Paraffin-embedded Histologic Specimens Research Articles

Cytology techniques for minimally invasive molecular autopsies: An opportunity not to be missed.

Cytology techniques for minimally invasive molecular autopsies: An opportunity not to be missed.

Use of Panitumumab‐IRDye800 to Image Cutaneous Head and Neck Cancer in Mice

To assess the feasibility of panitumumab in real-time fluorescent imaging and histologic processing of cutaneous squamous cell carcinoma (cSCC) in mice. A near-infrared (NIR) fluorescent probe (IRDye800CW) was covalently linked to a monoclonal antibody-targeting epidermal growth factor receptor (panitumumab) or nonspecific IgG and injected into mice bearing flank xenografts from a cSCC cell line (SCC-13 or SRB-12; n = 7), human split-thickness skin grafts (STSGs; n = 3), or a human tumor explant (n = 1). The tumor and lymph nodes were imaged and dissected using fluorescence guidance with the SPY imaging system and verified with a charge-coupled NIR system. An NIR scanning device (Odyssey) was used to measure fluorescence intensity in histological sections. Immunodeficient mice. In vivo and in vitro imaging lab. Tumor tissue could be delineated from the human STSG with tumor-to-background ratios of 4.5 (Pearl) and 3.4 (SPY). Tumor detection was substantially improved with panitumumab-IRDye800 compared with IgG-IRDye800. Biopsies positive for fluorescence were assessed by histology and immunohistochemistry (n = 18/18) to confirm the presence of tumor, yielding a 100% sensitivity. Biopsies of nonfluorescent tissue negative for malignancy (n = 18/18) yielded a specificity of 100%. Furthermore, the SPY system was able to detect residual disease as small as 200 µm in diameter. In addition, the Odyssey confirmed fluorescence of microscopic disease (in tumor samples of frozen and paraffin-embedded histologic specimens) but not in adjacent noncancerous tissue. These data suggest panitumumab-IRDye800 may have clinical utility in detection and removal of subclinical cSCC using Food and Drug Administration-approved imaging hardware.

Imaging Cutaneous Head and Neck Cancer with Panitumumab‐IRDye

Objective: To determine if intraoperative, real-time fluorescence imaging hardware (SPY System, Novadaq, Toronto, Canada) and lab-based fluorescence optical imaging of histological sections (Odyssey scanner, LiCor) are capable of detecting micrometastatic cutaneous squamous cell carcinoma (SCC) in preclinical models. Method: An NIR fluorescent probe (IRDye800) was covalently linked to a monoclonal antibody targeting EGFR (panitumumab) or non-specific IgG and injected into mice bearing flank xenografts derived from a cutaneous SCC cell line (SCC-13). The primary tumor and regional lymph nodes were imaged and dissected using fluorescence guidance with the SPY system and verified with a charge-coupled NIR system (Pearl). An NIR charged-coupled device (Odyssey) was used to measure fluorescence intensity of cut sections of tumor and were confirmed with immunohistochemical staining (cytokeratin, CD147). Results: Tumors were clearly delineated from normal tissue with tumor-to-background ratios of 4.5 (Pearl) and 3.4 (SPY). Disease detection was significantly improved with panitumumab-IRDye compared to IgG-IRDye800 ( P < .05). Tissue biopsies positive for fluorescence were confirmed for pathologic disease by histology and immunohistochemistry (n = 10/10); while biopsies of non-fluorescent tissue were proven to be negative for malignancy (n = 12/12). The SPY system was able to detect regional lymph node metastasis (<1.0 mm) and microscopic areas of disease as small as 200 micrometers in diameter. Additionally, the Odyssey successfully detected residual microscopic disease in both frozen and paraffin-embedded histologic specimens. Conclusion: These data suggest that panitumumab-IRDye800 may have clinical utility in detection and removal of cutaneous SCC using existing optical imaging hardware. This technique may prove useful in the detection of microscopic disease grossly and in histological sections.

