Beta-2 microglobulin is an amyloidogenic protein in man.

Curvilinear fibrils with the tinctorial properties of amyloid were isolated from a patient with bone and joint involvement complicating chronic dialysis for renal disease. Subunit fractions of 24,000 and 12,000 mol wt were identified after gel filtration under dissociating conditions, the latter containing a significant amount of a dimer of the former. This was confirmed by Edman degradation of each fraction, which yielded the amino terminal sequence of normal human beta-2 microglobulin (B2M) to residues 20 and 30, respectively. The size of the subunit protein (12,000 mol wt) and the amino acid composition make it likely that intact B2M is a major constituent of the fibrils. B2M is thus another example of a low molecular weight serum protein, with a prominent beta-pleated sheet structure, that may adopt the fibrillar configuration of amyloid in certain pathologic states.

Hepatosplenic alphabeta T-cell lymphomas: a report of 14 cases and comparison with hepatosplenic gammadelta T-cell lymphomas.

Hepatosplenic gammadelta T-cell lymphoma is a distinct entity, characterized by occurrence in young adult males with hepatosplenomegaly, B-symptoms, peripheral blood cytopenias, and no lymphadenopathy; lymphomatous infiltrates in the splenic red pulp, hepatic sinusoids, and bone marrow sinuses; T-cell receptor (TCR) gammadelta chains and a cytotoxic T-cell phenotype; isochromosome 7q; and an aggressive clinical course. In comparison, this study describes the clinicopathologic features of 14 hepatosplenic T-cell lymphomas expressing TCR alphabeta chains. They occurred in 11 women and 3 men with a median age of 36 years. Clinical presentation was similar to that described previously for hepatosplenic gammadelta T-cell lymphomas, except for the female preponderance and age distribution (5 patients younger than 13 years of age and 5 patients older than 50 years of age). Disease distribution was primarily in the splenic red pulp and hepatic sinusoids, although liver infiltrates were largely periportal in four cases. Bone marrow involvement, observed in eight patients, was usually interstitial and/or within the sinuses. Lymph nodes were involved in five patients, although lymphadenopathy was demonstrable in only two. Ten cases were composed of intermediate-size tumor cells with round/oval nuclei, slightly dispersed chromatin, inconspicuous nucleoli, and scant to moderate amounts of cytoplasm. Four lymphomas contained primarily large cells with irregular nuclei, dispersed chromatin, discernible nucleoli, and moderate to abundant cytoplasm. Tumor cells in all 14 lymphomas were cytotoxic alphabeta T-cells; 13 co-expressed natural killer cell-associated antigens and showed T-cell clonality. Three lymphomas were associated with Epstein-Barr virus. Two of four cases had an isochromosome 7q. Eleven patients are dead, eight within a year of diagnosis, and two patients have maintained complete remissions after combination chemotherapy. These data show that hepatosplenic T-cell lymphomas include an alphabeta-subtype. This group, along with the previously recognized gammadelta group, should be recognized as phenotypically heterogeneous subtypes of the same disease entity.

Tumoral amyloidosis of bone of beta 2-microglobulin origin in association with long-term hemodialysis: a new type of amyloid disease.

Amyloid lesions of bone are rare and limited almost exclusively to patients with amyloidosis secondary to plasma cell dyscrasias. The present report describes the cases of two patients receiving long-term hemodialysis (nine and 12 years) who had multiple lytic lesions of bone proved by biopsy to contain an unusual type of amyloid. Results of serum protein electrophoreses and immunoelectrophoreses, as well as bone marrow examinations, were normal. In both cases the amyloid displayed characteristic Congo red affinity and birefringence on polarized light microscopy that was inhibited by potassium permanganate treatment of sections prior to staining. Although this staining reaction was described previously exclusively in AA amyloid (i.e., the material associated with classic secondary amyloidosis), immunoperoxidase staining for AA protein in these cases was negative. Transmission electron microscopy revealed the amyloid fibrils to have unusual curvilinear configurations. Immunoperoxidase staining for beta 2-microglobulin (beta 2m) was positive in the amyloid lesions of both patients at the light microscopic level. Ultrastructural immunohistochemical studies for beta 2m, performed in one case, were positive. Both patients had markedly elevated serum beta 2m levels. By Ouchterlony immunodiffusion, purified beta 2m demonstrated partial identity with purified amyloid protein fractions and a serum constituent. Bone lesions composed of amyloid related to beta 2M probably represent a new subgroup of amyloid disease that may be linked to renal failure and long-term hemodialysis.

