Vinod B. Shidham

Evaluation of micrometastases in sentinel lymph nodes of cutaneous melanoma: Higher diagnostic accuracy with Melan-A and MART-1 compared with S-100 protein and HMB-45

Accurate diagnosis of micrometastases in sentinel lymph nodes of cutaneous melanoma is critical for proper clinical management. S-100 protein and HMB-45 are the traditional immunomarkers widely used for this purpose. However, the interpretation of micrometastases by these markers is difficult with significant reduction in the diagnostic accuracy. S-100 protein demonstrates immunoreactivity for other nonmelanoma cells and obscures nuclear details, which are crucial for the interpretation of single cell metastases. We compared the new melanoma markers, Melan-A (clone A103) and MART-1 (clone M2–7C10), with S-100 protein and HMB-45, by examining 77 formalin-fixed paraffin-embedded sections of sentinel lymph nodes from 13 cases of primary cutaneous melanoma. CD68 (PG-M1) and hematoxylin–eosin-stained sections were also studied. Four pathologists interpreted the staining pattern after concealing the identity of each immunomarker. Az values (area under receiver operating characteristic curve) with receiver operating characteristic curve were higher with Melan-A (0.9742) and MART-1 (0.9779) compared with S-100 protein (0.8034) and HMB-45 (0.8651), demonstrating a higher diagnostic accuracy with Melan-A and MART-1 with superior detection of melanoma micrometastases. Melan-A and MART-1 showed sharp cytoplasmic immunoreactivity, almost exclusively restricted to the melanoma cells. Therefore, Melan-A and MART-1 are recommended for the evaluation of micrometastases in sentinel lymph nodes of cutaneous melanoma as a routine alternative to S-100 protein and HMB-45.

Immunohistochemical comparison of gastrointestinal stromal tumor and solitary fibrous tumor

CONTEXT The differential diagnosis of gastrointestinal stromal tumors (GIST) and solitary fibrous tumors (SFT) may be a diagnostic challenging because of overlapping clinicopathologic features. Many studies have shown consistent immunoreactivity for CD117 (c-Kit) in GIST. However, only a few studies have evaluated CD117 expression in SFT, and these studies have used an antibody from Santa Cruz Biotechnology. In non-GIST lesions, reactivity with this antibody has been shown to differ from that with a CD117 antibody from Dako Corporation. The immunoreactivity of SFT with the Dako CD117 antibody has not been reported. Conversely, CD99 is a marker for SFT, and its expression in GIST has not been evaluated. OBJECTIVE To study the immunohistochemical profiles of GIST and SFT to evaluate their diagnostic overlap. DESIGN We studied the immunoreactivity of 27 unequivocal GIST and 19 unequivocal extra-abdominal SFT for CD117, CD34, CD99, alpha-smooth muscle actin, vimentin, CD31, S100 protein, and muscle-specific actin. All antibodies, including CD117, were from Dako Corporation. RESULTS We found positive immunoreactivity for CD117 in 100% of GIST and none of SFT; for CD34 in 89% of GIST, and 100% of SFT; for CD99 in 89% of GIST and 100% of SFT; for alpha-smooth muscle actin in 48% of GIST and 31% of SFT; for vimentin in 89% of GIST and 90% of SFT; and for muscle-specific actin in 22% of GIST and none of SFT. None of the GIST or SFT showed immunoreactivity for CD31 and S100 protein. CONCLUSIONS The major difference between GIST and SFT was strong CD117 immunoexpression in all GIST and an absence of this expression in all SFT. With the exception of muscle-specific actin, the prevalence of immunoreactivity for the markers studied did not differ substantially between these 2 tumors. We conclude that GIST and SFT show distinctly divergent immunoprofiles with respect to CD117 and muscle-specific actin.