Use of panitumumab-IRDye800 to image microscopic head and neck cancer in an orthotopic surgical model.

Fluorescence imaging hardware (SPY) has recently been developed for intraoperative assessment of blood flow via detection of probes emitting in the near-infrared (NIR) spectrum. This study sought to determine if this imaging system was capable of detecting micrometastatic head and neck squamous cell carcinoma (HNSCC) in preclinical models. A NIR fluorescent probe (IRDye800CW) was covalently linked to a monoclonal antibody targeting epidermal growth factor receptor (EGFR; panitumumab) or nonspecific IgG. HNSCC flank (SCC-1) and orthotopic (FADU and OSC19) xenografts were imaged 48-96h after systemic injection of labeled panitumumab or IgG. The primary tumor and regional lymph nodes were dissected using fluorescence guidance with the SPY system and grossly assessed with a charge-coupled NIR system (Pearl). Histologic slides were also imaged with a NIR charged-coupled device (Odyssey) and fluorescence intensity was correlated with pathologic confirmation of disease. Orthotopic tongue tumors were clearly delineated from normal tissue with tumor-to-background ratios of 2.9 (Pearl) and 2.3 (SPY). Disease detection was significantly improved with panitumumab-IRDye compared to IgG-IRDye800 (P<0.05). Tissue biopsy samples (average size 3.7mm) positive for fluorescence were confirmed for pathologic disease by histology and immunohistochemistry (n=25 of 25). Biopsy samples of nonfluorescent tissue were proven to be negative for malignancy (n=28 of 28). The SPY was able to detect regional lymph node metastasis (<1.0mm) and microscopic areas of disease. Standard histological assessment in both frozen and paraffin-embedded histologic specimens was augmented using the Odyssey. Panitumumab-IRDye800 may have clinical utility in detection and removal of microscopic HNSCC using existing intraoperative optical imaging hardware and may augment analysis of frozen and permanent pathology.

Fluorescence in situ hybridization for identification of Tritrichomonas foetus in formalin-fixed and paraffin-embedded histological specimens of intestinal trichomonosis

In the present study a highly species-specific oligonucleotide sequence of Tritrichomonas foetus 18S rRNA was used to design an antisense probe for identification of T. foetus in formalin-fixed and paraffin-embedded histological specimens by means of fluorescence in situ hybridization (FISH). Using archival histological specimens from several species with light microscopic evidence of intestinal trichomoniasis, and under optimized hybridization conditions, the probe positively identified trichomonads in colonic specimens from piglets and a kitten with PCR-confirmed T. foetus infection. Neither positive hybridization of the probe or PCR amplification of T. foetus DNA was observed in histological specimens from hamster (Tritrichomonas muris), turkey, nor mouse (Entamoeba muris) intestinal protozoal infections. Sequence-specific binding of the probe was further verified by successfully out-competing the hybridization with 10 x molar excess unlabeled probe and failure of a labeled sense probe to hybridize. The FISH assay described here enables simultaneous location and molecular identification of T. foetus in formalin-fixed and paraffin-embedded histological specimens of intestinal trichomoniasis. The methods employed are likely to also be applicable to probes designed for specific recognition of other trichomonad species, especially in mammalian tissue where red blood cell auto-fluorescence can be easily differentiated from the hybridization signal of trichomonads.

Application of multiplex PCR with histopathologic features for detection of familial breast cancer in formalin-fixed, paraffin-embedded histological specimens

Breast cancer is the most common malignancy among females in the world. Age and familial history are the major risk factors for the development of this disease in Iran. Mutations of BRCA1 and BRCA2 genes are associated with a greatly increased risk for development of familial breast cancer. Frequency of BRCA mutations was identified in familial breast cancers (FBC) and non-familial breast cancers (NFBC) by molecular genetics, morphological and Immunohistochemical methods. Thirty forth formalin-fixed, paraffin-embedded breast tissue tumors were analyzed from 16 patients with FBC and 18 patients with NFBC. Three 5382insC mutations detected by multiplex PCR in 16 familial breast cancers. Immunohistochemical method was used to detect estrogen receptor (ER) and progesterona receptor (PR) and TP53. Comparison of ER, PR and TP53 exhibited high difference (P < 0.0001) in familial breast cancers and non-familial breast cancers. Our results demonstrated that 5382insC mutation, ER, PR, TP53, mitotic activity, polymorphism, necrosis and tubules can serve as the major risk factors for the development of FBC.

