Chemiluminescent detection of gene rearrangements in hematologic malignancy.

A chemiluminescent based Southern blot assay is described for the detection of gene rearrangements in human hematologic malignancies. The DNA probes for regions of rearrangements within the immunoglobulin genes were labeled with digoxigenin-11-dUTP and detected by an antibody conjugated to alkaline phosphatase with Lumiphos 530 as a substrate. This assay has proven more sensitive than colorimetric assays and provides rapid turn-around times without the hazards of radioactive isotopes.

Primary lymphoproliferative disorders of the breast.

Primary breast lymphoproliferative disorders are rare lesions and include both the malignant lymphomas and the benign pseudolymphomas. We reviewed 4,491 consecutive cases of breast cancer diagnosed and treated between 1973 and 1988. Patients with lymphoma in other sites and those with lymphomas limited to axillary nodes were excluded. RESULTS. Five patients (0.11%) presented with primary lymphoreticular lesions, of which three were primary non-Hodgkin's lymphoma and two were pseudolymphomas. Patients were followed clinically through to the present time or until death occurred. Surgical procedures included incisional or excisional biopsy in four patients and modified radical mastectomy in one. Two patients received chemo-therapy and one received radiotherapy. One patient with pseudolymphoma subsequently developed infiltrating ductal carcinoma of the same breast. Three patients with primary breast non-Hodgkin's lymphoma died within the follow-up period, with a mean survival of 33 months. CONCLUSIONS. We conclude that primary breast lymphoma is a rare and aggressive breast malignancy with a poor prognosis despite different treatment options.

Methods in pathology. Identification of T-cell lymphomas in paraffin-embedded tissues using polyclonal anti-CD3 antibody: comparison with frozen section immunophenotyping and genotypic analysis.

While L26 (CD20) is now well established as a B-cell marker of high specificity for use in paraffin-embedded tissues, paraffin-reactive T-cell antibodies (UCHL1, MT1, Leu-22, DF-T1, and MT2) have not shown comparable lineage specificity. A new commercially available polyclonal antibody directed against a synthetic peptide sequence of the CD3 (T-cell) antigen has recently become available for use on paraffin sections. In order to evaluate the utility of this antibody, we studied CD3 expression in conjunction with L26 and leukocyte common antigen (LCA) in 15 T-cell and 20 B-cell non-Hodgkin's lymphomas (NHL), all genotypically confirmed by DNA hybridization and immunophenotyped by immunoperoxidase studies in frozen tissue. Ten of 15 T-cell NHLs (67%) showed unequivocal immunolabeling of neoplastic cells with anti-CD3 in paraffin-embedded tissue. Of the five negative cases, three were lymphoblastic lymphomas, and two were peripheral (postthymic) lymphomas (one anaplastic large cell, Ki-1 positive and one large cell, immunoblastic). CD3 expression was identical in paraffin and cryostat sections (100% concordance). Twenty of 20 B-cell NHLs were positive with L26 and LCA but were negative with anti-CD3. Other neoplasms examined, including three granulocytic sarcomas and 45 nonhematopoietic tumors, were similarly negative with anti-CD3. We conclude that polyclonal anti-CD3 is a sensitive and highly specific T-cell marker in paraffin-embedded tissue and, when used in conjunction with LCA and L26, that it can determine cell lineage in the majority of non-Hodgkin's lymphomas.

Arsenic intoxication presenting as a myelodysplastic syndrome: a case report.

A case of arsenic intoxication presenting as a myelodysplastic syndrome is reported. A 41-year-old woman with a 6-month history of gastrointestinal and neurological symptoms was noted to be pancytopenic at presentation. A bone marrow aspirate revealed dysmyelopoietic changes involving all three marrow cell lines. Subsequent analysis of urine for heavy metals demonstrated very high levels of arsenic. Treatment with British anti-Lewisite (BAL) resulted in the resolution of gastrointestinal symptoms and hematological abnormalities although the neurological complications progressed. This case emphasizes that heavy metal intoxication should be considered in the differential diagnosis of any individual presenting with the hematological features of myelodysplasia especially when accompanied by clinical features considered atypical for primary or secondary myelodysplasia.

The role of cellular maturation in neutrophil heterogeneity.