Rapid immunocytochemical analysis of acute leukemias.

Immunophenotypic analysis of acute leukemias is time consuming and often requires flow cytometric analysis. A 1-hour alkaline phosphatase-labeled streptavidin-biotin immunocytochemical procedure was evaluated as an alternative. Seventeen cases of acute leukemia, including 10 acute lymphocytic (ALL) and 7 acute nonlymphocytic, were phenotyped by the rapid immunocytochemical procedure and the results were compared with standard analyses. In all 17 cases, the diagnoses made using standard cytochemical and immunologic methods were the same as obtained in blinded reviews by rapid immunocytochemical analysis. Nine cases of precursor B-cell ALL were positive for CD19 and/or CD22. Five CD19 + cases of ALL reacted with anti-myeloperoxidase, with one case also positive for CD15. CD15 positivity was confirmed on repeated study as well as with plastic section immunoperoxidase staining. Nine cases of ALL were positive for CD10 and eight were positive for terminal deoxynucleotidyl transferase. One case of ALL marked as T-cell ALL with CD1, CD2, CD3, and CD7. All cases of acute nonlymphocytic leukemia were positive for CD15, CD13, and/or CD33; anti-myeloperoxidase was positive in all but one case of monocytic leukemia. All cases of acute nonlymphocytic leukemia were negative for CD10 and one was positive for terminal deoxynucleotidyl transferase. Acute leukemias apparently may be phenotyped easily and accurately in 1 hour with this immunocytochemical technique, and slides may be stored permanently for review. There was in these 17 cases high correlation of the diagnoses with standard flow cytometric and cytochemical results. This rapid method allows a coordinated evaluation of morphologic features and immunophenotype; the latter features facilitated confirmation of unexpected reactivity of myeloid markers CD15 and MPO-7 in some cases of ALL.

Differentiation of reactive from neoplastic small-cell lymphoid aggregates in paraffin-embedded marrow particle preparations using L-26 (CD20) and UCHL-1 (CD45RO) monoclonal antibodies.

The pathologic findings in 1,390 consecutive patients who had bone marrow examinations at Nashville Veterans Administration Hospital from 1977 through 1979 were reviewed. Seventy-three patients who did not meet diagnostic criteria for a small-cell lymphoid neoplasm (SCLN) and 11 patients with SCLN had, on marrow particle preparations: (1) at least three lymphoid aggregates and (2) suitable material available for immunoperoxidase studies with monoclonal antibodies UCHL-1 (CD45RO, pan T cell) and L-26 (CD20, pan B cell). Staining with UCHL-1 was difficult to interpret due to high background positivity in myeloid elements. With L-26, three distinct patterns of lymphocyte marking were identified within aggregates: (1) homogeneous--uniform marking of almost all lymphocytes; (2) mixed--even distribution of marking and nonmarking lymphocytes; (3) and focal homogeneous--collections of uniformly marking lymphocytes either surrounding or surrounded by nonmarking lymphocytes. A homogeneous marking pattern was the predominant pattern in 8 of 11 patients (73%) with SCLN. Only 6 of 73 patients without overt SCLN marked in a homogeneous pattern, and these were always associated with aggregates with other staining patterns. All patients with apparently non-neoplastic lymphoid infiltrates had mixed (67 of 73) or focal homogeneous (32 of 73) patterns of aggregate marking, whereas only 5 of 11 patients (45%) with extramarrow SCLN had aggregates with these patterns. The size and number of aggregates with a homogeneous marking pattern further helped discriminate between the patients with SCLN and those with apparently non-neoplastic lymphoid aggregates. These findings suggest that a homogeneous pattern of lymphoid aggregate staining with L-26 is more common in patients with SCLN than in those patients with lymphoid aggregates and no evidence of neoplasia. Paraffin immunoperoxidase staining with L-26 may be a helpful adjunct to histopathologic examination in evaluating marrow lymphoid aggregates.