Application of multiplex PCR with histopathologic features for detection of familial breast cancer in formalin-fixed, paraffin-embedded histological specimens

Application of multiplex PCR with histopathologic features for detection of familial breast cancer in formalin-fixed, paraffin-embedded histological specimens

P1984 Utility of the Gen-Probe amplified MTD test for the tuberculosis diagnosis in formalin-.xed, paraffin-embedded histologic specimens

P1984 Utility of the Gen-Probe amplified MTD test for the tuberculosis diagnosis in formalin-.xed, paraffin-embedded histologic specimens

Basal cell carcinomas with unusual histologic patterns

Uncommon histologic variants of basal cell carcinoma (BCC) can present a diagnostic challenge. In this case series, we describe 3 patients with unusual BCCs encountered in a dermatologic surgical unit over a 1-year period from September 2003 to September 2004. The formalin-fixed, paraffin-embedded histologic specimens were initially examined microscopically after staining with hematoxylin and eosin. Additional stains, including diastase periodic acid-Schiff, colloidal iron, carcinoembryonic antigen, and cytokeratin-20, were subsequently performed as appropriate. Of the 3 lesions, one exhibited apocrine differentiation and two demonstrated a trabecular growth pattern. Although BCCs demonstrating apocrine differentiation have previously been described, a trabecular growth pattern, to our knowledge, has not been previously reported for BCC.

Comparison of polymerase chain reaction with histopathologic features for diagnosis of tuberculosis in formalin-fixed, paraffin-embedded histologic specimens.

Objective.-To investigate the relationship between various histopathologic features and the results of the tuberculosis (TB)-polymerase chain reaction (PCR) method in routinely submitted histologic specimens for the histopathologic diagnosis of TB. Design.-We used 95 formalin-fixed, paraffin-embedded tissue blocks from 81 patients who were clinically suspected of having TB. We assessed the presence of histopathologic features including well-formed granuloma, poorly formed granuloma, caseous necrosis, and Langhans-type giant cells. We performed nested PCR for IS6110 and Ziehl-Neelsen staining for acid-fast bacilli (AFB). Results.-Of the 81 patients studied, 53 patients had chronic granulomatous inflammation, whereas 28 patients had only chronic inflammation without definite granulomatous inflammation. Of the 53 cases with chronic granulomatous inflammation, 17 (32%) were AFB positive and 36 (68%) were TB-PCR positive. Among cases with chronic granulomatous inflammation, the percentage that were positive and negative by TB-PCR differed significantly with the presence of various histopathologic features. All of the 13 cases with well-formed granuloma, caseous necrosis, and Langhans-type giant cells were TB-PCR positive; however, 10 (36%) of the 28 cases with chronic inflammation without granulomatous lesions were also TB-PCR positive. Conclusions.-TB-PCR is a rapid, sensitive method for the diagnosis of TB in routinely processed formalin-fixed, paraffin-embedded histologic specimens and is readily available in histopathology laboratories. We recommend use of TB-PCR when TB is suspected clinically, especially in cases of chronic inflammation without definite evidence of granulomatous inflammation.

