Wei-Sing Chu

Splenic Inflammatory Myofibroblastic Tumor (Inflammatory Pseudotumor) A Clinicopathologic and Immunophenotypic Study of 12 Cases

CONTEXT Inflammatory pseudotumor is an uncommon and enigmatic lesion. The spindle cells found in this tumor have features of myofibroblasts. Because of the indefinite relationship of these lesions with inflammatory fibrosarcoma and their indefinite biologic behavior, inflammatory pseudotumor is currently classified as inflammatory myofibroblastic tumor (IMT). To date, only case reports or small series have been published on these tumors, which are primary in the spleen. DESIGN In this study, we describe the clinical, morphologic, and immunophenotypic findings of 12 cases of splenic IMT and examine their relationship to Epstein-Barr virus (EBV). RESULTS The patients included 8 women and 3 men, ranging from 19 to 77 years of age (mean, 53 years; median, 60 years). Demographic data were unavailable for 1 patient. Patients generally presented with abdominal pain (n = 5) and fever (n = 4). Associated lesions included renal cell carcinoma (n = 2), colonic adenocarcinoma (n = 1), and cholecystitis (n = 1). All tumors were composed of a bland spindle cell proliferation in association with a variable mixed inflammatory component. There were 2 growth patterns, namely, a cellular spindle cell pattern and a hypocellular fibrous pattern. An immunohistochemical panel confirmed the myofibroblastic nature of the spindle cells. The spindle cells of 2 cases were immunoreactive for EBV latent membrane protein 1, whereas 6 of 10 cases were positive for EBV-encoded RNA using in situ hybridization. Follow-up was available for 8 patients; 6 were alive with no evidence of recurrence and 2 were dead of other causes. CONCLUSION Splenic IMTs are uncommon lesions that can be distinguished from other conditions using a combination of clinical, histologic, and immunophenotypic findings. Epstein-Barr virus may play a role in the pathogenesis of splenic IMT, and there may be an association of splenic IMT with concomitant disease or malignancy. Most splenic IMTs have an excellent long-term prognosis.

Differential Expression of Cyclin D1 in Mantle Cell Lymphoma and Other Non-Hodgkin's Lymphomas

Mantle-cell lymphomas are associated with a characteristic chromosomal translocation, t(11;14)(q13;q32). This translocation involves rearrangement of the bcl-1 proto-oncogene from chromosome 11 to the immunoglobulin heavy chain gene on chromosome 14, resulting in an overexpression of cyclin D1 mRNA (also known as bcl-1 and PRAD1). In the current study performed on paraffin-embedded tissue, cyclin D1 mRNA could be detected in 23 of 24 mantle-cell lymphomas by reverse transcription polymerase chain reaction (RT-PCR) whereas only 9 of 24 demonstrated a t(11;14) by PCR. However, we also found that cyclin D1 mRNA could be detected in the majority (11 of 17, 65%) of non-mantle-cell lymphomas and in a minority of atypical lymphoid hyperplasias (3 of 7, 43%). Cyclin D1 mRNA expression was not observed in floridly reactive lymph nodes (0 of 9) or in unstimulated lymph nodes (0 of 20), suggesting that it is a sensitive adjunct marker for malignant lymphoproliferative processes, but not specific for mantle-cell lymphoma. A semiquantitative RT-PCR assay was developed that compared the ratio of cyclin D1 to the constitutively expressed gene beta2-microglobulin. Using this assay on a limited number of our specimens, cyclin D1 overexpression in mantle-cell lymphoma could be reliably distinguished from its expression in other non-Hodgkins lymphomas. This assay for cyclin D1 expression, designed for formalin-fixed, paraffin-embedded tissue, was a very sensitive and specific marker for mantle-cell lymphoma.