Previous studies have shown that many neutrophil (PMN) characteristics are heterogeneous but the origin of PMN heterogeneity is unknown. It is unclear if PMN functional heterogeneity is secondary to maturational differences or due to distinct subpopulations of cells that possess different functional capacities. The PMN 31D8 antigen is a useful probe for evaluation of PMN subpopulations. The majority of PMNs (approximately 85%) exhibit a high intensity fluorescence after 31D8 monoclonal antibody (MoAb) labeling (31D8 enriched or "bright" PMNs) as determined by flow cytometric analysis. These cells are more functional than cells with low intensity fluorescence (31D8 diminished or "dull" PMNs). Various immunologic, clonogenic and functional techniques were used to study the expression of the 31D8 antigen in HL-60 cells and myeloid cells in order to evaluate antigenic and functional heterogeneity during morphologic maturation. The results of this study indicate that the percentage of 31D8 antigen positive (31D8 antigen enriched and diminished) bone marrow cells increases from 20 +/- 11% in myeloblast cells to 68 +/- 10% in promyelocytes, 93 +/- 2% in myelocytes and 99 +/- 1% in bands and PMNs. 31D8 antigen enriched cells first appear at the myelocyte stage (32 +/- 10%) and increase in bands (52 +/- 13%), marrow PMNs (62 +/- 13%) and peripheral blood PMNs (88 +/- 4%). These data indicate that the heterogeneous expression of 31D8 antigen in PMNs is due, at least in part, to maturational differences within the PMN population and raise the possibility that other heterogeneously expressed PMN characteristics are also maturationally derived. They also suggest that 31D8 antigenic expression may be a more precise indicator of myeloid functional maturation than maturation as identified by cellular morphology.

DNA ploidy in breast lesions. A comparative study using two commercial image analysis systems and flow cytometry.

DNA ploidy determinations on a series of 24 breast specimens were performed independently utilizing flow cytometry (FCM) and two separate commercially available computerized image analysis systems for image cytometry (ICM). The tissues analyzed were obtained from 20 carcinomas, 2 benign neoplasms and 2 benign reductive procedures. The results showed a close correlation between the DNA indices (DIs) obtained by all methods in 14 of the 24 cases. In four cases, all methods showed aneuploid peaks, but with differing DIs. In six cases (two benign and four malignant) FCM showed diploidy while ICM showed peridiploid cell populations. The results obtained with the two image analysis systems were in agreement for 20 of the 24 cases. ICM is an acceptable alternative to FCM for reproducible ploidy analysis. ICM-based measurements have the advantage of the visual discrimination of abnormal cells and therefore may have a greater sensitivity in identifying small aneuploid populations. Populations with DIs in the range of 1.0 to 1.3 need to be assessed carefully in ICM-based determinations due to the potential that these "aneuploid" peaks may represent shifted diploid populations.

Hodgkin's disease, lymphocyte-predominant type: immunoreactivity with B-cell antibodies.

Utilizing the monoclonal antibodies L26 (a new antibody possessing immunoreactivity with B-lymphocytes in paraffin-embedded tissue), LN1, LN2, and Leu-M1, 44 cases of Hodgkin's disease (HD) were examined for the presence of immunoreactivity in Reed-Sternberg (R-S) cells by the avidin-biotin-peroxidase complex (ABC) technique. In 16 cases of lymphocyte-predominant Hodgkin's disease (LPHD), the L&H variants of R-S cells exhibited a different pattern of staining compared to R-S cells in other histologic types (total, 28 cases: 11, mixed cellularity; 8, nodular sclerosing; 6, lymphocyte depleted; 3, unclassified). L&H variants in LPHD were immunoreactive for L26 and LN1 in 15 and 14 cases, respectively, whereas R-S cells in the remaining types were negative or rarely positive (3, L26; 2, LN1). Leu-M1 was strongly positive in 27 of 28 cases of non-LPHD versus only 4 of 16 in LPHD. LN2 was reactive in virtually all cases (43 of 44). These findings suggest the possibility that the R-S cells of LPHD are derived from a different lineage than R-S cells in other histologic types of HD or that the latter have somehow lost the ability to express the antigens defined by L26 and LN1. Finally, based on immunologic and morphologic findings in this study, the similarities seen between the nodular and diffuse subtypes of LPHD are felt to favor a close relationship between the two subtypes.