The role of constitutive NF-κB activity in PC-3 human prostate cancer cell invasive behavior

The purpose of this study was to determine if increased NF-κB activity of highly invasive PC-3 cells contributed to their invasive behavior. Increased NF-κB activity has been observed in several malignant tumors and it may have an important role in tumorigenesis, progression and chemotherapy resistance. By serial selection, we obtained invasion variant PC-3 cell sublines. The PC-3 High Invasive cells invade readily through a Matrigel® reconstituted basement membrane while PC-3 Low Invasive cells have low baseline invasion activity. In these studies, we discovered that NF-κB DNA binding activity was increased in PC-3 High Invasive cells when compared to PC-3 Low Invasive cells by electrophoretic mobility shift assay (EMSA). Gel supershift assays showed a 4-fold increase in p65 containing complexes and a 2.2-fold increase in the p50 containing complexes in the PC-3 High Invasive cells. Luciferase reporter assays showed that NF-κB dependent transcription activity was increased 10.2±2.5-fold in the highly invasive cells (P<0.002). The PC-3 High Invasive cells showed a constitutive increase in phospho-IκBα and introduction of the super-repressor IκBα S32/36A inhibited NF-κB activity to 19.2±2.5 percent of control transfected cells (P≤0.001). The IκBα super-repressor reduced the basement membrane invasion of PC-3 High Invasive cells from 6.2±1.1 to 3.8±0.4 percent (P<0.002) with no decrease in cell viability or proliferation. These results demonstrate that increased NF-κB activity contributed directly to the invasive behavior of PC-3 High Invasive prostate cancer cells.

Immunophenotypic profile of myeloid cells in granulocytic sarcoma by immunohistochemistry. Correlation with blast differentiation in bone marrow.

The present study was designed to evaluate the lineage differentiation (particularly monocytic differentiation) of immature myeloid cells in granulocytic sarcoma (GS) by immunohistochemistry and correlate the results with lineage differentiation of blasts in the bone marrow and to determine the degree of maturation of the infiltrating myeloid cells in GS by immunohistochemistry using CD34 and HLA-DR. Immunohistochemical stains were performed on paraffin-embedded tissue from 17 GS lesions with lineage-associated markers: myeloperoxidase, CD68 (KP1), CD68 (PG-Ml), glycophorin A, factor VIII, and CD56; and with markers for blasts and immature myeloid cells: CD34 and HLA-DR. Our results show that positive staining with PG-M1, but not KP1, suggests monocytic differentiation of myeloid cells in GS and correlates with the monocytic differentiation of blasts in the bone marrow. Expression of CD56 is frequent in GS, especially when the marrow blasts have monocytic differentiation, and should not be interpreted as a primary natural-killer cell process. The immature myeloid cells in GS are frequently HLA-DR positive. However, CD34 positivity of the immature myeloid cells is relatively uncommon, except in cases with underlying myelodysplastic syndrome or chronic myelogenous leukemia.

Primary bilateral mucosa-associated lymphoid tissue lymphoma of the breast with atypical ductal hyperplasia and localized amyloidosis. A case report and review of the literature.

Primary non-Hodgkin lymphoma of the breast is a rare disease. Primary mucosa-associated lymphoid tissue lymphoma is even rarer, and bilateral involvement is exceptional. We describe a case of primary bilateral breast mucosa-associated lymphoid tissue lymphoma with bilateral atypical ductal hyperplasia and bilateral localized amyloidosis in a 64-year-old woman with a history of arthritis and systemic lupus erythematosus and its clinical, histologic, and immunohistochemical features. Microscopic examination of the breast lesion showed dense periductal and perilobular small and plasmacytoid lymphocytes with eosinophilic amyloid in the vessels and the stroma. Bilateral single foci of atypical ductal hyperplasia were also noted. Fine needle aspiration showed small and large lymphocytes and plasma cells. Molecular analysis demonstrated a heavy chain immunoglobulin H gene rearrangement. Flow cytometry studies showed an abnormal B-cell population. The combined histologic, paraffin immunohistochemistry, flow cytometry, and molecular results were considered diagnostic for low-grade mucosa-associated lymphoid tissue lymphoma. The patient underwent bilateral local breast radiation without other organ or site involvement.

