Hiroshi Hojo

Intergroup Rhabdomyosarcoma Study: Update for Pathologists

ABSTRACT Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, and 75% of such cases in the United States are reviewed at the Pathology Center for the Intergroup Rhabdomyosarcoma Study Group (IRSG). The first four generations of IRSG therapeutic trials (IRS I–IV) and supportive pathologic studies have generated a new International Classification of Rhabdomyosarcoma (ICR) that offers new morphologic concepts to the practicing pathologist. The objective of this report is to clearly define emerging histopathologic categories of RMS as defined by the ICR, and to emphasize correlative immunohistochemical or molecular studies. Emerging ICR variants of RMS place the patient in widely divergent prognostic categories (superior, botryoid or spindle cell variants; poor, solid alveolar or diffusely anaplastic variants). The cardinal histopathologic features of the ICR combined with results of studies of fusion genes seen with t(1;13) and t(2;13) will help delineate therapeutic subgroups of RMS for the fifth generation (IRS V) of IRSG studies. Consequently, it is imperative for the practicing pathologist to be familiar with the practical workup and diagnosis of RMS in childhood.

Pseudosarcomatous myofibroblastic tumor of the urinary bladder in children: a study of 11 cases with review of the literature. An Intergroup Rhabdomyosarcoma Study.

Pseudosarcomatous myofibroblastic tumor (PMT) is the result of reactive proliferation of myofibroblasts. In children, PMT of the urinary bladder can be mistaken for embryonal rhabdomyosarcoma clinically, radiologically, and by light microscopy. We are reporting the clinical, histological, and immunohistological features of 11 patients with childhood PMT of urinary bladder that were diagnosed initially as a sarcoma, usually rhabdomyosarcoma. The morphologic spectrum of PMT is broad, with mixtures of myxoid, leiomyomatous, and sclerosing matrix patterns, the myxoid type being the most common. The proliferating cells consist of three forms of myofibroblastic cells: long spindle cells (type I), intermediate spindle cells (type II), and ganglion-like cells (type III), together with various types of inflammatory cells. The immunohistologic profile of the proliferating cells was characterized by positive reactions to vimentin, muscle-specific actin, alpha-smooth-muscle actin, polyclonal desmin, and keratin. Ultrastructural studies showed myofibroblastic differentiation of the tumor cells. No patients have had metastases or local recurrence. Histologic, immunohistochemical, and clinical data from 71 cases of PMT, including the 11 cases in this report, confirm the benign behavior of these lesions. The etiology of these lesions is unclear, including the absence of surgical or other trauma in all of the children.

Tumor-associated macrophages correlate with vascular space invasion and myometrial invasion in endometrial carcinoma.

OBJECTIVE This study was conducted to determine whether tumor-associated macrophages (TAMs) correlate with clinicopathological features in endometrioid adenocarcinoma. METHODS 76 cases of endometrioid adenocarcinoma treated initially by hysterectomy with pelvic lymphadenectomy were retrospectively retrieved, and their histological features were evaluated. Immunohistochemical staining for CD68, CD34, and Ki-67 was performed on paraffin-embedded sections. TAMs were counted in two areas: in the invasive margin (margin TAMs) and in the tumor (intratumor TAMs). RESULTS Margin TAMs were significantly associated with FIGO stage (P=0.033), histological grade (P=0.008), myometrial invasion (P=0.0001), pelvic lymph node metastasis (P=0.027), and vascular space invasion (P=0.0001). Intratumor TAMs were significantly associated with intratumor Ki-67 (P=0.006) and microvessel density (P=0.020). Patients with high margin TAMs (> or = 20) had significantly worse progression-free survival (PS) and overall survival (OS) than those with low margin TAMs (< 20) (log rank test, P=0.0031 and P=0.0085, respectively). On multivariate analysis, high margin TAMs were significantly associated with vascular space invasion (P=0.013; HR, 6.05; 95% confidence interval [CI], 1.468-24.938) and myometrial invasion (P=0.041; HR, 4.03; 95% CI, 1.06-14.71). Vascular space invasion was only associated with PFS. CONCLUSION Although on univariate analysis TAMs are associated with other poor prognosticators, on a multivariate analysis, TAMs appear only to be associated with MI and VI. TAMs may play a significant role in the biology of tumor progression of endometrial adenocarcinoma, but do not appear to be independent prognostic indicators of patients survival.

Rhabdoid papillary meningioma

We have studied an uncommon case of rhabdoid papillary meningioma in a 15-year-old boy with a dura-based mass arising in the left posterior fossa. The patient exhibited prominent extracranial extension during the past 6 years, consisting of a mixture of both perivascular pseudopapillary growth and rhabdoid cytologic features of neoplastic meningothelial cells. The meningothelial features were evidenced by the focal whorl formation of tumor cells, coexpression of epithelial membrane antigen and vimentin, and ultrastructural findings of interdigitated cytoplasmic process and intercellular junction. However, the regional and histologic resemblances to ependymoma were further complicated by unexpected focal expression of glial fibrillary acidic protein, neurofilament, and &agr;-smooth muscle actin of the tumor cells. The rhabdoid morphology was characterized by sheets of tumor cells with eccentric nuclei and abundant eosinophilic cytoplasm with often recognizable intracytoplasmic hyaline inclusions. These inclusions revealed ultrastructural paranuclear whorls of intermediate filaments, ruling out the other forms of intracytoplasmic eosinophilic inclusions resembling rhabdoid morphology. Diagnosis of an unusual rhabdoid papillary meningioma with aggressive behavior is resoluble by immunohistochemical and ultrastructural analyses.

