ICSH guidelines for the standardization of bone marrow immunohistochemistry.

Bone marrow (BM) tissue biopsy evaluation, including trephine biopsy and clot section, is an integral part of BM investigation and is often followed by ancillary studies, in particular immunohistochemistry (IHC). IHC provides in situ coupling of morphological assessment and immunophenotype. The number of different IHC tests that can be applied to BM trephine biopsies and the number of indications for IHC testing is increasing concurrently with the development of flow cytometry and molecular diagnostic methods. An international Working Party for the Standardization of Bone Marrow IHC was formed by the International Council for Standardization in Hematology (ICSH) to prepare a set of guidelines for the standardization of BM IHC based on currently available published evidence and modern understanding of quality assurance principles as applied to IHC in general. The guidelines were discussed at the ICSH General Assemblies and reviewed by an international panel of experts to achieve further consensus and represent further development of the previously published ICSH guidelines for the standardization of BM specimens handling and reports.

Expression of protein gene product 9.5 (PGP9.5)/ubiquitin-C-terminal hydrolase 1 (UCHL-1) in human myeloma cells.

The neuron cytoplasmic protein gene product 9.5 (PGP9.5)/ubiquitin-C-terminal hydrolase 1 (UCHL-1) protein is a thiol protease that recognizes and hydrolyzes a peptide bond at the C-terminal of ubiquitin, and is involved in the processing of ubiquitin precursors and ubiquinated proteins. Although this molecule is known as a specific tissue marker for the neuroendocrine system, many reports have indicated that PGP9.5 is a marker for certain tumour types, such as cancer of the lung, colon, and pancreas. The expression of PGP9.5 in myeloma cells was examined. PGP9.5 seemed to be expressed specifically in myeloma cells as compared with other haematological malignant cells. In addition, in myeloma cells subjected to growth-factor starvation, the upregulation of PGP9.5 was observed in association with that of p27(Kip1), a cyclin-dependent-kinase inhibitor, although the upregulation caused by irradiation was milder. In contrast, the hypoxic culture of myeloma cells induced down-regulation of PGP9.5. These results suggested that PGP9.5 may be a good marker for myeloma among haematological malignancies. In addition, it may indicate certain cellular features of myeloma cells, such as sensitivity to proteasome inhibitors.

Hepatosplenic T cell lymphoma with no expression of cytotoxic molecules.

Hepatosplenic T cell lymphoma is defined as an extranodal and systemic neoplasm derived from cytotoxic T cells. This report describes a postmortem case of T cell lymphoma that showed histological features of hepatosplenic T cell lymphoma but did not express cytotoxic molecules. The patient was a 57 year old man who presented with severe icterus and hepatosplenomegaly, followed by an aggressive clinical course. The liver and spleen were enlarged, weighing 2000 g and 360 g, respectively. Histologically, the liver, spleen, and bone marrow were entirely affected by lymphoma, comprising pleomorphic small and large cells, which displayed sinusoidal infiltration in the liver, diffuse infiltration in the splenic cord, and interstitial/diffuse infiltration with fibrosis in the bone marrow. Lymphoma cells showed positivity for CD3 epsilon, CD8, and CD45RO and clonal rearrangement of the TCRgamma gene by the polymerase chain reaction on paraffin wax embedded sections. However, they were negative for TIA-1 and granzyme B, in addition to betaF1, CD4, and CD56. Few neoplastic cells were stained for Epstein-Barr virus encoded mRNA 1. These findings indicate that this case might represent a variant of hepatosplenic T cell lymphoma despite the absence of cytotoxic molecules.

Post-transplant malignant lymphoma with monoclonal immunoglobulin gene rearrangement and polyclonal Epstein-Barr virus episomes.

This report describes the case of an 8 year old boy who developed ileocecal B cell lymphoma after liver transplantation. The patient underwent orthotopic liver transplantation for biliary atresia and had been given immunosuppressive drugs--cyclosporin A and tacrolimus hydrate. Six years after the liver transplantation, the patient had a sudden onset of fever and abdominal pain. Necropsy revealed an ileocecal mass that was a B cell lymphoma. Epstein-Barr virus (EBV) encoded RNA 1 was demonstrated in lymphoma cells and hyperplastic follicular germinal centre cells in various tissues. Although monoclonal immunoglobulin gene rearrangement was detected in the liver, EBV episomes were of polyclonal origin and lytic forms of EBV were also demonstrated by Southern blotting. Immunohistochemically, lymphoma cells were positive for p53 but negative for latent membrane protein 1 and EBV nuclear antigen 2. These findings suggested that this B cell lymphoma might have occurred sporadically, regardless of EBV infection.