Colon biopsies for evaluation of acute graft-versus-host disease (A-GVHD) in allogeneic bone marrow transplant patients

BackgroundProper histomorphological interpretation of intestinal acute graft versus host disease (A-GVHD) associated with allogeneic bone marrow transplantation (BMT) is critical for clinical managaement. However, studies methodically evaluating different histomorphological features of A-GVHD are rare.MethodsColonic biopsies from 44 allogeneic BMT patients having biopsy-proven cutaneous A-GVHD were compared with colon biopsies from 48 negative controls.ResultsA-GVHD showed intra-cryptal apoptosis in 91% and pericryptal apoptosis in adjacent lamina propria in 70% (p < 0.002). Nonspecific apoptosis along the surface epithelium was observed in all groups with comparable frequency. The number of apoptotic cells in mucosa were approximately four times (5.3 per 10 HPF) the negative controls (p < 0.002) in A-GVHD group. 48% of cases with A-GVHD showed decreased number of lymphocytes in lamina propria. Some features, including intraepithelial lymphocytes in surface or crypt epithelium; and neutrophils, eosinophils, and edema in lamina propria, did not demonstrate significant difference in A-GVHD and negative controls. Pericryptal apoptosis, dilated crypts, irregular distribution of crypts, decreased lymphocytes, increased microvessel network, focal fibrosis, presence of muciphages, reactive changes in surface epithelium with mucin depletion, mucosal ulceration, and/or reduced mucosal thickness showed higher association with A-GVHD group.ConclusionsIntracyptal apoptosis is a reliable indicator of A-GVHD. Its diagnostic significance was improved if intracyptal apoptosis was associated with features which were observed more frequently in A-GVHD group as mentioned above.

Evaluation of crystals in formalin-fixed, paraffin-embedded tissue sections for the differential diagnosis of pseudogout, gout, and tumoral calcinosis.

Hematoxylin-eosin (H&E)–stained sections may not allow proper evaluation of birefringence properties of the crystals in the lesions of pseudogout, gout, and tumoral calcinosis. This study was undertaken to verify the application of a special stain that could facilitate the evaluation of the birefringence properties of these crystals for definitive diagnosis. We evaluated previously described nonaqueous alcoholic eosin staining (NAES) method based on the principle of using alcoholic eosin without hematoxylin and any other aqueous reagents for staining of formalin-fixed, paraffin-embedded tissue sections. Two observers, in a blinded fashion, evaluated the sections stained with routine H&E and NEAS method without the knowledge about clinical diagnosis. All pseudogout (nine sections from seven cases) and gout (eight sections from five cases) lesions demonstrated birefringence in the sections stained with NAES method. H&E–stained sections showing the respective diagnostic histomorphology failed to demonstrate the birefringent crystals by polarizing microscopy in all the eight sections from gout and in seven of nine sections from pseudogout. Only two H&E–stained sections showed scant calcium pyrophosphate dihydrate (CPPD) crystals in pseudogout. None of the three sections from two cases of tumoral calcinosis showed birefringence with either stain. We conclude that CPPD in pseudogout and monosodium urate in gout may not polarize in the routine H&E–stained sections. However, polarizing microscopy of sections stained with NAES method allowed demonstration of CPPD crystals with positive birefringence in pseudogout, MSU crystals with negative birefringence in gout, and calcium hydroxyapatite crystals without birefringence in tumoral calcinosis. Section stained with NAES method is a significantly useful adjunct to the routine H&E stain for proper evaluation of the crystals under polarizing microscope in these lesions.

Low-grade Fibromyxoid Sarcoma: Clinicopathologic Case Report With Review of the Literature