Leu-22 (L60). A more sensitive marker than UCHL1 for peripheral T-cell lymphomas, particularly large-cell types.

Paraffin-embedded sections of 77 peripheral T-cell lymphomas (PTCLs) were stained with several monoclonal antibodies, including the preferential T-cell markers Leu-22 (L60[CD43]) and UCHL1 (CD45RO). The staining characteristics of L60 and UCHL1 were compared to determine the value of each in the immunophenotypic analysis of PTCLs. Lineage specificity was evaluated among 39 B-cell lymphomas and 33 cases of Hodgkin's disease (HD). L60 and/or UCHL1 stained 95% of PTCLs, whereas L60 and UCHL1 alone stained 90% and 69% of cases, respectively. L60 demonstrated significantly greater numbers of immunopositive tumor cells than UCHL1 in 37% of the PTCL cases, principally because of enhanced marking of large, neoplastic cells. UCHL1 was a better marker in only 10% of the PTCL cases. L60 stained 33% of B-cell lymphomas, usually small lymphocytic or lymphoplasmacytic types. UCHL1 stained only 8% of B-cell lymphomas, all large-cell types. L60 and UCHL1 stained Reed-Sternberg cells and variants in three cases of nodular sclerosing HD. These results suggest that both L60 and UCHL1 are useful markers of PTCLs in routinely processed tissue. L60 is a more sensitive marker of large neoplastic T-cells than UCHL1 but is less lineage-specific. These antibodies are most effective when used as part of a panel of monoclonal antibodies.

Latent membrane protein antibody reacts with normal hematopoietic precursor cells and leukemic blasts in tissues lacking Epstein-Barr virus genome by polymerase chain reaction.

Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP) has been detected in various reactive and neoplastic lymphoproliferations and in some epithelial malignancies. However, data are lacking on LMP immunoreactivity in normal and neoplastic hematopoietic cells. Therefore, the authors studied LMP staining in 29 paraffin-embedded tissues containing these cells and correlated the findings with polymerase chain reaction (PCR) analysis for a EBV-BAM HI W genome sequence in 15 of these cases. Latent membrane protein immunostains showed strong uniform marking of normal early myeloid and erythroid precursors, whereas neutrophils, bands and late normoblasts were negative. Leukemic myeloblasts and lymphoblasts were also strongly positive for LMP. Polymerase chain reaction analysis showed no evidence of EBV-BAM HI W genome in 13 cases with amplifiable DNA. This study indicates normal hematopoietic precursor cells and leukemic blasts mark strongly with monoclonal LMP antibody. Furthermore, the absence of EBV genome in these tissues suggests a lack of specificity of monoclonal LMP for EBV-infected cells in the marrow.

Stratified diagnostic approach to fine needle aspiration of the breast.