Expression of p53 and Ki-67 antigen in bone marrow giant proerythroblasts associated with human parvovirus B19 infection.

Giant proerythroblasts are hallmarks of human parvovirus B19 infection. We attempted to characterize these cells in 5 patients with parvovirus B19-induced pure red cell aplasia using immunostaining of paraffin-embedded bone marrow sections with antibodies against erythroid-lineage-specific proteins, viral capsid antigen VP-1, and apoptosis- and cell-cycle-related proteins. Giant proerythroblasts are immunohistochemically consistent with early erythroid precursors of cells in the differentiation stage of CD34-, cytoplasmic spectrin+, glycophorin A-, and band-3-. VP-1 was expressed in the nucleus and cytoplasm of small- to medium-sized spectrin+ erythroid cells but not in giant proerythroblasts. The giant proerythroblasts displayed nuclear staining for p53 (41%+/-16%) and Ki-67 antigen (100%+/-0%) and cytoplasmic staining for Bax (65%+/-11%) and procaspase-3 (78%+/-10%), whereas they were not stained for p21Wafl/Cip1, active form of caspase-3, or terminal deoxynucleotidyltransferase-mediated deoxyuridine nick-end labeling (TUNEL). Antiapoptotic proteins, Bcl-2 and Mcl-1, were not expressed in the giant cells, and Bcl-x was infrequently expressed in these cells (11%+/-4%). These immunohistochemical findings suggest that giant proerythroblasts are proliferating erythroid precursors with accumulation of nonfunctional p53.

Cell cycle analysis and expression of cell cycle regulator genes in myeloma cells overexpressing cyclin D1.

Among the recently discovered myeloma-specific gene alterations associated with chromosomal translocations, cyclin D1/PRAD1/Bcl-1 overexpression caused by t(11;14)(q13;q32) is considered to be the most frequent in myeloma patients and cell lines, and may be a prognostic factor clinically. To elucidate the cellular biological role of overexpressed cyclin D1 in myeloma cells, we examined the mRNA expression levels of cell cycle regulators including three cyclin Ds, cyclin-dependent kinase inhibitors (CDK-Is) and accelerators. Cyclin D1 overexpression was clearly demonstrated in the lines with abnormal 11q13 and associated with overexpression of S and G2 accelerator genes. The cyclin D1-overexpressing lines tended to have a shortened G1 phase compared with the non-expressing lines. In addition, artificial silencing using antisense oligonucleotides for cyclin D1 suppressed the growth rate of some but not all cyclin D1-overexpressing cells. These results indicate that overexpression of cyclin D1 caused by cytogenetic abnormalities may make cells progress through the cell cycle rapidly, but it seems that other factors such as cyclin D2 and translocation-related genes affect the cell cycle progression in myeloma cells.

Comparative ultrastructural study of cytotoxic granules in nasal natural killer cell lymphoma, intestinal T-cell lymphoma, and anaplastic large cell lymphoma.

Comparative immunohistochemical and ultrastructural studies were performed on five nasal natural killer (NK) cell lymphoma cases, two intestinal T-cell lymphoma cases, and eight anaplastic large cell lymphoma (ALCL) cases to clarify morphological differences in cytotoxic granules among these cytotoxic lymphomas. Nasal NK-cell lymphomas and intestinal T-cell lymphomas had fine azurophilic granules and displayed dot-like immunostaining of granzyme B- and T-cell intracellular antigen 1 (TIA-1), predominantly in the central area of the cytoplasm. Ultrastructurally, these NK-cell lymphomas and intestinal T-cell lymphomas had two types of cytotoxic granules, type-I granules (dense core granules) and type-II granules (multivesicular bodies), which have been demonstrated in normal large granular lymphocytes in peripheral blood. However, ALCLs did not have azurophilic granules, and only type-II cytotoxic granules were found ultrastructurally, even though they showed similar dot-like immunostained patterns of granzyme B and TIA-1, as seen in NK-cell lymphomas and intestinal T-cell lymphomas. Immunoelectron microscopy revealed that TIA-1 was primarily located at the periphery of the cytoplasmic granules in the NK-cell lymphoma and ALCL cases. These findings suggest that malignant lymphomas with a cytotoxic phenotype can be divided into two types, (azurophilic granule)+, (type-I granule)+, (type-II granule)+ lymphomas and (azurophilic granule)-, (type-I granule)-, (type-II granule)+ lymphomas.