A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section

In clinical practice, molecular analysis of tumor specimens is often restricted by available technology for sample preparation. Virtually all current methods require homogenization of tissues for molecule extraction. We have developed a simple, rapid, nondestructive molecule extraction (NDME) method to extract proteins and nucleic acids directly from a single fixed or frozen tissue section without destroying the tissue morphology. The NDME method is based upon exposure of micron-thick tissue section to extraction buffer with the help of heating and/or intact physical forces (ultrasound and microwave) to facilitate release of macromolecules into the buffer. The extracted proteins and nucleic acids can be used directly without further purification for downstream SDS-PAGE analysis, immunoblotting, protein array, mass spectra protein profiling, PCR, and RT-PCR reactions. Most importantly, the NDME procedure also serves as an antigen retrieval treatment, so that after NDME, the same tissue section can be used for histopathological analyses, such as H&E staining, immunohistochemistry, and in situ hybridization. Thus, the NDME method allows, for the first time, both histological diagnosis and molecular analysis on a single tissue section, whether it is from frozen or fixed tissue specimens.

c-Kit (CD117) Reactivity in Extramedullary Myeloid Tumor/Granulocytic Sarcoma

CONTEXT c-kit, a proto-oncogene, encodes the transmembrane tyrosine kinase receptor CD117 and is detected by flow cytometry in the majority of cases of acute myeloid leukemia. The prognostic significance of the presence of c-Kit in acute myeloid leukemia is debated. Recently, c-kit inhibitors have been studied as possible therapies against hematopoietic malignancies; therefore, c-Kit detection may have important implications for treatment. OBJECTIVES In this study, we investigated the expression of c-Kit in granulocytic sarcoma (GS) using paraffin-embedded tissue. DESIGN Routinely formalin-fixed, paraffin-embedded tissues from 30 cases of GS were studied using immunohistochemistry. c-Kit (C-19) (a polyclonal antibody against carboxy terminal domain of c-Kit p145 or CD117) reactivity was compared with myeloperoxidase and lysozyme. The immunohistochemical panel also included CD34, CD68, CD43, Bcl-2, CD45RB, CD20, CD3, CD10, terminal deoxynucleotidyl transferase (TdT), and CD79a. RESULTS The morphologic patterns included well-differentiated (5 cases), poorly differentiated (19 cases), and blastic forms (6 cases). Clinical data were obtained from 28 of 30 patients. Granulocytic sarcoma presented in lymph nodes in 10 cases, whereas in 20 cases it presented in extranodal sites. c-Kit reactivity was found in 87% (26/30) of the GS cases. There was no significant difference in c-Kit positivity between the nodal (90%, 9/10) and extranodal (85%, 17/20) neoplasms. c-Kit expression was not associated with the degree of the myeloid maturation. Two of 13 lymphoblastic lymphoma control cases and 1 of 28 of the large B-cell lymphomas were weakly immunoreactive with c-Kit. CONCLUSIONS c-Kit reactivity can be demonstrated in a high percentage of GS cases; its presence may be useful not only in diagnosis, but also in the treatment of GS with new modalities.

T-cell lymphoma presenting in the breast: a histologic, immunophenotypic and molecular genetic study of four cases.

Primary non-Hodgkins lymphoma of the breast is uncommon. Most primary breast lymphomas are of B-cell phenotype, with only rare cases showing a T-cell phenotype. In this study, we report the clinicopathologic features of four cases of T-cell lymphoma in the breast. The patients all were female with a mean age of 48 years (range, 13 to 77 years). All cases showed immunoreactivity in paraffin-embedded tissue for T-cell markers CD3, CD45RO, and CD43. βF1 was positive in three of four cases. The four cases were further subclassified as anaplastic large cell lymphoma (CD30 positive) of T-immunophenotype; natural killer/T-cell lymphoma; peripheral T-cell (CD4 positive), large cell type; and peripheral T-cell (CD8 positive, T-cell intracellular antigen positive), medium cell type. Three of the four cases were monoclonal for T-cell receptor β and/or T-cell receptor γ. The one case of natural killer/T-cell lymphoma was negative for monoclonality with both T-cell receptor β and γ by molecular diagnostic studies. In all cases, IgH was negative. Follow-up was obtained in three cases. Two patients died within less than 1 year after the diagnosis. The third patient died within 18 months of the diagnosis. Our results suggest an aggressive clinical course for T-cell lymphomas that present in the breast.