Increased microvessel density in adenomyosis uteri

Objective: To investigate microvessel density in adenomyosis compared to the endometrium in patients with adenomyosis and in normal controls. Design: Uterine paraffin-embedded histologic specimens were immunostained for CD34. The area with the highest microvessel density in adenomyosis and in the endometrium was evaluated. All microvessels in a specific field of view (×200 magnification) were counted. Setting: The Department of Gynecological Endocrinology and the Institute of Clinical Pathology, Department of Gynecopathology, in an university hospital. Patient(s): Specimens of 53 patients with adenomyosis, who had undergone hysterectomy. Endometrial specimens of 17 women without uterine pathology were investigated as normal controls. Main Outcome Measure(s): Microvessel density in adenomyosis and in the endometrium. Result(s): The mean microvessel density was significantly higher in adenomyosis than in the endometrium of the same patients (33.5 ± 14.6 vs. 19.5 ± 12.5 microvessels/field; P<.001 sign test). No significant difference between the endometrium of patients and of normal controls was observed ( P=.805). Conclusion(s): Adenomyosis exhibits angiogenic properties. However, the endometrium of patients with adenomyosis is not more prone to express angiogenic activity compared to the endometrium of normal controls.

Utility of rush paraffin-embedded tangential sections in the management of cutaneous neoplasms.

Mohs micrographic surgery (MMS) modified by the use of tangential, formalin-fixed, paraffin-embedded histologic specimens is advantageous in treating selected skin neoplasms. To review the use of our experience with a modification of MMS to treat lentigo maligna melanoma (LMM), lentigo maligna (LM) and other melanoma in situ (MIS) lesions, dermatofibrosarcoma protuberans (DFSP), atypical fibroxanthoma (AFX), and angiosarcoma. Our experience utilizing a modification of MMS in the treatment of 77 patients with LM or other MIS, 23 patients with LMM, 11 patients with DFSP, 1 patient with AFX, and 1 patient with angiosarcoma was reviewed. Length of follow-up and rate of recurrence were examined. A literature review of this pertinent modification of the Mohs technique was performed. One hundred fourteen patients underwent MMS for melanocytic (LM, MIS, LMM), spindle cell (DFSP, AFX), and vascular malignant neoplasms. One patient developed locally recurrent LM and one patient with LMM developed satellite metastasis. Regional lymph node metastasis occurred in one patient with LMM and in a patient with angiosarcoma. The use of Mohs micrographic surgery in conjunction with rush formalin-fixed, paraffin-embedded tangential histologic sections provides the accuracy and tissue conservation of the Mohs procedure while ensuring more confident interpretation of histology in cases of lentigo maligna, lentigo maligna melanoma, dermatofibrosarcoma protuberans, atypical fibroxanthoma, and angiosarcoma.

Detection of SYT-SSX fusion transcript in synovial sarcoma using archival cytologic specimens.

Identification of the SYT-SSX fusion transcript can be a useful diagnostic aid for synovial sarcoma. However, there is no report on the detection of the specific transcript in archival cytologic specimens. We performed a rapid reverse transcription-polymerase chain reaction assay to detect the fusion transcript using such specimens as RNA sources and compared its usefulness with the assay using formalin-fixed paraffin-embedded histologic specimens. Eleven cytologic and 12 histologic specimens stored for 0.1 to 20 years were selected from 9 patients. The SYT-SSX transcript was identified in 10 (91%) of 11 of cytologic and 9 (75%) of 12 histologic cases. One of the transcript-positive cytologic cases was obtained by fine-needle aspiration. SYT-SSX1 fusion was found in 4 patients and SYT-SSX2 in 5. Cytologic specimens will be an excellent RNA source for the reverse transcription-polymerase chain reaction diagnosis of synovial sarcoma with a sensitivity of more than 90%, comparable to that in frozen materials. Histologic materials also may be useful when cytologic or frozen materials are not available.

