differently labeled and applied to interphase-FISH in seven normal controls and eight T-cell neoplasms with t(10;14)(q24;q11) or t(7;10)(q35;q24). In over 1600 nuclei of controls, a considerable split defined as separation of each one signal for the proximal and distal probe by more than three times the signal diameter was observed in only one cell. In contrast, all T-cell neoplasms with t(10;14) or t(7;10) contained at least 47% of nuclei with a signal split indicating a breakpoint in the HOX11/TCL3-locus. Thus, the established double-color FISH approach provides a new reliable and routinely applicable tool for diagnosing breakpoints in the HOX11/TCL3-locus.     

Gold-facilitated in situ hybridization: a bright-field autometallographic alternative to fluorescence in situ hybridization for detection of Her-2/neu gene amplification

Fluorescence in situ hybridization (FISH) represents an excellent method for profiling gene amplification in situ, but correlation with tissue morphology is difficult because of dark-field visualization. Validation of a bright-field assay for assessment of HER-2/neu gene amplification was investigated. Streptavidin-Nanogold was used to generate bright-field gene copy signals using GoldEnhance gold-based autometallography, catalyzed reported deposition, and a biotin-labeled probe. One hundred cases of invasive breast carcinoma were evaluated for which FISH gene copy results, and mRNA and oncoprotein gene expression, were known. Autometallographic signals were qualitatively evaluable without the use of oil immersion microscopy. Results correlated well with indirect and direct label FISH. Autometallographic gold-based in situ hybridization represents a promising bright-field assay for the assessment of HER-2/neu gene amplification.     

Characterization of the breakpoints in a t(8;13)(p11;q12) translocation from a patient with myeloproliferative disease using fluorescence in situ hybridization

We used fluorescence in situ hybridization to characterize the molecular position of the breakpoints in a t(8;13)(p11;q12) reciprocal translocation from a patient with an atypical myeloproliferative disorder. This structural chromosome abnormality is characteristic of this specific disease and occurs often as the only chromosome abnormality in the malignant cells. Yeast artificial chromosome (YAC) analysis has demonstrated that the 8p11 breakpoint lies within a region defined by YAC 959A4 and that the 13q12 breakpoint is spanned by YAC 769F9. Identifying the position of the breakpoints in this rearrangement provides the means to search for candidate genes rearranged by this highly specific structural chromosome abnormality. Genes Chromosomes Cancer 21:160–165, 1998. © 1998 Wiley-Liss, Inc.     

Interphase fluorescence in situ hybridization on selected plasma cells is superior in the detection of cytogenetic aberrations in plasma cell dyscrasia

Interphase fluorescence in situ hybridization (FISH) detects nonrandom cytogenetic abnormalities in plasma cell (PC) dyscrasia according to PC burden. However, when performed on cultured whole bone marrow (BM), it often fails to detect these aberrations. We have compared this interphase FISH technique with FISH after PC purification or identification to detect recurrent aberrations. In this study, 235 BM samples were collected from patients with multiple myeloma (MM) or related PC disorders regardless of disease status. All samples were analyzed in parallel. Clonal abnormalities were detected in 34.9% of cultured samples compared with 71.0% PC selected samples (P < 0.001). Moreover, FISH on PCs allowed to detect more abnormalities per case (P < 0.001) and identified higher percentages of abnormal nuclei (P < 0.001). This study indicates that FISH on PCs is the preferred technique for routine cytogenetic investigation of MM. © 2010 Wiley‐Liss, Inc.     

