Aberrant methylation of the major breakpoint cluster region in chronic myeloid leukemia.

Isolated hypomethylated sites exist in the major breakpoint cluster region (M-bcr) where most Philadelphia chromosome (Ph) breakpoints are located. Twenty of 50 (40%) chronic myeloid leukemia (CML) patients were found to have aberrant hypermethylation of these sites on the rearranged M-bcr when compared with control marrows. The aberrancy correlated strongly with M-bcr breakpoint location; 19 of 20 cases had breakpoints located 5' of the M-bcr Sca I site, and 28 of 30 cases with normal M-bcr methylation had breakpoints located 3' of the M-bcr Sca I site. Sequence analysis of the Ph M-bcr breakpoints failed to find an M-bcr nucleotide position that delineated the transition between abnormally and normally methylated cases, indicating that the translocation of a critical M-bcr sequence was not responsible for the methylation abnormality. In 3 of 8 CML patients, cells without the t(9;22) were found to have abnormally methylated, unrearranged M-bcrs. The data indicate that abnormally methylated rearranged M-bcrs are present in CML cases with Ph breakpoints 5' of the M-bcr Sca I site and that the M-bcr in Ph- cells of patients with CML may also be abnormally methylated.

Paternal origin of the rearranged major breakpoint cluster region in chronic myeloid leukemia.

The Philadelphia chromosome, t(9;22), is present in virtually all cases of chronic myeloid leukemia (CML). It has previously been shown by cytogenetic studies that the rearranged chromosome 22 in patients with CML is exclusively maternal in origin. To address this issue at a molecular level, the major breakpoint cluster region (M-bcr) on chromosome 22 was examined using Southern blot assays and M-bcr Pvu II and Mae II restriction site polymorphisms in three CML patients. In all three cases, the rearranged allele was paternal in origin. These results indicate that the paternally derived M-bcr allele may also be involved in the M-bcr rearrangement.

Duplication of small segments within the major breakpoint cluster region in chronic myelogenous leukemia.

The t(9;22) in chronic myelogenous leukemia (CML) may be reciprocal or, in a minority of cases, may result in an extensive deletion of a portion of the major breakpoint cluster region (M-bcr) of the BCR. This report provides evidence of the duplication of small segments within the M-bcr in a small group of patients with CML. Southern blots of Bgl II and Bgl II/BamHI double-digested DNA from the blood or bone marrow of 46 patients with CML were probed with a 5' 1.4-kb Taq I/HindIII M-bcr probe and a 3' 2-kb HindIII/BamHI M-bcr probe. In three patients, rearrangements were noted with both probes in Bgl II-digested DNA, but were not present in Bgl II/BamHI-digested DNA with either probe. Southern analysis of DNA samples double-digested with Bgl II and BspHI from two of these three cases showed no rearrangements with either probe; the M-bcr BspHI site is located 26 bp 3' of the BamHI site in the second intron of the M-bcr. The presence of a rearranged M-bcr with both probes in Bgl II-digested DNA and the lack of rearrangement in Bgl II/BamHI and Bgl II/BspHI double-digested DNA suggest the presence of M-bcr BamHI and BspHI sites on both 9q+ chromosome (9q+) and the Philadelphia chromosome (Ph). This implies a duplication of at least the 26-bp M-bcr BamHI/BspHI fragment in these two samples. Sequence data from one of these two cases confirmed the M-bcr breakpoints to be staggered; the Ph M-bcr breakpoint occurred 258 bp downstream from the 9q+ M-bcr breakpoint. It is concluded that a duplication of small segments within the M-bcr occurs in a small group of patients with CML, which may lead to pseudogermline patterns on Southern blot. Such a duplication may provide insight into the mechanism of some chromosomal translocations in neoplasia.

Infectious mononucleosis in lymphoid tissue. Histopathology, in situ hybridization, and differential diagnosis.

