SNP analysis of minimally evolved t(14;18)(q32;q21)-positive follicular lymphomas reveals a common copy-neutral loss of heterozygosity pattern.

Follicular lymphoma (FL) cases with a t(14;18)(q32;q21) and minimal or no additional karyotypic alterations, such as copy number gains and losses and/or chromosomal rearrangements, may exhibit pathologic features and a clinical behavior similar to those with more complex karyotypes. This study sought to investigate whether the copy-neutral loss of heterozygosity (cnLOH) profiles of these minimally evolved t(14;18)(q32;q21)-positive follicular lymphoma (MEV-FL) cases are similar to or different from the majority of FL cases with more karyotypic alterations. Affymetrix SNP 6.0 array analysis was applied to the tumor genomes of 23 MEV-FL biopsy samples to assess for the presence of cnLOH. These cases carried either a single or no chromosomal abnormality in addition to t(14;18)(q32;q21) as determined by karyotyping. We found that, although these MEV-FL cases had simple karyotypes, they showed very similar cnLOH profiles as compared to cytogenetically complex cases. The most frequent regions affected by cnLOH were 1p (17%), 6p (17%), 12q (13%) and 16p (13%). Our study suggests that cnLOH alterations may serve as important contributors to the pathological and clinical manifestations of FL.

Distinctive patterns of BCL6 molecular alterations and their functional consequences in different subgroups of diffuse large B-cell lymphoma.

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B-cell-like (ABC) and primary mediastinal (PM) DLBCL. The BCL6 gene is often translocated and/or mutated in DLBCL. Therefore, we examined the BCL6 molecular alterations in these DLBCL subgroups, and their impact on BCL6 expression and BCL6 target gene repression. BCL6 translocations at the major breakpoint region (MBR) were detected in 25 (18.8%) of 133 DLBCL cases, with a higher frequency in the PM (33%) and ABC (24%) subgroups than in the GCB (10%) subgroup. Translocations at the alternative breakpoint region (ABR) were detected in five (6.4%) of 78 DLBCL cases, with three cases in ABC and one case each in the GCB and the unclassifiable subgroups. The translocated cases involved IgH and non-IgH partners in about equal frequency and were not associated with different levels of BCL6 mRNA and protein expression. BCL6 mutations were detected in 61% of DLBCL cases, with a significantly higher frequency in the GCB and PM subgroups (>70%) than in the ABC subgroup (44%). Exon-1 mutations were mostly observed in the GCB subgroup. The repression of known BCL6 target genes correlated with the level of BCL6 mRNA and protein expression in GCB and ABC subgroups but not with BCL6 translocation and intronic mutations. No clear inverse correlation between BCL6 expression and p53 expression was observed. Patients with higher BCL6 mRNA or protein expression had a significantly better overall survival. The biological role of BCL6 in translocated cases where repression of known target genes is not demonstrated is intriguing and warrants further investigation.

A comprehensive genetic and histopathologic analysis identifies two subgroups of B-cell malignancies carrying a t(14;19)(q32;q13) or variant BCL3-translocation.

The biologic and pathologic features of B-cell malignancies bearing a translocation t(14;19)(q32;q13) leading to a fusion of IGH and BCL3 are still poorly described. Herein we report the results of a comprehensive cytogenetic, fluorescence in situ hybridization (FISH), molecular and histopathological survey of a large series of B-cell malignancies with t(14;19) or variant translocations. A total of 56 B-cell malignancies with a FISH-proven BCL3 involvement were identified with the translocation partners being IGH (n=51), IGL (n=2), IGK (n=2) and a non-IG locus (n=1). Hierarchical clustering of chromosomal changes associated with the t(14;19) indicated the presence of two different groups of IG/BCL3-positive lymphatic neoplasias. The first group included 26 B-cell malignancies of various histologic subtypes containing a relatively high number of chromosomal changes and mostly mutated IgVH genes. This cluster displayed three cytogenetic branches, one with rearrangements in 7q, another with deletions in 17p and a third one with rearrangements in 1q and deletions in 6q and 13q. The second group included 19 cases, mostly diagnosed as B-cell chronic lymphocytic leukemia (B-CLL), and characterized by few additional chromosomal changes (e.g. trisomy 12) and unmutated IgVH genes. In conclusion, our study indicates that BCL3 translocations are not restricted to B-CLL but present in a heterogeneous group of B-cell malignancies.