Low-grade fibromyxoid sarcoma is a rare, benign-appearing soft tissue neoplasm with an aggressive clinical course characterized by multiple local recurrences over several years, with ultimate spread to lung and occasionally to bone. Thus far, a total of 24 cases of low-grade fibromyxoid sarcoma have been reported in the literature. The authors present an additional case that grossly and microscopically emphasizes a pronounced lobular pattern of contrasting areas of cellularity showing high proliferative activity, as demonstrated by a proliferation marker, Ki 67 with MIB-1, and hypocellular areas with prominent myxoid component and abundant collagen fibrils. There was predominance of delicate capillary-sized stromal vessels with collagenized walls in both cellular and myxoid areas. The unusual features in this case were osseous metaplasia, prominent intranuclear pseudoinclusions, DNA tetraploidy, and membrane-bound intracytoplasmic fat vacuoles. The immunoprofile and cytologic and ultrastructural features are described. After the excision of the tumor, the patient was treated with radiotherapy without chemotherapy. The patient has been observed for 26 months and is alive without the evidence of disease. The postoperative follow-up with axial computed tomography at 24 months showed no evidence of disease, except postsurgical fibrotic changes.

Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

This video demonstrates Shidhams method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually scattered cells and small cell groups. This technique uses HistoGel (Thermo Scientific) with conventional laboratory equipment. The use of cell block sections is a valuable ancillary tool for evaluation of non-gynecologic cytology. They enable the cytopathologist to study additional morphologic specimen detail including the architecture of the lesion. Most importantly, they allow for the evaluation of ancillary studies such as immunocytochemistry, in-situ hybridization tests (FISH/CISH) and in-situ polymerase chain reaction (PCR). Traditional cell block preparation techniques have mostly been applied to non-gynecologic cytology specimens, typically for body fluid effusions and fine needle aspiration biopsies. Liquid based cervicovaginal specimens are relatively less cellular than their non-gynecologic counterparts with many individual scattered cells. Because of this, adequate cellularity within the cell block sections is difficult to achieve. In addition, the histotechnologist sectioning the block cannot visualize the level at which the cells are at the highest concentration. Therefore, it is difficult to monitor the appropriate level at which sections can be selected to be transferred to the glass slides for testing. As a result, the area of the cell block with the cells of interest may be missed, either by cutting past or not cutting deep enough. Current protocol for Shidhams method addresses these issues. Although this protocol is standardized and reported for gynecologic liquid based cytology specimens, it can also be applied to non-gynecologic specimens such as effusion fluids, FNA, brushings, cyst contents etc for improved quality of diagnostic material in cell block sections.

ASC-H in Pap test- definitive categorization of cytomorphological spectrum

Objective The American Society for Colposcopy and Cervical Pathology (ASCCP) guidelines for management of ASC-H is colposcopic examination followed by biopsy. HPV testing (HPVT) is recommended after a negative biopsy result. More definitive interpretation of ASC-H could prevent discomfort and minimize the cost. The purpose of this study was to evaluate association of various cytomorphological patterns of ASC-H with various clinical scenarios. Methods We reviewed SurePath™ (TriPath Imaging, Inc. Burlington, NC, USA) cervical smears interpreted as ASC-H in 161 women (mean age, 37 {15 to 78} years), over 24 months (2002 to 2003). HPVT (Digene, Hybrid Capture® II HPV test, Digene Corporation, Gaithersburg, MD, USA) was performed in 20% of cases (33/161) and biopsy results were available in 54 cases (19 with and 35 without HPVT). Results HPVT was positive in 64% (21/33) cases, and negative in 36% (12/33) cases. In the follow-up biopsies of 71% (15/21) of cases with positive HPVT, 27% showed HPV changes or CIN1, 27% showed CIN2-3, and 46% were negative for epithelial abnormality. Follow-up biopsies from cases with negative HPVT (33%, 4/12 cases), 8% showed CIN1 and 25% were negative for any epithelial abnormality. Six cytomorphological patterns of ASC-H correlated with different clinical categories in relation to HPVT and biopsy results. 35% (19 out of 54 ASC-H cases in which biopsy results were available) could be interpreted definitively as HSIL by cytopathology, 11% (6/54) cases as LSIL with cyanophilic atypical parakeratotic pattern, and 31% (17/54) cases as reactive, with HPV status. The interpretation had to be continued as ASC-H in 22% (12/54) cases. Conclusion ASC-H demonstrated a spectrum of cytomorphological patterns. Some of these patterns in liquid-based cervical smears may be more specifically interpreted as LSIL, HSIL, or benign if HPV status is known.