The histopathologic features of tissue specimens from 16 patients with acute Epstein-Barr virus-induced infectious mononucleosis, which was confirmed by clinical and serologic methods, are described. The clinical course was usually self-limited (14 patients), but it resulted in the death of two patients, one of whom (patient with renal transplantation) was immunosuppressed. Each lymphoid tissue specimen, including those obtained from the lymph nodes (n = 9), tonsils (n = 5), spleen (n = 1), and appendix (n = 1), showed a nonuniform expansion of nonfollicular areas by a polymorphous population of lymphocytes, including transformed lymphocytes and immunoblasts. In situ hybridization demonstrated Epstein-Barr virus-infected lymphocytes in four of eight tissue specimens that were studied. Other histologic features included Reed-Sternberg-like cells, plasma cells, histiocytes, frequent mitoses, abundance of postcapillary venules, and necrosis. These histologic features should suggest a diagnosis of infectious mononucleosis rather than other processes, either benign or malignant, that can mimic it.

Detection of Epstein-Barr virus by in situ hybridization with a commercially available biotinylated oligonucleotide probe.

Epstein-Barr virus (EBV) nucleotide sequences can be detected in routinely processed tissues by in situ hybridization (ISH) with either radiolabeled or nonisotopic EBV probes. In this paper, we describe our nonisotopic ISH protocol for EBV detection with use of a triple-biotinylated oligonucleotide NotI/PstI EBV probe. We compared this technique with our radioisotopic ISH technique that uses a 35S-labeled EBV probe and found that the nonisotopic technique was as sensitive as the radioisotopic technique for detecting EBV-infected cells in tissues from patients with posttransplantation lymphoproliferative disorders. Furthermore, use of the biotinylated probe has several advantages over the 35S-labeled probe, including commercial availability of labeled probe, increased probe stability, decreased technical and development time, and ease of interpretation. We conclude that the nonisotopic ISH procedure is practical for use in diagnostic surgical pathology.

Adenovirus in the gastrointestinal tracts of immunosuppressed patients.

Adenovirus was cultured from various gastrointestinal sites and one lung specimen from seven immunosuppressed patients, four of whom were bone marrow transplant recipients. Histologic examination and in situ hybridization studies of the lung demonstrated adenovirus in the bronchial epithelium and alveolar lining cells. In contrast, none of the 34 gastrointestinal biopsies performed within 30 days of the positive adenovirus culture showed histologic or molecular evidence of invasive adenovirus in the gastrointestinal mucosa. These results suggest that isolation of adenovirus from gastrointestinal biopsy specimens taken from immunosuppressed patients probably does not indicate an invasive adenoviral infection.

B-cell lymphoproliferative disorders in solid-organ transplant patients: detection of Epstein-Barr virus by in situ hybridization.

B-cell lymphoproliferative disorders (BLPDs) occur in approximately 2% of transplant recipients and are frequently fatal. Indirect serologic evidence has implicated Epstein-Barr virus (EBV) as an etiologic factor in these lesions. Direct evidence of the presence of EBV in these lesions has been obtained in relatively few cases. We used in situ hybridization (ISH) with a probe for the BamHI-W region of the EBV genome to study 52 tissue specimens from 28 solid-organ transplant patients who had BLPD. Epstein-Barr virus-infected lymphoid cells were identified in 26 of these 28 patients. The two patients without ISH evidence of EBV infection showed no distinctive clinical, morphologic, or serologic features. Previous filter-hybridization studies of these two patients had demonstrated evidence of EBV infection. Seven additional transplant patients without evidence of BLPD were studied as controls and showed no evidence of EBV in their lymphoid cells by ISH. These data provide further support for the etiologic role of EBV in the pathogenesis of posttransplantation lymphoproliferative disorders.

B-cell lymphoproliferative disorders after bone marrow transplant. An analysis of ten cases with emphasis on Epstein-Barr virus detection by in situ hybridization.