Analysis of the immunoglobulin heavy chain gene variable region of 101 cases with peripheral B cell neoplasms and B cell chronic lymphocytic leukemia in the japanese population.

We have analyzed the immunoglobulin heavy chain (VH) gene variable regions (CDR2 and FW3) of 101 Japanese cases with peripheral B cell neoplasms. When all except one case with a deletion were graphed by frequency of replacement mutation, the 100 cases could be separated into two groups: 24 cases with zero, one and two mutations (germline or low frequency of somatic mutation); and 76 cases with three or more mutations (medium to high frequency of somatic mutation). While most mantle cell lymphoma cases (11/13) showed germline or low frequency of somatic mutation, all cases of mucosa‐associated lymphoid tissue (MALT) lymphoma (11/11), follicular lymphoma (three of three cases), plasma cell myeloma (seven of seven cases) and most cases of diffuse large B cell lymphoma (DLBCL; 42/47) belonged to the latter group. These 76 cases, therefore, may be considered to show somatic hypermutation. More than half of chronic lymphocytic leukemia/small lymphocytic lymphoma cases (CLL/SLL; eight of 13) showed a hypermutated VH gene and the ratio of replacement mutation : silent mutation in CDR2 of CLL/SLL was considerably higher compared with DLBCL and MALT lymphoma, showing somatic hypermutation. When comparing VH gene type of B cell‐CLL (B‐CLL) among our series and those in the literature, more cases of CD5+ B‐CLL in the Western literature have the VH5 and VH6 family types, while more cases in Japan are reported to have VH4 family. The occurrence of VH families in B‐CLL between Japanese and Western people seems to be comparable.

Characterization of expression of protein kinase C isozymes in human B-cell lymphoma: Relationship between its expression and prognosis

Protein kinase C (PKC) enzymes play a major role in signal transduction and contribute to the regulation of cellular differentiation and proliferation. However, little is known about subtype‐specific intracellular expression of PKC in human malignant lymphoma. To characterize the relationship between expression of PKC and B‐cell lymphomas based on the different subspecies, we investigated the expression of four subspecies (α, βII, γ and δ) in five cases of reactive lymphoid tissues, 77 cases of human B‐cell lymphoma and 17 human lymphoma cell lines. In the reactive lymphoid tissues, PKC βII‐positive cells were found in the mantle zones and marginal zones, and centroblasts and centrocytes in the germinal centers showed cytoplasmic staining with strong intensity against PKC δ. The present study is the first report to examine the expression of PKC δ in reactive lymphoid tissues. In interfollicular areas, a small number of T‐cells were positive for PKC α. Protein kinase C γ‐positive cells were not found in these lymphoid tissues. Eight cases of Burkitt lymphoma (BL) (8/10; 80%) showed the overexpression of PKC α (P < 0.01), but other B‐cell lymphoma cases except three cases of diffuse large B‐cell lymphoma did not express PKC α. In addition, six and eight out of nine BL cell lines expressed the protein and mRNA of PKC α, respectively. These results indicate that PKC α was predominantly expressed on BL in comparison with other types of lymphoma. The expression of PKC γ was observed in only five cases of BL. The overall survival of PKC γ‐positive BL was significantly better than that of PKC γ‐negative BL (P < 0.05). The expression of PKC γ seems to be associated with a better prognosis in the limited number of BL cases in the present study.

CYTOLOGICAL CHARACTERS, TRANSFORMATION, AND ONTOGENESIS OF DERMAL HISTIOCYTES AND FIBROBLASTS OF RATS

Cell morphology of histiocytes and fibroblasts was observed light‐ and electron microscopically in the subcutaneous connective tissue of unstimulated adult rats. Striking ultrastructural differences between them were noted in the cell surface characters, extent and development of endoplasmic reticula, especially of rough‐surfaced ones, and volumes of lysosomal components. Rosetting assays revealed that histiocytes were positive for EA‐ and EAC‐rosette formation and capable of engaging in immunophagocytosis. However, fibroblasts were negative for both rosettings. In the experiments of vital staining with lithium carmine, Fesin phagocytosis, carrageenin granuloma formation, in vivo culture by intraperitoneal implantation of difision chambers, and subculture, cytological changes of histiocytes or macrophages and fibroblasts, as well as their transformation, were investigated. Although fibroblasts often simulated histiocytes under certain stimulated conditions, no transformation of fibroblasts into histiocytes was confirmed. Ontogenetically, histiocytes already existed in the subepidermal mesenchyme of rat fetuses before differentiation of undifferentiated mesenchymal cells into fibroblasts and showed no relationship to the flbroblasts. Ultrastructural cell morphology and cultural characteristics of the fetal histiocytes were presented and their possible origin was discussed briefly.