Leukocyte count as a predictor of death during remission induction in acute myeloid leukemia.

Acute myeloid leukemia (AML) presenting with a high leukocyte count has been associated with an increase in induction mortality and poor results in a number of other survival measures. However, the level at which an elevated leukocyte count has prognostic significance in AML remains unclear. In this report on a series of 375 adult (non-M3) AML patients undergoing induction chemotherapy at a single institution, leukocyte count analyzed as a continuous variable is shown to be a better predictor of induction death (ID) and overall survival (OS) than a leukocyte count of > or = 100 x 10(9)/L, a value characteristically associated with "hyperleukocytosis" (HL). In this patient cohort, a presenting leukocyte count of > or = 30 x 10(9)/L had high sensitivity and specificity for predicting ID, and both performance status (PS) and leukocyte count more accurately predicted for ID than age. Considering these parameters in newly-diagnosed AML patients may facilitate the development of strategies for reducing induction mortality.

Concurrent translocation of BCL2 and MYC with a single immunoglobulin locus in high-grade B-cell lymphomas.

B-cell leukaemia or lymphoma with a combination of t(8;14)(q24;q32) of Burkitt leukaemia/lymphoma and t(14;18)(q32;q21) of follicular lymphoma may present clinically as de novo acute lymphoblastic leukaemia or transformation of follicular lymphoma to aggressive histology diffuse lymphoma. A number of cell lines have been reported with a complex t(8;14;18) with fusion of MYC, IGH and BCL2 on the same derivative 8 chromosome. The objective of this study was to determine the frequency and chromosomal features of this der(8)t(8;14;18) in a series of acute leukaemias and malignant lymphomas. A database of 1350 leukaemia and lymphoma karyotypes was searched for cases with structural alterations affecting both 8q24 and 18q21. A total of 55 cases were identified, of which eight revealed a complex der(8)t(8;14;18) with an MYC-IGH-BCL2 rearrangement resulting from translocation of BCL2 and MYC with a single disrupted IGH allele. Molecular cytogenetic investigation is essential to identify cases of high-grade leukaemia/lymphoma with concurrent translocations affecting the BCL2 and MYC loci.

Karyotypic changes associated with loss of prolactin dependency of rat Nb2 node lymphoma cell cultures.

The parent line of cultured "Nb2 node" lymphoma cells is dependent on the hormone prolactin (PRL) for growth and is widely used for the in vitro bioassay of lactogenic hormones. As reported previously, PRL-independent sublines have been developed in vitro from the parental line by lactogen deprivation. The present study describes the G-banded karyotypes of the Noble (Nb) strain of rat (in which the original lymphoma developed), the PRL-dependent cell line (157th generation), and two of its PRL-independent sublines (1220th and 2372nd generations). The karyotype of the Nb rat was determined to be the same as that of Rattus norvegicus. The stemline karyotype of the PRL-dependent cells contains a number of well-defined chromosomal abnormalities. The PRL-independent sublines examined have the same chromosomal abnormalities as the PRL-dependent cells plus a few additional changes indicative of clonal evolution from the PRL-dependent stemline. The development of PRL independence (as seen in the 1220th generation) was associated with only two karyotypic changes, i.e., loss of the Y chromosome and a translocation involving chromosomes 14 and 17. The recently reported mapping of the rat PRL gene and other PRL-related genes to chromosome 17 suggests that rearrangement of chromosome 17 could be involved in the development of the PRL independence. The PRL-dependent and PRL-independent Nb2 cell lines provide a useful system for studying chromosomal and molecular genetic events associated with the malignant progression of polypeptide hormone-dependent cancers.