The clinical value of fine needle aspiration (FNA) of the breast is enhanced by incorporating into the cytologic diagnosis explicit comments on the level of diagnostic certainty. This stratification of diagnostic certainty is based predominantly on the cytologic features but occasionally also takes into consideration the clinical situation. Strong clinical and mammographic suspicion of mammary carcinoma associated with FNA, diagnostic of typical, intermediate to high-grade mammary carcinoma, warrants proceeding to definitive therapy without further diagnostic studies. False-positive results are virtually eliminated by placing cases with any uncertainty into a "probable" category, which does not support definitive therapy. In addition, oversimplified "benign versus malignant" approaches to FNA diagnoses ignore the heterogeneity of breast masses, with in situ and low-grade carcinomas warranting special clinical management and usually being placed in the "probable" category. Thus, malignant diagnoses are stratified into "definite" and "probable," with only the former supporting definitive therapy. Within our recent series of 1,005 FNAs of the breast, we were able to confirm the diagnosis in all 62 patients with a "definite" carcinoma diagnosis, and only 3 of 25 "probable" cancer diagnoses were benign at tissue biopsy. Thus, false-positive results were successfully avoided in the "definite" category. Furthermore, a much greater incidence of unusual and good prognosis tumor types were identified by the "probable" category. If the clinical setting is relatively suspicious only, a definitive diagnosis of cancer by FNA is rare and not necessary because the clinical question to be addressed is only whether to biopsy. This approach to FNA diagnosis, unlike the oversimplified "benign versus malignant" scheme, provides an approach that is more likely to result in optimal therapy for breast neoplasms, with low-grade or in situ carcinomas requiring special clinical management since these types of cancers are found predominantly in the "probably malignant" category. It also provides additional security against false-positive diagnoses by incorporating clinical level of certainty statements into FNA diagnostic categories, which more closely reflect the diversity and inherent complexity in the appropriate diagnosis and therapy of mammary carcinomas.

Plastic section immunohistochemistry in the diagnosis of hematopoietic and lymphoid neoplasms.

Plastic section immunohistochemistry is an important method in the multiparameter approach to the diagnosis of hematopoietic and lymphoid neoplasms. Morphologic, histochemical, and immunophenotypic data may be correlated even on small fragments of tissue by this technique. A variety of differentiation markers is detectable in plastic sections for the delineation of lymphocytic, histiocytic, and myeloid neoplasms, as well as Hodgkin's disease. Plastic section immunohistochemistry should also be useful in the diagnosis of epithelial and stromal neoplasms caused by enhanced antigen preservation in those neoplasms. For these reasons, we anticipate that the plastic section technique will have a wide variety of research and investigative applications.

OKT4 epitope deficiency in significant proportions of the black population. A cause for underestimation of helper/suppressor lymphocyte ratios.

Monoclonal antibodies such as OKT4, OKT4A, Coulter T4, and Leu 3a are commonly used to define subsets of human peripheral blood lymphocytes having helper activity in vitro. Analysis of peripheral blood lymphocyte populations is a useful aid in the diagnosis of immunodeficiency syndromes. Peripheral blood lymphocytes of certain black and oriental patients do not mark with the conventional OKT4 antibody but do mark with OKT4A and Leu 3a antibodies. We determined helper subset populations of peripheral blood lymphocytes as delineated by OKT4, OKT4A, and Coulter T4 antibodies in 103 unselected patients from two tertiary care hospitals. Twenty percent of black patients without clinical immunodeficiency demonstrated marked reduced numbers of cells marking with OKT4 antibody, but normal numbers of cells marking with OKT4A and Coulter T4 antibodies. No white patients demonstrated this trait to the degree seen in black patients. Use of OKT4 antibody to define helper cell percentages in black patients may lead to underestimation of these percentages in certain hospital populations.

Tonsillar granular cell tumour in a cat.

Granular cell tumour was diagnosed in a cat based on light and electron microscopic findings. Immunohistochemical findings for S-100 protein and neuron-specific enolase were negative, unlike its human counterpart.

A simplified plastic embedding and immunohistologic technique for immunophenotypic analysis of human hematopoietic and lymphoid tissues.

Routine fixation and paraffin embedding destroys many hematopoietic and lymphoid differentiation antigens detected by flow cytometry or frozen section immunohistochemistry. On the other hand, morphologic evaluation is difficult in flow cytometric or frozen section studies. A simplified three-step plastic embedding system using acetone-fixed tissues embedded in glycol-methacrylate (GMA) resin has been found to provide both excellent morphologic and antigenic preservation. With our system, a wide variety of antigens are detected in plastic sections without trypsinization or prolonged embedding procedures; pan-B (CD19, CD22), pan-T (CD7, CD5, CD3, CD2), T-subset (CD4, CD8, CD1, CD25) markers as well as surface immunoglobulin and markers for myeloid and mononuclear-phagocyte cells are preserved. In summary, modifications of plastic embedding techniques used in this study simplify the procedure, apparently achieve excellent antigenic preservation, and facilitate evaluation of morphologic details in relation to immunocytochemical markers.