A role for fluorescence in situ hybridization detection of chromosome 22q dosage in distinguishing atypical teratoid/rhabdoid tumors from medulloblastoma/central primitive neuroectodermal tumors

It has been postulated that infants with medulloblastomas/central primitive neuroectodermal tumors (MB/PNET) may fare worse than older patients because some of them harbor unrecognized atypical teratoid/rhabdoid tumors (AT/RT), rare intracranial neoplasms that are typically unresponsive to therapy and rapidly fatal. Although small primitive cells are common to both entities, chromosome 22q11.2 deletions are common only in AT/RTs. Using fluorescence in situ hybridization (FISH) on archival, paraffin-embedded biopsy tissue with commercially available probes to 22q11.2, the region associated with RTs, we studied 8 cases of AT/RT, 12 cases of MB/PNET, and 4 cases of primitive central nervous system (CNS) neoplasms, which were difficult to classify. 22q Deletions were identified in 6 of 8 (75%) conventional AT/RTs and 0 of 12 (0%) children with classic MB/PNET. Of the 4 originally "difficult to classify" cases, 3 had deletions of 22q. In light of the FISH results, review of the morphology and immunophenotype resulted in 3 tumors being reclassified as AT/RTs and 1 as a large cell MB. These 4 cases highlight the potential diagnostic use of FISH for selected cases of primitive CNS malignancies in children and substantiate the notion that misdiagnosed AT/RTs may, in part account for the worse prognosis associated with "MB/PNET" in children younger than 2 years of age.     

Burkitt lymphoma (BL): reclassification of 39 lymphomas diagnosed as BL or Burkitt-like lymphoma in the past based on immunohistochemistry and fluorescence in situ hybridization

Burkitt lymphoma (BL) is a well characterized entity. For atypical findings a term Burkitt-like lymphoma (B-LL) was applied in the past, but the interpretation of the morphological appearances was subjective and poorly reproducible. We used a combined approach (morphology using classical histological staining; immunohistochemistry-IHC; fluorescence in situ hybridization-FISH on interphase nuclei; cytogenetics) to perform a retrospective study on 39 patients diagnosed as BL and B-LL at our department in the years 1982 to 2002. By FISH we demonstrated t(8;14)(q24;q32) in 31 patients; in further two we found a break at 8q24, suggestive of a variant translocation. In three patients with the cytogenetic investigation available we confirmed the findings of FISH--two lymphomas had the t(8;14)(q24;q32), one had t(2;8)(p12;q24). IHC showed CD20, CD10, BCL-6, p53 expression, and Ki-67 antigen in > 95% of the tumor cell population in a majority of the patients. There was a group of 4 patients in whom the t(8;14)(q24;q32) or a break at 8q24 were not found (FISH). These cases were reclassified within the WHO defined grey zone subgroup of B-cell lymphoma unclassifiable with features intermediate between diffuse large cell lymphoma (DLBCL) and Burkitt lymphoma--I-DLBCL/BL. Two further cases were reclassified as DLBCL based on a combined IHC and FISH findings. A lymphoma of one of these patients had breaks at 3q27 (BCL6) and at 14q32 (IGH) suggestive of t(3;14)(q27;q32). The overall survival estimate of 33 patients with the diagnosis of BL was 54%. Most of deaths occurred within 6 months after the tumor diagnosis. The unfavorable clinical outcome appears to be associated with a strong expression of the p53 protein in the tumor cell population. Individually utilized methods in the diagnosis of BL may lead to false diagnostic conclusions. A combined approach helps to establish a more reliable diagnosis of BL and to separate grey zone lymphomas I-DLBCL/BL and DLBCL with morphological mimics of BL to start adequate treatment. I-DLBCL/BL is a non-homogenous group of lymphomas necessitating further analysis in a prospective study.     