Iatrogenic implantation of malignant meningioma to the abdominal wall.

We report a case of malignant meningioma that occurred in the abdominal operation scar of a 71-year-old woman. This tumor was a 13 x 8 cm gray-tan soft tumor, consisting of multiple nodules. Histologically, tumor cells proliferated in the subcutaneous tissue, displaying mostly a storiform pattern and a focal whorl formation with high mitotic figures. The immunohistochemical positivity for epithelial membrane antigen and negativity for CD34 enabled us to differentiate this tumor from a dermatofibroma protuberance or hemangiopericytoma. The patient had a history of operation for a recurrent orbital lesion of a malignant meningioma that initially developed in the frontal skull base. The present case probably resulted from iatrogenic transplantation of the orbital malignant meningioma to the lower abdominal wall, which had served as a donor site for adipose tissue used to pack the orbital defect.

Impaired splenic erythropoiesis in phlebotomized mice injected with CL2MDP-liposome: an experimental model for studying the role of stromal macrophages in erythropoiesis.

Erythropoiesis occurs in the presence of erythropoietin (EPO) without macrophages in vitro. In hematopoietic tissues, however, erythroid cells associate closely with stromal macrophages, forming erythroblastic islands via interactions with adhesion molecules. To elucidate the role of macrophages in erythropoiesis, we selectively abrogated stromal macrophages of splenic red pulp of phlebotomized mice by injection with dichloromethylene diphosphonate encapsulated in multilamellar liposomes (CL2MDP-liposome). In the spleen, no erythropoietic activity occurred until 5 days after the treatment. Colony assay revealed that the erythropoiesis was suppressed at the level of CFU-E. The splenic erythropoietic activity gradually developed from day 6 after the treatment, when F4/80+ macrophages began to appear in the red pulp. EPO mRNA was expressed in kidney but not in liver or spleen of phlebotomized mice injected with CL2MDP-liposome, and the serum EPO concentration in these mice was higher than that in phlebotomized mice. These findings suggest that abrogation of stromal macrophages by injection with CL2MDP-liposome impairs the splenic microenvironment for erythropoiesis induced by hypoxic stress, and this may be an excellent experimental model for further characterization of the in vivo role of splenic macrophages in erythropoiesis.

Nasal NK/T cell lymphoma presenting as transverse myelopathy.

A case of nasal NK/T cell lymphoma with central nervous system (CNS) involvement is reported. A 56-year-old man presented with eyelid edema and transverse myelopathy. Cerebrospinal fluid examination revealed atypical lymphoid cells with azurophilic granules, which were positive for CD2, CD8, and CD56, and negative for CD3 and CD5 by flow cytometry. Because a tumor mass was found involving the ethmoid and maxillary sinuses, CNS involvement was considered to have resulted from local invasion by the nasal lymphoma. In spite of intensive chemotherapy including intrathecal infusion, the patient died 6 months after the initial diagnosis. Autopsy revealed that lymphoma cells were positive for cytotoxic molecules, granzyme B and TIA-1, and EB virus-encoded RNA-1 (EBER-1), and they showed no rearrangement of TCR-beta, -gamma, or -delta genes, suggesting an NK-cell origin of the lymphoma cells. They showed an angiocentric and angiodestructive pattern in the subarachnoid space, focally extending to the cerebral cortex and cranial and spinal nerve roots. Marked demyelination was found in the lateral and posterior funiculi of the spinal cord. Thus, the pathogenesis of this spinal demyelination might be attributed to ischemia secondary to angiocentric and angiodestructive infiltration by lymphoma cells.

Giant lamellar bodies in pulmonary MALT lymphoma. A case report.