Utilization of monoclonal antibody L26 in the identification and confirmation of B-cell lymphomas. A sensitive and specific marker applicable to formalin-and B5-fixed, paraffin-embedded tissues.

Immunophenotypic analysis of paraffin-embedded tissues of lymphoproliferative disorders has been facilitated by recent developments of monoclonal antibodies that react with epitopes that survive histologic processing. Leukocyte common antigen (LCA) antibody has made a significant contribution to the immunocytochemical separation of non-Hodgkin's lymphomas from nonlymphoid neoplasms. However, a small percentage of lymphomas, particularly some large cell or immunoblastic B-cell tumors, will not label with LCA antibody. Other antibodies, directed against B lymphocytes, experience problems of specificity and a lack of sensitivity when applied to formalin-fixed specimens. The authors recently investigated a monoclonal antibody (L26) that demonstrates excellent specificity and sensitivity for B lymphocytes, and tumors derived from them, in formalin- and B5-fixed, paraffin-embedded tissue. The avidin-biotin peroxidase complex (ABC) technique was utilized for immunostaining 95 cases of malignant lymphoproliferative disorders and a variety of normal and neoplastic nonlymphoid tissues. When applied to sections of benign lymphoid tissue, the L26 antibody labeled germinal center cells, mantle zone and scattered interfollicular lymphocytes, but not histiocytes or plasma cells. L26 marked 100% (44/44) of the large cell and immunoblastic B-cell lymphomas, along with 1 case of pre-B cell lymphoblastic lymphoma. This included 8 cases that were LCA-negative. None of the T-cell lymphomas or plasma cell tumors studied demonstrated L26 immunostaining. No normal, benign, or neoplastic nonlymphoid tissues examined stained with this antibody. L26 successfully labels B lymphocytes and B-cell lymphomas in routinely processed tissues, often with greater sensitivity and intensity than LCA. This antibody should prove invaluable in the investigation of atypical lymphoid proliferations and the identification of B-cell derived lymphomas, when fresh or frozen tissue is unavailable for analysis.

Polymorphonuclear leukocyte heterogeneity in neonates and adults.

We have used a mouse monoclonal antibody (31D8) to determine whether differences in neutrophil (PMN) subpopulations might help explain decreased PMN chemotaxis in neonates compared with that of adults. 31D8 has been shown to bind heterogeneously to adult PMNs. Approximately 80% of the PMNs that strongly bind 31D8 (31D8 "bright") are the same cells that depolarize and migrate chemotactically when stimulated with the chemoattractant N-formyl-methionylleucylphenylalanine, while the 20% that weakly bind 31D8 fail to similarly respond. All neonatal PMNs bound 31D8 heterogeneously. There was a smaller population of 31D8 "bright" cells in neonates at birth (76% +/- 6%, n = 45) compared with that of neonates at three to 15 days of age (82% +/- 5%, n = 10, P less than 0.002) and both were smaller than that of adults (88% +/- 4%, n = 45, P less than 0.001 and P less than 0.001). Neonatal cord PMNs, which traversed a micropore filter in a modified Boyden chemotaxis chamber in the presence of a chemoattractant, had an increased percentage of 31D8 "bright" cells (89% +/- 7%) than did PMNs which remained above the filter (82% +/- 7%, n = 10, P = 0.034). PMN chemotaxis was less in neonates at birth (32.7 +/- 4.5 micron) than at three to six days of age (36.8 +/- 11.3 micron) and both were decreased compared with that of adults (69.1 +/- 12.4 micron, P less than 0.001 and P less than 0.001). These findings indicate that decreased PMN chemotaxis in neonates may be due in part to a smaller PMN subpopulation of highly motile cells.

Fulfilling Medicare's criteria for consultative services.

Consultative clinical pathology services must fulfill certain conditions to be reimbursed by Medicare. This article explores how pathologists can best comply with the requirements. This article is adapted from Dr. Pastuszak's presentation at the Bridgeport Symposium entitled "Performance Requirements for Clinical Laboratories Under Prospective Reimbursement and DRGs." The Nov. 15-16, 1984 symposium was sponsored by Bridgeport Hospital, Bridgeport, Conn., and supported by contributions from 20 companies (listed on page 29).