Partial deletions of the long and short arm of chromosome 3 point to two tumor suppressor genes in uveal melanoma.

Uveal melanoma is the most common form of primary eye cancer. Monosomy 3, which is an unusual finding in tumors but is present in approximately 50% of uveal melanomas, is significantly correlated with metastatic disease. To obtain positional information on putative tumor suppressor genes on this chromosome, we have investigated tumors from 333 patients by comparative genomic hybridization, microsatellite analysis, or conventional karyotype analysis. A partial deletion of the long arm was found in eight tumors, and the smallest region of deletion overlap (SRO) spans 3q24-q26. We found six tumors with a partial deletion of the short arm and were able to define a second SRO of about 2.5 Mb in 3p25. This SRO does not overlap with the VHL gene. Our finding suggests a role for two tumor suppressor genes in metastasizing uveal melanoma and may explain the loss of an entire chromosome 3 in these tumors.

Peripheral primitive neuroectodermal tumors in adults: documentation by molecular analysis.

PURPOSE: The Ewing tumor (ET) family of peripheral primitive neuroectodermal tumors (pPNETs) are primitive small round-cell tumors (SRCTs) of the bone and soft tissue that occur predominantly in children and adolescents. However, pPNETs only rarely enter the differential diagnosis of bone and soft tissue SRCTs in adults. Recently, gene fusions between the EWS gene and different members of the ETS transcription factor family have been shown to occur in virtually all pPNETs and thus constitute a pathognomonic marker for this tumor subclass. The aim of the present study was to document EWS/ETS fusion gene expression in suspected pPNETs of adults as objective evidence for the existence of this tumor family in older patients. PATIENTS AND METHODS: The three contributing molecular diagnostic laboratories retrospectively compiled a cohort of all SRCT cases in which EWS/ETS gene fusions had been shown by molecular analysis. This cohort was surveyed for cases that occurred in patients aged 40 years or older, which were then analyzed for their clinical and pathologic features. RESULTS: Nine patients between 40 and 65 years of age were found to have tumors positive for EWS/ETS gene fusions. Standard histopathologic and clinical features of these cases, other than age, were similar to those of childhood pPNETs. Patients were initiated on appropriate therapy after molecular analysis confirmed the diagnosis of pPNET. CONCLUSION: Identification of an EWS/ETS gene fusion is useful in providing objective evidence of the diagnosis of pPNET in patients over the age of 40 years. This diagnosis should be considered in adults who present with bone and soft tissue SRCTs and appropriate biopsy specimens should be collected for molecular analysis at the time of diagnosis.

Establishment and comprehensive analysis of a new human transformed follicular lymphoma B cell line, Tat-1.

A spontaneously EBV transformed follicular lymphoma (FL) cell line, Tat-1, was established from the lymph node biopsy specimen of a patient with B cell FL, grade 1 in transformation to high grade disease. Tat-1 cells expressed lymphoid markers and developed tumor masses in immunodeficient mice. Bcl-2, Bcl-X(L), Bax and p53 protein expression was revealed by Western blotting. Flow cytometric analysis confirmed P-gp expression. Cytogenetically, the Tat-1 cell line showed identical chromosomal alterations to that of the initial biopsy specimen, among which the most notable were the t(14;18) typical of FL and additional abnormalities involving chromosomes 1, 8 and 13. Multicolor FISH analysis delineated all abnormalities, including a t(1p;8q), a der(8)(8q24::14q32::18q21) and a der(13)(13q32::8q24::14q32::18q21). Further FISH investigations using a locus-specific probe cocktail containing c-myc, IgH and bcl-2 revealed fusion of these three loci on the derivatives 8 and 13, in addition to the derivative 14 IgH/bcl-2 fusion and an extra copy of c-myc on derivative chromosome 1. These results demonstrate an additional example of the deregulation of bcl-2 and c-myc expression through recombination with a single IgH enhancer region. The unusual molecular features of the Tat-1 cell line render it a unique tool for studies focused on cytogenetic alterations, expression of multidrug resistance phenotype and expression of anti-apoptotic proteins in FL.