BACKGROUND: Giant lamellar bodies are laminated, scroll-like whorls seen within alveolar spaces and have been occasionally observed in sclerosing hemangioma of the lung. However, to the best of our knowledge, the cytologic findings of giant lamellar bodies have not been reported. We describe cytologic findings of giant lamellar bodies associated with pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma. CASE: A 72-year-old male had a pulmonary mass measuring 2.0 x 1.4 x 1.5 cm. Cytologic smears imprinted from a cut surface of the resected mass revealed a large number of concentrically laminated structures, giant lamellar bodies, measuring 15-40 microns in diameter. Necrotic cellular remnants were occasionally observed in the center of the structures. In the background, small to medium-sized lymphoid cells and plasmacytoid cells were observed. Histologic diagnosis of the tumor was IgG, kappa type, MALT lymphoma. An aggregate of giant lamellar bodies was observed within entrapped, dilated alveolar spaces lined with hypertrophied, type II pneumocytes. Immunohistochemically, the giant lamellar bodies were positive for KL-6. CONCLUSION: Giant lamellar bodies may be derived from surfactant and necrotic type II pneumocytes and may be observed cytologically in cases of pulmonary MALT lymphoma.

Role of the macrophage in erythropoiesis.

Macrophages, which are derived from precursor cells in the bone marrow, differentiate specifically under the influence of the local microenvironment. Resident macrophages in hematopoietic tissues can be distinguished from other stromal cells and monocytes by immunostaining with monoclonal antibody F4/80 and anti-Forssman glycosphingolipid antibody, respectively. Erythroid colony-forming units adhere to a resident macrophage and differentiate to erythroblasts in the presence of erythropoietin (EPO), resulting in the formation of an erythroblastic island. Resident macrophages play a supportive role in erythropoiesis, probably by preventing apoptosis of the erythroid precursors via adhesive interaction between very late activation antigen 4 and vascular cell adhesion molecule 1. Herein is proposed a model of erythropoiesis based on cooperative interaction between EPO and resident macrophages.

Bone marrow involvement in NPM-ALK-positive lymphoma: report of two cases.

Two cases of NPM-ALK-positive anaplastic large cell lymphoma (ALCL) with bone marrow involvement are reported. These cases were recognized within a group of NPM-ALK-positive ALCLs (n = 6) by using immunohistochemistry with the ALK1 monoclonal antibody. In case 1, the bone marrow showed diffuse infiltration of round to spindle-shaped lymphoma cells with moderate fibrosis. In case 2, lymphoma cells intermingling with hematopoietic cells could only be identified by immunohistochemical staining. In contrast to the four NPM-ALK-positive ALCL cases, which showed a cohesive growth pattern in the lymph nodes, the two cases reported here displayed lymphoma cells of smaller size, and they were classified as lymphohistiocytic variants histologically. ALK1 stained small-sized components more clearly than did CD30 (HRS-4). These results suggest that bone marrow involvement of NPM-ALK-positive ALCL may be frequently associated with a histological variant showing a small-sized cell component, and that ALK1 immunostaining is a useful tool to investigate lymphomas for bone marrow involvement.

Immunohistochemical assessment of human bone marrow macrophages in hematologic disorders.

Changes in bone marrow macrophages may be associated with abnormal hematopoiesis in various hematologic disorders. We immunohistochemically evaluated the density of macrophages in bone marrow trephine biopsies. In reactive erythroid hyperplasia (hemolytic anemia and megaloblastic anemia), the macrophages slightly increased in density, extending their cytoplasmic processes between hematopoietic cells. In erythroid hypoplasia (pure red cell aplasia), they became rounded and frequently had hemosiderin granules. There was no significant difference in the macrophage density in the hematopoietic area between erythroid hyperplasia and hypoplasia. The macrophages increased in density in myeloproliferative disorders (polycythemia vera, chronic myelogenous leukemia and primary thrombocythemia). In myelofibrosis, some macrophages became extremely elongated along the line of the fibroblastic cells. In contrast, in conditions in which myelopoietic activity is considerably impaired (aplastic anemia, acute leukemia and multiple myeloma), they significantly decreased in density. These results suggest that the morphologic change in bone marrow macrophages is associated with erythropoietic activity and that there is a correlation between macrophage density and myelopoietic activity.

Basaloid type adenoid cystic carcinoma of the breast.

We report a case of adenoid cystic carcinoma (ACC) of the breast with a prominent basaloid feature. The patient was a 62-year-old Japanese woman with a right breast mass, measuring 1.5 cm in diameter. Histologically, the tumor was composed of basal cell-like tumor cells, and it was originally diagnosed as invasive ductal carcinoma. The presence of PAS-positive basement membrane material around the tumor cell nests may be a diagnostic clue to ACC. The prognosis of ACC of the breast is considered to be favorable. However, basaloid type ACC may represent a poor prognosis, since our case revealed an aggressive behavior in spite of its small size.

[Primary splenic lymphoma with t(3;14)(q27;q32) chromosomal abnormality and rearrangement of BCL-6 gene].