Detection of chromosome 11q13 breakpoints by interphase fluorescence in situ hybridization. A useful ancillary method for the diagnosis of mantle cell lymphoma

We assessed cytologic specimens from 11 mantle cell lymphomas (MCLs) and 32 other B-cell non-Hodgkin lymphomas (NHLs) for 11q13 breakpoints using a 2-color fluorescence in situ hybridization (FISH) assay that uses an 11q13 probe centered on the CCND1 gene and a centromeric chromosome 11 probe (CEP11). The number of nuclei in 200 cells were counted, and results were expressed as an 11q13/CEP11 ratio. All MCLs showed a high percentage of interphase nuclei with 3 or more 11q13 signals (mean, 74.8%; range 57%-90%). In contrast, in other B-cell NHLs the mean percentage of cells with 3 or more 11q13 signals was 9.2%. All MCLs had an elevated 11q13/CEP11 ratio (mean, 1.38). The mean ratio for other B-cell NHLs was 0.99. Two non-MCL cases, 1 large B-cell and 1 B-cell unclassified NHL, had high 11q13/CEP11 ratios of 1.15 and 1.30, respectively. Conventional cytogenetic analysis performed on the former case revealed a t(5;11)(q31;q13). Interphase FISH analysis using 11q13 and CEP11 probes is a convenient ancillary method for assisting in the diagnosis of MCL. This commercially available assay is simple to use on cytology or imprint specimens, and results can be obtained within 24 hours.     

Deletion 7q in uterine leiomyoma: fluorescence in situ hybridization characterization on primary cytogenetic preparations.

Deletions of the long arm of chromosome 7 constitute one of the most common clonal chromosomal changes associated with uterine leiomyoma cells. Recently, the molecular cytogenetic refinement of 7q deletions in two myoma-derived cell lines, with the use of a panel of 39 ordered 7q DNA probes corresponding to 87 genetic markers, showed results in line with those obtained by loss of heterozygosity (LOH) analysis. Referring to this panel, we extended fluorescence in situ hybridization (FISH) analysis on primary cytogenetic preparations from three myomas with del(7q), thereby avoiding cell passages. This was fundamental in the maintenance of cells with del(7q) in the two cases showing mosaicism (i.e., the presence of an extra normal clone), which are prone to lose the abnormal clone in the very early passages. The data obtained, together with previously published findings on the two leiomyoma-derived cell lines, indicated a commonly deleted region of 11 cM. If the fact that the presence of normal cells may interfere with LOH analysis is taken into account, the FISH approach seems to be a reliable complementing tool for refining the deletion and analyzing the smallest overlapping region in cases with both normal and del(7q) cells.     

Novel regions of amplification on 8q distinct from the MYC locus and frequently altered in oral dysplasia and cancer

Genetic studies aimed at identifying key alterations in oral cancers have focused on analysis of tumors, with few such studies using early oral premalignant lesions (OPLs) because of limitations in both sample availability and size. In this study, we used a randomly amplified polymorphic DNA (RAPD)-PCR approach to fingerprint DNA from microdissected normal and dysplastic cells and identified two recurrent genetic alterations on the long arm of chromosome 8 in OPLs, one mapping to 8q22 and the other to 8q24 near the MYC locus. We constructed a high-resolution bacterial artificial chromosome (BAC) comparative genomic hybridization array consisting of 166 overlapping BAC clones that spans about 52 Mbp, from 8q21 to 8q24. Hybridization of DNA from microdissected oral tumors to the array revealed alteration at 8q24, with amplification of the BAC containing MYC. Strikingly, at least two other novel regions of amplification at 8q22 were identified. Microsatellite analysis of 93 oral dysplasias and tumors confirmed the presence of one of the alterations at 8q22. Loss of heterozygosity (LOH) at D8S1830, mapping within one of the regions of amplification, was observed in high frequency in both OPLs and tumors. Of the 37 cases with LOH at D8S1830, 23 (62%) showed retention at D8S1793, which maps 1.6 Mbp centromeric to MYC. This is further support for the alteration at 8q22 being distinct from MYC. These data raise the possibility of additional oncogenes on 8q near the MYC locus that are potentially involved in OPL disease progression.     