X chromosome aneuploidy in lymphocyte cultures from women with recurrent spontaneous abortions.

Occasional metaphases with X chromosome aneuploidy can be detected in short-term lymphocyte cultures from women with recurrent abortions. The significance of this finding is unknown. It has been suggested that it may reflect a genetic tendency to nondisjunction that predisposes these women to an increased risk of producing aneuploid offspring. We have investigated prospectively the frequency of X chromosome aneuploidy in lymphocyte cultures from 104 women with a normal chromosome constitution and a history of recurrent pregnancy loss defined as 2 or more spontaneous abortions. Seventeen women (16%) had a significant number (2-10%) of X aneuploid cells in cultured lymphocytes but no evidence of constitutional chromosome mosaicism, based on analysis of fibroblast cultures. A control group of age-matched fertile women without a history of recurrent abortions showed a similar level of X chromosome aneuploidy in lymphocyte cultures. No increased risk for production of liveborn children with aneuploidy was found on retrospective analysis of the reproductive histories of these women. A significant effect of culture conditions on X chromosome gain or loss has been demonstrated by comparison of medium 199 with Dulbecco's modified Eagle medium.

Bcl-6 and Bcl-2 protein expression in diffuse large B-cell lymphoma and follicular lymphoma: correlation with 3q27 and 18q21 chromosomal abnormalities.

The bcl-2 gene on chromosome 18 at q21 and the bcl-6 gene on chromosome 3 at q27 are both highly regulated during B-cell differentiation and show an inverse relationship of expression in the normal secondary lymphoid follicle. The objective of this study was to investigate the relationship between bcl-2 and bcl-6 protein expression and the relationship between protein expression and the corresponding chromosomal alterations in malignant lymphomas, including those associated with the germinal center. Expression of bcl-2 and bcl-6 proteins was studied in 55 cases of diffuse large B-cell lymphoma (DLBCL) and 21 cases of follicular lymphoma (FL), and the results correlated with the presence of t(14;18) and 3q27 abnormalities in a subset of 52 cases with cytogenetic analysis. These cases were selected to represent a spectrum of nodal and extranodal lymphomas, including those with and without a t(14;18). It was shown that the neoplastic cells in 71% of DLBCLs and 100% of FLs expressed bcl-6 protein. Expression of bcl-6 was seen more frequently in diffuse large B-cell lymphomas with large noncleaved morphology compared with immunoblastic morphology (82% v 27%, P = .0015), but failed to correlate with 3q27 abnormalities. Thirty-eight percent of cases with 3q27 abnormalities were bcl-6 protein negative, whereas 85% of cases without a 3q27 abnormalities were bcl-6 protein positive. Expression of bcl-2 protein was shown in 51% DLBCLs (nodal v extranodal, 71% v 30%, P = .012). bcl-2 protein was expressed in 89% of FLs with t(14;18), in contrast to 25% of FLs without t(14;18) (P = .016). In DLBCL and FL with t(14;18), the most common pattern of expression was bcl-2+/bcl-6+. In lymphomas without t(14;18), there was not an inverse relationship between bcl-2 and bcl-6 protein expression. In conclusion, these data suggest that mechanisms other than gene rearrangements can deregulate bcl-2 and bcl-6 expression in lymphomas, and there does not appear to be an inverse relationship between these two proteins as seen in the normal germinal center.

Testicular relapse of acute promyelocytic leukemia after allogeneic BMT.