Primary splenic lymphoma (PSL) is extremely rare, accounting for less than 1% of all reported cases of extranodal lymphoma. A 62-year-old woman was referred to our hospital because of general fatigue. A heterogenous mass with irregular margins was detected in the spleen by abdominal computed tomographic scan, and Gallium scintigraphy demonstrated abnormal accumulation only in the spleen. Malignant lymphoma was strongly suspected on the basis of histologic findings from an ultrasonically guided needle biopsy. The final diagnosis was established by splenectomy as PSL of diffuse large B-cell type. After 6 courses of CHOP chemotherapy, the patient recovered and has been disease-free more than a year. Chromosomal analysis of her tumor cells detected t(3;14)(q27;q32), an abnormality not reported in cases of PSL to date. The rearrangement of BCL-6 was also observed. We discuss the possibility of BCL-6 involvement in Japanese cases of PSL, with reference to case reports dating back over the past decade.

Cutaneous granulocytic sarcoma mimicking immunoblastic large cell lymphoma.

A peculiar case of cutaneous granulocytic sarcoma without leukemic manifestation (so-called aleukemic leukemia cutis) that developed in the skin of the back of a 69-year-old man is reported. A skin biopsy specimen showed atypical cells with a prominent nucleolus proliferating around dermal blood vessels and along adnexa without epidermotropism. Atypical cells similar to those of the skin had infiltrated diffusely into the interfollicular area of an inguinal lymph node. Flow cytometric and immunohistochemical studies with a panel of monoclonal antibodies revealed neoplastic cells that had a biphasic phenotype of myeloid and T cell precursors. They expressed CD13, CD15, CD33, lysozyme, CD3epsilon, CD4, CD7 and terminal deoxynucleotidyl transferase (TdT). Gene analysis showed no rearrangement of the immunoglobulin heavy chain or T cell receptor beta and gamma genes. Ultrastructurally, the tumor cells exhibited a few intracytoplasmic electron-dense granules and well-developed rough endoplasmic reticulum with an occasional whorling arrangement. The initial diagnosis was immunoblastic large cell lymphoma, and the patient was treated with six courses of ProMACE-CytaBOM. In spite of the high-grade cytological characteristics of this tumor, the patient has been free of disease for 5 years.

Immunohistochemical identification of erythroid precursors in paraffin embedded bone marrow sections: spectrin is a superior marker to glycophorin.

AIM: To investigate whether spectrin can be used as an immunohistochemical marker for erythroid precursors in routinely processed paraffin embedded bone marrow sections. METHODS: Bone marrow biopsies and clot sections were stained with rabbit antihuman erythrocyte spectrin antibodies, specific for erythroid cells as shown by western blotting and bone marrow smears, and compared to sections stained with antiglycophorin monoclonal antibodies (JC159 and Ret49f). RESULTS: Antispectrin antibodies resulted in diffuse cytoplasmic staining of early erythroblasts and membranous staining of late erythroblasts as well as erythrocytes. In haematopathological samples, immature erythroid cell clusters were clearly identified. In contrast, antiglycophorin monoclonal antibodies resulted in only membranous staining of late erythroblasts, and faint staining of early erythroblasts. CONCLUSIONS: Spectrin may be a superior marker to glycophorin for the identification of erythroid precursors in paraffin embedded sections.

Parvovirus B19-associated transient pure red cell aplasia with lymphadenopathy: a case report.

There have been few reports on lymph node swelling in human parvovirus (HPV) B19 infection. A report of a 42-year-old female, who developed HPV B19-associated transient red cell aplasia with lymphadenopathy, is presented. The lymph node swelling began with the appearance of atypical lymphocytes in the peripheral blood and it disappeared as the patient recovered from the aplasia. Microscopically, the patient's bone marrow showed characteristic giant proerythroblasts with no maturation of the erythroid series. An excised inguinal lymph node showed florid, reactive follicular hyperplasia with paracortex expansion, and neutrophil infiltration and hemophagocytosis in the medullary sinus. These findings were compatible with the histology of a viral infection. A polymerase chain reaction study revealed HPV B19 in her serum and lymph node, but an immunohistochemical study failed to demonstrate HPV B19 capsid antigen in the lymph node or bone marrow. Although the present case suggests that reactive lymphadenopathy is associated with HPV B19 infection, the mechanism of the lymph node swelling still remains to be elucidated.