B-cell lymphomas with MYC/8q24 rearrangements and IGH@BCL2/t(14;18)(q32;q21): an aggressive disease with heterogeneous histology, germinal center B-cell immunophenotype and poor outcome

B-cell lymphomas with MYC/8q24 rearrangement and IGH@BCL2/t(14;18)(q32;q21), also known as double-hit or MYC/BCL2 B-cell lymphomas, are uncommon neoplasms. We report our experience with 60 cases: 52 MYC/BCL2 B-cell lymphomas and 8 tumors with extra MYC signals plus IGH@BCL2 or MYC rearrangement plus extra BCL2 signals/copies. There were 38 men and 22 women with a median age of 55 years. In all, 10 patients had antecedent/concurrent follicular lymphoma. Using the 2008 World Health Organization classification, there were 33 B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (henceforth referred to as unclassifiable, aggressive B-cell lymphoma), 23 diffuse large B-cell lymphoma, 1 follicular lymphoma grade 3B, 1 follicular lymphoma plus diffuse large B-cell lymphoma, 1 B-lymphoblastic lymphoma, and 1 composite diffuse large B-cell lymphoma with B-lymphoblastic lymphoma. Using older classification systems, the 33 unclassifiable, aggressive B-cell lymphomas most closely resembled Burkitt-like lymphoma (n=24) or atypical Burkitt lymphoma with BCL2 expression (n=9). Of 48 cases assessed, 47 (98%) had a germinal center B-cell immunophenotype. Patients were treated with standard (n=23) or more aggressive chemotherapy regimens (n=34). Adequate follow-up was available for 57 patients: 26 died and 31 were alive. For the 52 patients with MYC/BCL2 lymphoma, the median overall survival was 18.6 months. Patients with antecedent/concurrent follicular lymphoma had median overall survival of 7.8 months. Elevated serum lactate dehydrogenase level, ≥2 extranodal sites, bone marrow or central nervous system involvement, and International Prognostic Index >2 were associated with worse overall survival (P<0.05). Morphological features did not correlate with prognosis. Patients with neoplasms characterized by extra MYC signals plus IGH@BCL2 (n=6) or MYC rearrangement with extra BCL2 signals (n=2) had overall survival ranging from 1.7 to 49 months, similar to patients with MYC/BCL2 lymphomas. We conclude that MYC/BCL2 lymphomas are clinically aggressive, irrespective of their morphological appearance, with a germinal center B-cell immunophenotype. Tumors with extra MYC signals plus IGH@BCL2 or MYC rearrangement plus extra BCL2 signals, respectively, appear to behave as poorly as MYC/BCL2 lymphomas, possibly expanding the disease spectrum.     

HER-2/neu detection in fine-needle aspirates of breast cancer: fluorescence in situ hybridization and immunocytochemical analysis

We evaluated HER-2 receptor status by immunocytochemical and immunohistochemical analyses and fluorescence in situ hybridization (FISH) in 51 fine-needle aspiration (FNA) specimens together with the corresponding formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from surgically resected breast cancers. Three fixation methods were compared: ethanol, formalin, and CytoLyt-ThinPrep (Cytyc, Boxborough, MA). HER-2 was overexpressed and amplified in 8 (16%) of 51 FFPE specimens. Of the 8 cases, gene amplification was observed in 8 FNA specimens (100%) and overexpression in 2 (25%) ethanol-, 4 (50%) CytoLyt-, and 5 (63%) formalin-fixed FNA specimens. Strong pairwise kappa association between FISH results performed on FNA specimens and FFPE tissue samples (ethanol fixation, kappa = 0.848; ThinPrep, kappa = 0.918) and moderate (ThinPrep, kappa = 0.692; formalin fixation, kappa = 0.667) to poor (ethanol, kappa = 0.300) pairwise kappa agreement between tissue immunohistochemical and FNA immunocytochemical results was demonstrated. We conclude that HER-2 protein expression on cytologic preparations was insufficiently reliable for clinical use, whereas HER-2 gene amplification determined by FISH demonstrated strong and consistent correlation with HER-2 status of FFPE tissue samples.