After treatment of acute leukemia (typically ALL and the monocytic variants of AML), relapse may occur at sites other than the marrow. Isolated extramedullary relapse of acute promyelocytic leukemia (APL) however, is rare. We describe such an event in a man who underwent allogeneic BMT for APL in second relapse and 4 years later presented with testicular relapse. The marrow was morphologically and cytogenetically normal, but RT-PCR analysis revealed the specific PML/RAR chimeric RNA transcript.

High incidence of extramedullary relapse of AML after busulfan/cyclophosphamide conditioning and allogeneic stem cell transplantation.

While allogeneic stem cell transplantation (SCT) is curative for a significant number of patients with AML, relapse of disease within the bone marrow and/or extramedullary (EM) sites following high-dose therapy continues to limit the success of this treatment. Between October 1985 and December 1996, 81 adults underwent allogeneic SCT for de novo AML at our centre. Forty-two patients remain alive and free of leukaemia with a median follow-up of 50 months. The 5-year actuarial event-free survivals (EFS) for all patients and for those undergoing SCT in CR1 or with advanced disease were 46% (95% confidence interval (CI) 34-58%), 63% (CI 46-76%), and 19% (CI 7-36%), respectively. Twenty-two patients relapsed at a median of 8 (range 1.6-54.5) months with the actuarial risk of relapse for all, CR1 and advanced disease patients being 38%, (CI 27-52%), 23% (CI 13-40%) and 68% (CI 46-88%), respectively. Ten patients relapsed at EM sites; six of these (27% of relapses) had an isolated EM relapse at a median of 31 (range 8.5-54) months. Three of the patients with isolated EM relapse survived > or =24 months following relapse and two patients remain disease-free at 29+ and 33+ months. BuCy conditioning followed by allogeneic SCT in AML results in satisfactory EFS although there is a significant risk of late isolated EM relapse.

Clinical utility of heteroduplex analysis of TCR gamma gene rearrangements in the diagnosis of T-cell lymphoproliferative disorders.

We conducted this study to assess the clinical utility of heteroduplex analysis of polymerase chain reaction (PCR) products for Tgamma gene rearrangements in the diagnosis of T-cell lymphoproliferative disorders (LPDs) using a single set of consensus primers. This method was used to study 103 cases using fresh or frozen tissue. These included 62 cases of T-cell LPDs, 25 cases of B-cell LPDs, 3 cases of anaplastic large cell lymphomas of undefined lineage, and 13 cases of reactive disorders. The latter group served as the negative control group. For all cases of T-cell LPDs, the results of Tbeta Southern analysis (SA) and heteroduplex PCR analysis were compared. We also studied 10 cases of postthymic T-cell lymphoma using paraffin-embedded tissue. Heteroduplex Tgamma PCR was positive in 51 (89%) of 57 cases that were rearranged by Tbeta SA. Eight of the 10 cases of T-cell lymphoma analyzed using paraffin-embedded tissue were positive. There were no false-positive results. These results demonstrate that this method is an effective screening test in the clinical laboratory. A positive result obviates the need to perform SA. Preliminary results suggest this method is useful using paraffin-embedded tissue.

Comparison of cytogenetic analysis, southern analysis, and polymerase chain reaction for the detection of t(14; 18) in follicular lymphoma.