Ultrasound-accelerated formalin fixation of tissue improves morphology, antigen and mRNA preservation

Formalin fixation and paraffin embedding are conventional tissue preservation and processing methods used for histologic diagnosis in over 90% of cases. However, formalin fixation has three disadvantages: (1) slow fixation (16–24 h) hinders intraoperative decision making, (2) slow quenching of enzymatic activity causes RNA degradation, and (3) extensive molecule modification affects protein antigenicity. Applying high-frequency, high-intensity ultrasound to the formalin fixative cuts fixation time to 5–15 min. Fixation of various tissues such as lymph node, brain, breast, and prostate suggests that, compared to the conventional method, implementation of ultrasound retains superior and more uniform tissue morphology preservation. Less protein antigenicity is altered so that rapid immunohistochemical reactions occur with higher sensitivity and intensity, reducing the need for antigen retrieval pretreatment. Better RNA preservation results in stronger signals in in situ hybridization and longer RNA fragments extracted from fixed tissues, probably due to rapid inhibition of endogenous RNase activity. Molecules extracted from ultrasound-fixed tissues are of greater integrity and quantity compared to conventionally fixed tissues, and thus better support downstream molecular analyses. Overall, ultrasound-facilitated tissue preservation can provide rapid and improved morphological and molecular preservation to better accommodate both traditional and molecular diagnoses.

Apoptosis, bcl-2 expression, and p53 expression in gastrointestinal stromal/smooth muscle tumors.

The authors investigated the relations between outcome and apoptosis, immunohistochemical demonstration of bcl-2 protein, and immunohistochemical staining for p53 protein in patients with gastrointestinal stromal/smooth muscle tumors (GIST). Patients whose tumors demonstrated cellular apoptosis using the terminal deoxynucleotidyl transferase-mediated 2´-deoxyuridine 5´-triphosphate-biotin nick end labeling (TUNEL) assay had an improved survival over those whose tumors did not improve. In contrast, patients whose tumors demonstrated staining for bcl-2 protein had a decreased survival compared with those whose tumors did not demonstrate bcl-2. There was no relation between p53 immunoreactivity and survival. These results suggest that inhibition of apoptosis may be associated with malignant behavior in patients with gastrointestinal stromal/smooth muscle tumors.

Antiapoptotic marker, Bcl-XL, expression on Reed-Sternberg cells of Hodgkin's disease using a novel monoclonal marker, YTH-2H12☆

Inhibitors of apoptosis may regulate tissue differentiation and promote cell survival in neoplasia. A new apoptosis inhibitor of the bcl-2 gene family, bcl-X(L), was recently found in some types human neoplasia but not in normal tissue. We investigated bcl-X(L) expression in 419 cases of normal and neoplastic lymphoid lesions using immunohistochemistry with the monoclonal antibody bcl-X(L) (YTH-2H12). Ninety-four percent (141/150) of classic Hodgkins disease (HD) were positive for bcl-X(L) with strong intensity in most Reed-Sternberg (RS) cells. Forty-eight percent (38/80) of nodular lymphocyte predominance (LPHD) were positive. In the non-Hodgkins lymphomas (NHL), bcl-X(L) was expressed in a low percentage of cases (< 20%), with the exception of follicle center lymphoma, grade III/III (78%). All reactive hyperplastic lesions were negative for bcl-X(L). RS cells, which expressed bcl-X(L), were not labeled by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). We found RS cells expressing bcl-X(L) were absent of DNA fragmentation (apoptosis). Our data provide evidence that bcl-X(L) is abnormally expressed in the RS cells of HD and some types of NHL raising speculation that inhibition of apoptosis may be important in the pathogenesis of lymphoma, specifically HD. In addition, the previously reported correlation between bcl-X(L) and Epstein-Barr virus expression in HD was not supported by this study.

Ultrasound-accelerated Tissue Fixation/Processing Achieves Superior Morphology and Macromolecule Integrity with Storage Stability

We demonstrate that high-frequency and high-intensity ultrasound (US) can be applied to both tissue fixation and tissue processing to complete the conventional overnight formalin-fixation and paraffin-embedding (FFPE) procedures within 1 hr. US-facilitated FFPE retains superior tissue morphology and long-term room temperature storage stability than conventional FFPE. There is less alteration of protein antigenicity after US-FFPE preservation so that rapid immunohistochemical reactions occur with higher sensitivity and intensity, reducing the need for antigen retrieval pretreatment. US-FFPE tissues present storage stability so that room temperature storage up to 7 years does not significantly affect tissue morphology, protein antigenic properties, RNA distribution, localization, and quantitation. In addition, during fixation, tissue displays physical changes that can be monitored and reflected as changes in transmission US signals. As far as we know, this is the first effort to monitor tissue physical changes during fixation. Further study of this phenomenon may provide a method to control and to monitor the level of fixation for quality controls. The mechanism of less alteration of protein antigenicity by US-FFPE was discussed. (J Histochem Cytochem 54:503-513, 2006)