Metastatic squamous cell carcinoma from the esophagus occurring as small bowel obstruction.

Metastatic tumors of the small bowel from extra-abdominal sites are rare. The primary tumors that have been reported are malignant melanoma, breast cancer, bronchogenic carcinoma, embryonal myosarcoma, and seminoma. We have presented a case of small bowel obstruction due to metastasis from esophageal cancer.

Immunophenotypes of Reed-Sternberg cells: a study of 19 cases of Hodgkin's disease in plastic-embedded sections.

The immunophenotype of Reed-Sternberg (RS) cells in Hodgkin's disease (HD) has not been clearly defined, partly owing to difficulties in studying RS cells in cell suspensions or identifying them with certainty in frozen sections. We studied the immunophenotype of RS cells with a recently developed plastic section immunohistochemical technique on acetone-fixed tissues that affords superior morphological detail while preserving a wide variety of lymphoid differentiation antigens. Nineteen cases of HD [16 nodular sclerosing (NS), 2 mixed cellularity (MC), and 1 lymphocyte depleted (LD)] were embedded in plastic and stained for pan-B, pan-T, and various T-subset markers, as well as leukocyte common antigen (CD45), interleukin-2 (IL-2) receptor (CD25), and RS cell markers CD15 and CD30. RS cells were positive for CD45, CD15, CD30, and CD25, except for 3 cases (2 NS, 1 MC) that were CD15 negative and 2 cases (NS) that were CD45 negative. In 10 cases (NS), RS cells were positive for at least two pan-T-cell markers and CD4; pan-B cell markers were uniformly negative. RS cells in 6 cases (3 NS, 2 MC, 1 LD) were positive for at least one T-cell marker (CD2) and one B-cell marker (CD22). Two cases of NSHD showed no T- or B-cell marking. These data provide further evidence that RS cells in some cases of NSHD have T-cell phenotypes and that RS cells are not homogeneous in their immunoreactivity.

Pathologic fractures associated with idiopathic amyloidosis of bone in chronic hemodialysis patients.

Amyloid bone lesions were found in 2 chronic hemodialysis patients presenting with pathologic hip fractures. These amyloid deposits were noted as lytic defects on plain skeletal radiographs. No evidence for disseminated amyloidosis was discovered on physical examination, skin biopsy, or bone marrow biopsy. Myeloma, other plasma cell dyscrasia, and preceding chronic inflammatory states were not found in either patient. The amyloid deposits had staining characteristics suggestive of secondary amyloid based on the potassium permanganate reaction. Isolated amyloid bone deposits should be included in the differential diagnosis of lytic bone lesions or pathologic fractures in chronic dialysis patients.

Transformation of cutaneous T cell lymphoma to large cell lymphoma. A clinicopathologic and immunologic study.