Malignant lymphoma of the kidney

Background Primary renal lymphoma (PRL) is a rare disease, making information including etiologic factors for PRL extremely limited. Methods Clinical and pathologic findings of PRL in Japan are presented and compared with those from Western countries. The presence of Epstein-Barr virus (EBV) genomes in the tumor was also evaluated. Eight cases of PRL were collected from a review of the “Annual of the Pathological Autopsy Cases in Japan (1976–1992)”. These cases fullfilled the following criteria: (1) presence of renal mass without extrarenal lymphomatous involvement at admission and (2) absence of a leukemic blood picture. For histologic and immunohistochemical studies, 10% formalin-fixed and paraffin-embedded histologic specimens were used. Presence of Epstein-Barr virus (EBV) genome was examined by polymerase chain reaction (PRC) and in situ hybridization (ISH). Results There were five males and three females; age at admission ranged from 15 to 79 years (median 57 yr). Abdominal and/or flank pain were the most common presenting symptoms. No particular past history was present in any of the patients. Histologically, tumor cells in all cases showed a diffuse pattern of proliferation: large cell type in six cases, mixed cell type and small lymphocytic type in 1 each. Immunohistochemistry revealed B-cell nature of lymphoma cells in all cases. Neither PCR nor ISH showed the presence of EBV genome in any cases. Conclusions PRL is non-Hodgkin's lymphoma of predominantly large cell type with a B-cell immunophenotype. EBV etiology is unlikely in PRL. J. Surg. Oncol. 64:207–211, 1997 © 1997 Wiley-Liss, Inc.

Detection and significance of glutathione-S-transferase pi in osteogenic tumors of dogs

In this study we describe a relationship between the percentage of tumor cells staining positive for GST pi and the response to cisplatin treatment in dogs with appendicular osteosarcoma. Clinical resistance to platinum compounds has been attributed to tumor cell resistance to the drug. An overexpression of glutathione-S-transferase pi has been implicated in the resistance of tumor cells in vitro to cisplatin. In this study, the relative concentration of GST pi was quantified immunohistochemically in formalin-fixed, paraffin-embedded histologic specimens obtained from dogs with appendicular osteosarcoma prior to cisplatin treatment. The percentage of tumor cells staining positive for GST pi was compared with patient response (remission and survival times) following treatment. Those osteosarcoma-bearing dogs having a low pre-treatment tumor expression of GST pi (<50%; n=13) had a significantly longer (p <0.01) median remission and survival time (24.0 and 33.7 weeks, respectively) than those dogs with a high pre-treatment tumor expression of GST pi (>50%; n=8; median remission and survival times 14.2 and 24.0 weeks, respectively). The relative staining of GST pi in primary canine bone tumors has therapeutic importance and warrants further evaluation as a therapeutic model for human osteosarcoma.

Detection of clonal T-cell populations in gastrointestinal lymphomas by analysis of cRNA conformational polymorphisms of rearranged T-cell-receptor-gamma genes.

Analysis of complementary RNA molecules of junctional regions of rearranged T-cell-receptor-gamma genes show a pattern of conformational polymorphisms which is specific for an individual lymphocytic clone. In a blinded study we analysed formalin-fixed, paraffin-embedded histological specimens from gastrointestinal lymphomas and control tissues (lymphomas: pleomorphic T-cell 10, anaplastic large cell [Ki1+] 9, centroblastic 5, immunocytoma 1, B-CLL 2, Hodgkin's 2, centroblastic-centrocytic 1, MALT [mucosa associated lymphoid tissue] 1, T-cell acute lymphoblastic leukaemia 1, non-lymphoid or polyclonal lymphoid tissues 5). Junctional regions of rearranged TCR-gamma genes were amplified by the polymerase chain reaction and the products were transcribed into cRNA. Conformational patterns of cRNA molecules were analysed by polyacrylamide gel electrophoresis. 13/20 T-lineage lymphomas and the T-cell acute lymphocytic leukaemia displayed a distinct cRNA band pattern, all B-lineage lymphomas and the non-lymphoid control tissues were negative. Only one case of nasopharyngeal (lymphoepithelial, Schmincke-Regaud) carcinoma showed a faint cRNA banding pattern. This novel and non-radioactive assay allows for the rapid detection and molecular characterization of clonal lymphoid populations in minute histological biopsy specimens.