Ten patients with B-cell lymphoproliferative disorders (BLPD) after bone marrow transplant were studied in a retrospective analysis of 81 specimens available from biopsy and autopsy material. Histologic review, immunophenotyping, and in situ hybridization (ISH) for Epstein-Barr virus (EBV) sequences were done. Sixty-four specimens showed morphologic evidence of BLPD, demonstrating a heterogeneous spectrum with various degrees of plasmacytoid differentiation. Immunophenotypic evidence of clonality was found in six patients. The ISH detected EBV sequences in all ten patients, including 60 of the 64 specimens with morphologic evidence of BLPD. In addition, ISH identified EBV-infected lymphoid cells in two of 17 sites without morphologic evidence of BLPD. These data demonstrate the utility of ISH for detecting EBV genome in this setting and provide further evidence for the etiologic role of EBV in the pathogenesis of BLPD.

Lack of evidence for involvement of Epstein-Barr virus in the development of the "Quilty" lesion of transplanted hearts: an in situ hybridization study.

We investigated the possible relationship of Epstein-Barr virus to the development of subendocardial lymphocytic infiltrates. In situ hybridization for Epstein-Barr virus genomic sequences was performed in 22 heart biopsy specimens with subendocardial infiltrates for 19 heart transplant patients. Epstein-Barr virus genomic sequences were not detected in the lymphocytes or myocytes in any of the heart biopsy specimens. We conclude that Epstein-Barr virus is not related to the development of subendocardial lymphocytic infiltrates.

In situ hybridization for the detection of Epstein-Barr virus in central nervous system lymphomas.

Epstein-Barr virus (EBV) has been implicated in the development of lymphomas in immunocompromised patients. To test this hypothesis, 26 lymphomas involving the central nervous system (CNS) (11 primary, 15 systemic) were studied for the presence of EBV. In situ hybridization (ISH) was performed on formalin-fixed, paraffin-embedded tissue using a sulfur 35 (35S)-labeled EBV probe (EBV BAMH1-W). The results were interpreted without knowledge of the patients' immunologic status. The EBV sequences were detected in 11 lymphomas, nine of which were mixed or large cell subtypes. Review of the clinical information revealed that nine of the 26 lymphomas occurred in immunocompromised patients secondary to renal transplantation, human immunodeficiency virus infection, leukemia, and Wiskott-Aldrich syndrome. The EBV sequences were detected in all nine lymphomas occurring in immunocompromised patients, whereas two of the 17 lymphomas occurring in immunocompetent patients expressed EBV sequences. The authors conclude that the presence of EBV sequences in CNS lymphomas is highly correlated with a history of compromised immune status supporting a pathogenetic role of EBV in the development of CNS lymphomas in immunocompromised patients.

In situ hybridization in hematopathology.

In situ hybridization (ISH) is one of the molecular techniques that has applications in diagnostic hematopathology. This procedure allows the detection of DNA or RNA in intact cells from various preparations, including cytology specimens and routinely fixed paraffin-embedded tissues. ISH is therefore analogous to detecting proteins (antigens) in intact cells with immunohistochemistry. The purpose of this article is to review the basic concepts and principles of ISH and to briefly discuss the important technical details of this procedure. Examples of potential applications of ISH in hematopathology are then discussed, including detection of Epstein-Barr virus, Y chromosome, and oncogene activation.

Comparison of in situ hybridization and immunohistochemistry for detection of cytomegalovirus and herpes simplex virus.