Some patients with cutaneous T cell lymphoma (CTCL) develop a high-grade, large-cell lymphoma associated with rapid deterioration of clinical status. This change in histologic appearance and clinical behavior of CTCL is similar to transformations of other hematopoietic and lymphoid neoplasms. From a group of 92 cases of CTCL, morphologic, immunologic and clinical features were studied in 17 cases of transformed CTCL. Transformation was noted, at presentation or subsequently, in either cutaneous or extracutaneous sites; remarkably, transformation was found at initial diagnosis of CTCL in 7 of 17 patients. T cell characteristics were maintained in all 17 cases of transformed CTCL; in 11 cases with complete phenotypes, there were 6 T-helper, 3 T-suppressor, and 2 aberrant T subtypes. The pre- and posttransformation phenotypes were similar in 3 of 7 cases tested over time (all T-helper); retention of T-suppressor phenotype was suggested in another case. T cell features were maintained in the other 3 cases, but the T subtypes were altered in 2 of these cases. Absent or diminished pan-T antigens (CD 5, CD 3, or UCHL1) were found in 9 of 17 cases. Leu-M1, Ki-1, or LN 2 antigens were expressed by transformed cells in 10 of 17 cases, often in patterns identical to Reed-Sternberg cells. Survival in patients with transformed CTCL was significantly shorter (median, 29 months) than in 44 CTCL patients without transformation (58 months, P = 0.015); survival after diagnosis of transformation was short (12 months). Patients with extracutaneous transformation had a shorter median survival after transformation (8 months) than those with transformation limited to skin (19 months). It is concluded that CTCL can transform morphologically to a large cell variant associated with aggressive behavior and shortened survival. Extracutaneous transformation apparently indicates a poorer prognosis than cutaneous transformation. Although transformed CTCL usually retains a T cell phenotype, some antigens are lost while other new antigens may be expressed. Recognition of transformed CTCL is facilitated by identification of the dysplastic cerebriform cell component, but often requires correlation of immunologic and clinical features.

Synthesis of transforming growth factor-beta 1 by megakaryocytes and its localization to megakaryocyte and platelet alpha-granules.

We have directly demonstrated that megakaryocytes are a major site of synthesis and storage of transforming growth factor-beta 1 (TGF/beta 1) by combined immunohistochemical, immunocytochemical, and in situ hybridization methods. The presence of TGF/beta 1 messenger RNA (mRNA) in mature megakaryocytes in adult rat spleen and bone marrow (BM) was established by in situ hybridization. Localization of TGF/beta 1 protein to intact alpha-granules of megakaryocytes, its putative storage site, was accomplished in glycol-methacrylate embedded porcine BM with an immunoperoxidase technique and light microscopy. The TGF/beta 1 was sequestered in intracytoplasmic granules in a pattern virtually identical to that of another alpha-granule marker protein, fibrinogen. This observation strongly suggests packaging of TGF/beta 1 into this organelle within megakaryocytes. That TGF/beta 1 mRNA was localized to megakaryocytes suggests that the TGF/beta 1 found in the alpha-granules in platelets originates with megakaryocyte synthesis. The alpha-granule localization of TGF/beta 1, as well as fibrinogen, was also demonstrated in isolated platelets at the ultrastructural level by electronmicroscopy (EM) and postembedding colloidal-gold immunocytochemistry, thus directly demonstrating that alpha-granules are the final storage site for TGF/beta 1 in mature platelets.

Clinical correlates of false-negative fine needle aspirations of the breast in a consecutive series of 1,005 patients.

Fine needle aspiration (FNA) of the breast is a useful diagnostic tool in the management of lesions of the breast. However, false-negatives invariably occur and can detract from the usefulness of the technique. The current study of 16 patients with false-negative FNA of the breast from a consecutive series of 1,005 patients was undertaken in an attempt to better understand the clinical correlates most often associated with false-negative diagnoses. Pre-FNA physical examination and mammographic findings were correlated with the gross and microscopic features of these 16 patients. All 16 patients had palpable findings. Mammographic abnormalities were divided into three categories--highly suspicious for malignant tumor (n = 7), indeterminate (n = 3) and negative (n = 4). Mammograms were not available for two patients. The carcinomas ranged in size from 0.8 to 6.5 centimeters (mean of 1.9 centimeter). Thirteen of 16 carcinomas were 2 centimeters or less. Of the small tumors, histologic factors revealed no special type (NST) in six patients and special type carcinoma in seven patients. The notably large tumor (6.5 centimeters) was of high grade and demonstrated an unusual diffusely infiltrative pattern histologically extending between normal mammary lobules. Overall, special type carcinomas comprised seven of 16 patients. All of these carcinomas, as well as six of nine NST were paucicellular, that is, more than 20 percent area containing tumor cells. The current study supports the findings of others that small tumor size, paucicellularity and special type histologic factors contribute to false-negative diagnoses of FNA of the breast.