This study was undertaken to compare the ability of cytogenetic analysis (CG), Southern analysis (SA) and the polymerase chain reaction (PCR) to detect the t(14; 18) in follicular lymphoma (FL). All methodologies were performed by standard techniques. The probes used for SA included major breakpoint region (mbr) and minor cluster region (mcr) probes. The primers for PCR were identical or similar to those used by other investigators. One hundred fifteen cases of FL were ascertained by morphologic criteria, from which sufficient fresh tissue was available for both CG and molecular analysis. Eleven cases failed by both methods (nonrepresentative sampling). One hundred four cases showed evidence of an abnormal clone by CG and/or immunoglobulin gene rearrangement (IgH) studies. Cytogenetic analysis failed in 2 cases, was positive for t(14; 18) in 91 of the remaining 102 cases (89%) and detected a non-t(14; 18) close in 11 cases. An IgH clonal rearrangement was confirmed in all 104 cases. Southern analysis detected a mbr or mcr rearrangement in 78 of 104 cases (75%). Polymerase chain reaction detected an mbr or mcr rearrangement in 68 of 104 cases (65%). The use of PCR as a clinical test to detect t(14; 18)-positive lymphomas, with single primer sets for the mbr and mcr, will result in a high false-negative rate. The use of additional primers to detect uncommon breakpoints sites will be required to enhance the sensitivity of PCR for detection of t(14; 18) in malignant lymphoma.

Comparative study of automated versus manual extraction of DNA from clinical specimens.

The authors have compared an automated method for DNA extraction with an established manual method using routine clinical specimens. The purity, yield, and quality of the extracted DNA was assessed by ultraviolet absorbency measurements, gel electrophoresis, and DNA probe hybridization. The automated DNA extractor can provide high molecular weight DNA equivalent in purity, yield, and quality to that obtained by the manual method with substantial savings in technologist time and overall costs associated with the procedure.

Molecular methods for detecting t(11;14) translocations in mantle-cell lymphomas.

The t(11;14)(q13;q32) and its molecular counterpart, bcl1/JH, are characteristic of mantle-cell lymphomas (MCL). Molecular detection of the translocation is useful in diagnosis and classification, and also shows promise in detecting minimal residual disease. The purpose of this study was to determine the frequency of detecting bcl1/JH by polymerase chain reaction (PCR) compared with Southern blot analysis in cases proven by cytogenetic analysis to harbor t(11;14). Southern blot analysis using two probes targeting the major translocation cluster (MTC) and a third probe targeting the p94 region was performed, along with PCR using two different bcl1 MTC primers, on 18 cases of MCL known to have t(11;14). Southern blot analysis revealed bcl1 rearrangement in 13 of 18 cases (72%), 12 with MTC breakpoints and 1 with a p94 breakpoint. The 2.1-kb MTC probe "b" was superior to the smaller 700-bp probe "a" in detecting these rearrangements. The MTC translocation was identified by PCR in 10 of 12 cases, and both primer sets that were tested performed equally well. This study illustrates the frequency with which molecular methods detect known t(11;14) translocations in MCLs. These results may help clinical laboratory scientists optimize their procedure for detecting bcl1 translocations by molecular methods at initial diagnosis and for purposes of detecting minimal residual disease.

Acquired Robertsonian translocations in hematologic malignancy. A rare mechanism of clonal evolution.

We identified three patients with an acquired Robertsonian translocation among 1,200 patients with hematopoietic malignancy. In each case the translocation served to produce partial chromosomal trisomy or tetrasomy within the malignant clone. Acquired Robertsonian translocation represents another, although rare, mechanism of clonal evolution in malignant cells.

Trisomy 4 in acute nonlymphocytic leukemia.

Trisomy 4 has recently been identified as a nonrandom chromosomal abnormality associated with acute nonlymphocytic leukemia with myelomonocytic morphology (AML M4). It has been suggested that it may be related to a new type of environmental toxin exposure. During the 1975-1987 period we have detected only one case of simple trisomy 4 in over 1200 patients successfully investigated for hematologic malignancy; this patient was diagnosed as having AML M2 and did not have a history of toxic chemical or drug exposure.

Monosomy 3 and isochromosome 8q in a uveal melanoma.

Chromosome analysis of a locally invasive uveal melanoma revealed monosomy 3 and i(8q). Sublines with multiple copies of the i(8q) were also present. Loss of heterozygosity for genes on chromosome 3 and duplication of genes on 8q may play an important role in progression of uveal melanoma.