In situ hybridization (ISH) and immunohistochemistry (IHC) were compared for detection of cytomegalovirus (CMV) and herpes simplex virus (HSV) in routinely processed tissue. Fifty-four formalin-fixed paraffin-embedded tissue samples infected with CMV (36 tissues) or HSV (18 tissues) from 30 autopsies were studied. All tissues had either positive viral cultures (38 of 54) or characteristic viral inclusions on hematoxylin and eosin examination (39 of 54). The tissues examined included lung (28), liver (nine), kidney (five), heart (three), adrenal (two), spleen (two), and thymus, pancreas, appendix, esophagus, and duodenum (one each). Studies by ISH were performed with two detection systems, using biotinylated probes to CMV and HSV (Enzo Biochem, New York, NY). Using ISH with an alkaline phosphatase detection system, infected cells were detected in 33 of 54 tissues (CMV: 23 of 36, HSV: 10 of 18). Using ISH with a peroxidase detection system, infected cells were identified in 30 of 54 tissues (CMV: 22 of 36, HSV: eight of 18). With IHC, antibodies to CMV and HSV stained the infected cells in 34 of 54 tissues (CMV: 24 of 36, HSV: 10 of 18). All infections detected with ISH were also detected with IHC. We conclude that these techniques for ISH and IHC are equally effective for detecting CMV and HSV in paraffin sections. The results of both techniques correlate better with viral inclusions than with culture results. The ISH stains are more difficult to prepare and in some cases are more difficult to interpret. Therefore, IHC may be preferable to ISH for detecting CMV and HSV in routine diagnostic work.

Comparison of "host cell infiltrates" in patients with follicular lymphoma with and without spontaneous regression.

The "host cell infiltrates" in five patients with low-grade follicular lymphoma who had spontaneous regression without therapy were studied with the use of immunohistochemical methods applied to frozen sections. These infiltrates were compared with the "host cell infiltrates" in six patients with follicular lymphoma with progressive disease. The group with progressive disease was selected to be similar to the group with spontaneous regression in age, sex, histologic characteristics, and stage of disease. The patients with spontaneous regression had significantly more T-helper cells in the host cell infiltrate than the control patients. There were no statistically significant differences between the two groups in numbers of cytotoxic/suppressor T-cells, macrophages, Tac-positive cells, Leu-7-positive cells, or proliferating cells.

Large-cell hematolymphoid neoplasms of uncertain lineage.

In almost every large study attempting to characterize non-Hodgkin's lymphomas, there is a small subset of tumors for which the lineage remains poorly defined. The investigators studied a series of 20 hematolymphoid neoplasms that could not be clearly assigned to the B or T cell lineage by phenotypic criteria. Histologically, 12 cases had an appearance suggesting a histiocytic origin, seven cases resembled a pleomorphic immunoblastic lymphoma, and one had a sarcomatoid appearance. By immunologic studies, a variety of B cell, T cell, and monocyte/macrophage markers were expressed on the neoplasms, often with coexpression of markers for different lineages. Twelve cases expressed the Ki-1 antigen. In immunogenotyping studies of T cell receptor (TCR) and immunoglobulin genes, 13 cases showed clonal rearrangements of the beta or gamma TCR gene; one of these cases also had clonal rearrangements of a light chain immunoglobulin gene. Seven cases showed a germline configuration with all combinations of probes and enzymes used. We conclude that a small subset of hematolymphoid neoplasms shows a pattern of diverse immunologic marker expression that does not appear to reflect normal differentiation. However, a majority of these cases contain clonal TCR gene rearrangements, suggesting a frequent relationship to the T lineage.

Immunophenotypic differences between plasmacytoma/multiple myeloma and immunoblastic lymphoma.

Four cases of plasmacytoma (PC), six cases of multiple myeloma (MM), and nine cases of immunoblastic lymphoma (IL) of B-cell phenotype were studied with a large panel of monoclonal antibodies applied to frozen tissue sections. There were no significant differences in the immunophenotypes of plasmacytomas and multiple myelomas. However, significant immunophenotypic differences were noticed between the plasmacytoma/multiple myeloma cases (PC/MM) and the immunoblastic lymphoma specimens. The PC/MM cases characteristically stained with alpha (or gamma) and T10 and did not usually stain with mu, leukocyte common antibodies, certain B-lineage antibodies (B1, T015, 4G7, 6A4), or Ia. In contrast, IL sections usually did not stain with alpha or T10 and generally did stain with mu (or gamma), leukocyte common antibodies, B-lineage antibodies, and Ia. Ki-67, an antibody to proliferating cells, stained significantly fewer cells in PC/MM than in IL and stained significantly fewer cells that had a good clinical outcome. We conclude that although no one antibody is useful in distinguishing PC/MM from IL, the application of a panel of antibodies may be helpful in making this distinction. The prognosis may correlate with the numbers of proliferating cells as measured by reactivity with Ki-67.