Small cell neuroendocrine carcinoma of the urinary bladder: report of three cases with ultrastructural analysis.

Three cases of small cell neuroendocrine carcinoma of the urinary bladder were studied by light and electron microscopic techniques. Dense-core, membrane-bound granules were identified in the cytoplasm of all 3 cases, substantiating the light microscopic impression of a morphologic similarity to other neuroendocrine carcinomas, such as small cell (oat cell) carcinoma of the lung. Two of the three cases showed clinical evidence of distant metastases, suggesting an aggressive biologic potential of this tumor similar to oat cell carcinoma of the lung. A partial remission was induced in these 2 cases using chemotherapy protocols similar to the drug regimens for small cell (oat cell) carcinomas of the lung. Recognition of this distinct entity has important clinical implications regarding therapeutic approach.

Lymph node biopsy for early diagnosis in Kawasaki disease.

Kawasaki disease or mucocutaneous lymph node syndrome is an acute exanthematous illness of childhood of unknown etiology with a recent marked increase in incidence. Occasional fatalities occur usually as a result of coronary thromboarteritis. Diagnosis is based on a spectrum of clinical signs and symptoms, some of which occur late in the acute phase of the illness. We recently examined cervical lymph node biopsies from two children during the early stage of illnesses which subsequently met the clinical criteria of Kawasaki disease and in which electron microscopy, cultures, serology, and other special studies failed to identify an etiologic agent. Both lymph node biopsies revealed multiple foci of necrosis and fibrin thrombi within the microvasculature, changes which have received little attention in the pathologic literature. These pathologic alterations are distinctive and probably characteristic. Early presumptive diagnosis of Kawasaki disease in our second case was based on the histopathologic findings and resulted in prompt institution of antithrombotic therapy. Lymph node biopsy has been underutilized in the diagnosis, and characteristic acute pathologic changes which may occur have been underpublicized.

Gastric lymphoma: a clinicopathologic reappraisal.

Histologic material from 42 cases diagnosed as gastric lymphoma at Hartford Hospital was reviewed, confirming the diagnosis in 37. Three cases of pseudolymphoma were found. The incidence of gastric lymphoma has increased steadily over the past 50 years: 35 percent of cases occurred during the past decade. Most patients with gastric lymphoma are in the seventh or eighth decade of life. Resection offered the best chance for long-term survival, either alone or with radiation therapy. Nodal status was correlated with length of survival of survival; 60 percent of patients with negative nodes survived 5 years or more. Cases were classified according to the Rappaport classification and the Working Formulation of Non-Hodgkin's Lymphomas. The formulation was more useful than the Rappaport classification in assessing prognosis in various types of lymphoma and better reflects our current understanding of neoplasms of the lymphoid system.

Coexistence of a primary immunodeficiency disorder and Hodgkin's disease: evidence against a B-lymphocyte origin for the Reed-Sternberg cell.

A 44-year-old woman with a life-long history of recurrent sinopulmonary infections developed Hodgkin's disease with characteristic Reed-Sternberg cells in a biopsy specimen of a mediastinal lymph node. Hypogammaglobulinemia was documented on several serum determinations and plasma cells were absent from biopsy specimens of the lymph node and bone marrow. Immunochemical studies failed to demonstrate any B lymphocytes bearing surface immunoglobulin or Fc-receptors for IgG in the peripheral blood. Pokeweed mitogen stimulation of the patient's peripheral blood lymphocytes in vitro resulted in the development of virtually no plasma cells. Peripheral blood T-lymphocyte number and function were defective initially. Following chemotherapy and radiotherapy, peripheral blood E-rossette-forming cells returned to normal, but T-cell function remained defective and B lymphocytes remained undetectable. These findings are compatible with the presence of two separate immune disorders: a primary hypogammaglobulinemia and Hodgkin's disease. The absence of lymphocytes bearing surface Ig or Fc-receptors for IgG in this patient adds further support against a B-lymphocyte origin for the Reed-Sternberg cell.

Angioimmunoblastic lymphadenopathy: pleural-pulmonary disease.

A 48-year-old female with angioimmunoblastic lymphadenopathy is described. Her disease was complicated by pleuritic chest pain, an exudative pleural effusion and pulmonary infiltrates attributable to underlying pleural-pulmonary angioimmunoblastic lymphadenopathy.