A histogenesis of malignant lymphoma, small cleaved cell of the B cell type and intermediate lymphocytic lymphoma (mantle zone lymphoma). An immuno- and enzymehistochemical study.

We have studied the histogenesis of malignant lymphoma (ML), small cleaved cell of the B-cell type and intermediate lymphocytic lymphoma (mantle zone lymphoma) by comparing immunophenotypes and ALP-activity of neoplastic cells with those of germinal center cells (follicular center cells) anti mantle zone (MZ) cells of secondary follicles in non-neoplastic lymphoid tissues. The neoplastic cells in 3 cases of ML, follicular, small cleaved cell and 1 case of ML, small cleaved cell expressed the phenotypes similar to those of germinal center (GC) B lymphocytes (SIgM+, B1+, B2+, CALLA+, SigD−, IL-2R−, Leu-1− and ALP−). The neoplastic cells in 2 cases of ML, follicular, small cleaved cell and 12 cases of ML, diffuse, small cleaved cell displayed the characteristic phenotypes of MZ B lymphocytes (SIgM+, SIgD+, BA-1+, IL-2R+, Leu-1+ and ALP+). The phenotypes of 2 cases of mantle zone lymphoma were closely comparable with those of MZ B lymphocytes. These findings indicate that the histogenesis of ML, small cleaved cell of the B-cell type is heterogeneous and can be divided phenotypically into 2 types (GC B lymphocyte origin and MZ B lymphocyte origin). It is also apparent that intermediate lymphocytic lymphoma (mantle zone lymphoma) is derived from MZ B lymphocytes of secondary follicles.

Identification and Metastatic Potential of Tumor-Initiating Cells in Malignant Rhabdoid Tumor of the Kidney

Purpose: Malignant rhabdoid tumor of the kidney (MRTK) is a rare and highly aggressive malignancy of infanthood. In an effort to delineate MRTK progression, we investigated the metastatic fate of some MRTK cells using xenotransplantation animal models and the tumor-initiating potential of CD133+ MRTK cells. Experimental Design: We established two MRTK cell lines (JMU-RTK-1 and JMU-RTK-2) from patients with MRTK. We generated five luciferase-expressing MRTK cells for in vivo luminescent imaging and evaluated the metastatic fate in an orthotopic xenotransplantation model. Capacities of MRTK-initiating cells were examined in nonobese diabetic/severe combined immunodeficient mice after antibody-mediated magnetic bead sorting. Use of chemokine receptor CXCR4 expression as a metastatic marker was evaluated by flow cytometry and Western blotting. Results: MRTK cell lines showed distant organ metastasis. JMU-RTK-1, JMU-RTK-2, and G401 cells showed considerable aggressiveness compared with SWT-1 and SWT-2 cells (P < 0.05). Moreover, as few as 1,000 CD133+ MRTK cells initiated tumor development in nonobese diabetic/severe combined immunodeficient mice by 21 days (60-100%) in all examined cell lines, although the same number of CD133− MRTK cells could not form tumors (0%). Interestingly, the metastatic potential of the CD133+ population remained unaffected compared with a nonenriched population. The potential metastatic marker CXCR4 was expressed in CD133+ and CD133− MRTK cells, and CD133− cells seemed to play a cooperative role in terms of tumorigenicity and metastasis. Conclusions: These results suggest that CD133+ cells may determine the metastatic fate of MRTK cells and that CD133− cells may play an auxiliary role in tumor progression and metastasis.

KIT expression in normal and neoplastic renal tissues : Immunohistochemical and molecular genetic analysis

Renal chromophobe cell carcinomas (ChCC) and oncocytomas express KIT. This character seems to reflect their common histogenesis from distal nephrons. In the normal kidney, however, the expression and localization of KIT are unclear. KIT expression in angiomyolipoma and congenital mesoblastic nephroma (CMN), is still controversial. c‐kit mutations are reportedly rare in ChCC, but there is little information in other renal neoplasms, and no reported data on mutations of platelet‐derived growth factor receptor (PDGFR). In order to address these issues the authors examined five ChCC, five oncocytomas, seven papillary cell carcinomas, two collecting duct carcinomas, 12 angiomyolipomas, and three CMN, as well as 10 normal renal tissues. In the normal kidney KIT was specifically expressed in the distal nephrons. Nine of 12 (75%) angiomyolipomas contained scattered KIT‐positive cells, whereas all three CMN were completely negative for KIT. The presence of KIT‐positive cells in angiomyolipomas was likely to correspond to that of melanocytic marker‐positive cells, which mainly showed epithelioid morphology. Polymerase chain reaction‐single‐strand conformation polymorphism showed no evidence of mutations of c‐kit or PDGFR in any of the tumors examined.