Intermediate lymphocytic lymphoma: an immunophenotypic study with comparison to small lymphocytic lymphoma and diffuse small cleaved cell lymphoma.

Sixteen cases of intermediate lymphocytic lymphoma (ILL), including eight cases with mantle zone architecture, were studied using cryostat sections, a biotin-avidin immunoperoxidase technique, and a large panel of monoclonal antibodies. The neoplastic cells invariably expressed IgM, most B lineage antigens (B1, TO15, Leu-12, 6A4, 41H, BA-1, and LN-2), and Ia. IgD was expressed in 12 cases. Leu-1 and Leu-8 were weakly expressed by the tumor cells in 12 and 11 cases, respectively. The neoplastic cells did not express common acute lymphoblastic leukemia antigen (CALLA) or the T10 antigen in any case. Because ILL is difficult to differentiate from small lymphocytic lymphoma (SLL) and diffuse small cleaved cell lymphoma (DSCCL) on the basis of light microscopic criteria, the immunologic findings of ILL were compared to 31 cases of B cell SLL and 11 cases of B cell DSCCL previously studied in the laboratory to determine if immunologic findings might aid in the distinction. No absolute, and five statistically significant, differences were found; IgD in combination with IgM was seen more commonly in cases of ILL and DSCCL than in SLL (P less than .01), IgG was found more often in SLL than in ILL and DSCCL (P less than .05), Leu-8 was more commonly expressed in ILL and SLL than in DSCCL (P less than .05), T9 expression was less frequent in ILL as compared with SLL (P less than .05) and more proliferating cells were seen in ILL and DSCCL than in SLL (P less than .01). The investigators conclude that these three classes of lymphoma are remarkable much more for their immunologic similarities than for their differences and that immunologic studies are of limited usefulness in differentiating the three neoplasms. Their results also support the concept that these lymphomas are closely related to each other. In particular, DSCCL immunologically appears to be more closely related to SLL and ILL than to follicular small cleaved cell lymphoma.

Monoclonal antibodies reactive in routinely processed tissue sections of malignant lymphoma, with emphasis on T-cell lymphomas.

The immunoreactivity of eight monoclonal antibodies was evaluated on 45 routinely processed lymphomas (22 T-cell lymphomas, 11 B-cell lymphomas, and 12 cases of Hodgkin's disease). Two antibodies reactive with leukocyte common (T200) antigens (PD7/26 and 2B11) stained most of the B- and T-cell lymphomas but did not stain the Reed-Sternberg cells and variants in Hodgkin's disease. Two antibodies known to stain B cells (LN-1 and LN-2) reacted with some of the B-cell lymphomas, but LN-2 also reacted with the neoplastic cells in six of 22 T-cell lymphomas and with the Reed-Sternberg variants in eight of 12 cases of Hodgkin's disease. The granulocyte antibody anti-Leu M1 reacted with most cases of Hodgkin's disease but also reacted with two of 11 B-cell non-Hodgkin's lymphomas. An antibody to epithelial membrane antigen (anti-EMA) stained some cases of T-cell lymphoma, B-cell lymphoma, and Hodgkin's disease. Leu 7 was expressed in one T-cell lymphoma and in one case of Hodgkin's disease. A novel antibody reactive with T cells (L60) stained all cases of T-cell lymphoma but also stained some cases of B-cell lymphoma and one case of Hodgkin's disease. We conclude that none of these antibodies, when used alone on routinely fixed paraffin-embedded material, is completely sensitive and specific for T-cell lymphoma, B-cell lymphoma, or Hodgkin's disease. However, a panel of antibodies is useful in distinguishing Hodgkin's disease from non-Hodgkin's lymphoma and in suggesting the B- or T-cell phenotype of non-Hodgkin's lymphomas.