Archival Paraffin Blocks Research Articles

Molecular Confirmation of Parelaphostrongylus tenuis Infection in a Horse With Verminous Encephalitis

>> Editor: Advances in molecular diagnostic technology have enabled us to identify and confirm an etiologic agent that was not possible when we published a case report of verminous encephalitis in a colt in Veterinary Pathology. 2 Although we presumed that the cause of the encephalitis was Parelaphostrongylus tenuis—a meningeal helminth that commonly parasitizes white-tailed deer (Odocoileus virginianus)—we could identify only the parasites as members of the family Protostrongylidae. We recently applied a polymerase chain reaction–(PCR) based approach to the DNA isolated from archival paraffin blocks. Using genus-specific primers and sequence analysis, we confirmed the infection specifically as P tenuis. Thin sections of formalin-fixed, paraffin-embedded brain tissue containing protostrongylid larvae were processed for DNA extraction with a QIAamp 1 DNA Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The Parelaphostrongylus spp–specific primers used for PCR were described previously (PTP1, 5 0 -CCGTCGAATACATGT CATCC-3 0 ; PTP2, 5 0 -TCGTCAAGACGATGATTCCC-3 0 ). 1 Specificity of these primers for Parelaphostrongylus spp was confirmed by BLAST comparison with nonredundant sequences in the BLASTn database (National Center for Biotechnology Information). These primers amplify a 248–base pair product covering a portion of the second internal transcribed spacer of the ribosomal RNA gene in Parelaphostrongylus spp. The PCR reaction mixture included 10mM Tris-HCl (pH 9.0), 50mM KCl, 0.1% Triton X-100, 1.5mM MgCl2, 250mM deoxynucleotide triphosphates, 0.5mM of each primer, and 1 U of Taq DNA polymerase (Promega, Madison, WI). Cycling parameters included denaturation at 94 � Cf or 3 minutes, followed by 40 cycles of denaturation at 94 � Cf or 1 minute, annealing at 60 � C for 1 minute, and extension at 72 � C for 1 minute. Reaction products were examined by electrophoresis in a 1% agarose gel and were subsequently purified with a QIAquick 1 Gel Extraction Kit (Qiagen) according to

Genetic Polymorphisms in the Fibrinolytic System of Placentas with Massive Perivillous Fibrin Deposition

Massive perivillous fibrin deposition (MPFD) and maternal floor infarction (MFI) of the placenta are rare related conditions associated with poor perinatal outcome including antepartum stillbirth. The diseases are characterized by pathologic accumulation of fibrinoid deposits that surround the placental villi (in the case of MFI predominantly in the basal regions adjacent to the decidual plate). These findings suggest either overproduction and/or defective clearance of fibrinoid within the intervillous space. Recently genetic polymorphisms of the plasminogen activator inhibitor-1 (PAI-1) gene have been found in association with impaired fibrinolysis in the pelvis predisposing to endometriosis. We hypothesized that polymorphisms in one or more of four genes that regulate fibrinolysis were associated with MPFD and MFI placentas. We retrospectively identified 20 consecutive cases of MPFD/MFI from our placental pathology database and generated 2 random gestational age-matched controls for each case. Clinical charts were reviewed. DNA was extracted from archived paraffin blocks of placental tissue from cases and controls. Single nucleotide repeat polymorphisms (SNPs) in loci within PAI-1 gene, thrombin activated fibrinolysis inhibitor (TAFI) gene, plasminogen activator urokinase (u-PA) gene and plasminogen activator tissue (t-PA) gene were studied using PCR methods. Outcomes in the study group included perinatal death (8), preterm IUGR (6), preeclampsia (4) and only 3 normal term deliveries. A spectrum of placental ultrasound abnormalities was observed. No SNP polymorphism was found to associate with MPFD/MFI. MPFD/MFI are associated with significant abnormal perinatal outcomes but have not been shown to be mediated by polymorphisms in candidate genes that are predicted to impair fibrinolysis in our study.

Abstract 1757: Patterns of clonal chromosomal aberrations in invasive breast cancer compared to adjacent DCIS lesions detected by a ten-probe FISH panel

Abstract Ductal carcinoma in situ (DCIS) refers to lesions with malignant epithelial cells that have not invaded the surrounding stroma. These lesions are confined to the ducts of the breast. It is thought that DCIS represents a stage that precedes invasive ductal carcinoma (IDC). However the linkage between these two lesions is still obscure. In order to analyze the patterns of genetic alterations as an approach to understand the correlation between DCIS and IDC, we developed four panels of FISH probes consisting of a total of eight gene specific probes and two centromeric probes and conducted a systematic FISH evaluation of breast tumor specimens containing both lesions. The gene probes tested for oncogenes and tumor suppressor genes specific for breast tumorigenesis while the centromeric probes were included to reflect and adjust for the ploidy of the cells. Objectives Determine gain and loss patterns of the oncogene and tumor suppressor gene probes within DCIS and IDC Compare similarities in the genetic make-up of DCIS and corresponding IDC lesions from the same case Characterize clonal evolution from DCIS lesions to IDC The database of the “Breast Susceptibility Study” conducted at the Naval Medical Center, Bethesda, Maryland was searched for patients whose pathology reports were positive for breast carcinoma with neighboring DCIS. Archived paraffin blocks were analyzed by a pathologist for the presence of invasive cancer and DCIS. Representative areas were marked on thick sections. The material was micro-dissected and disintegrated using the Hedley method. The resulting single cell suspensions were cytospun onto slides. The best cytospin for each lesion was. hybridized with four FISH probe panels subsequently. An Imaging relocation software allowed us to assess the same cells for all four sets of probes. For each lesion, manual counting of 150-200 nuclei was performed and tabulated into an excel spreadsheet to allow analysis of signal patterns. In collaboration with NCBI, algorithms were developed to compare clonal patterns. Preliminary data show that there are often major clones both in the DCIS and the IDC that present strong similarities to each other. However, the clones can differ in specific gains or losses of the oncogenes and tumor suppressor genes tested. The signals depicted in the table represent an attempt to delineate major clones within the DCIS and IDC populations. This study will improve our understanding of the molecular events that are involved in the progression from pre-invasive to invasive cancer and may lead to the development of prognostic biomarkers that can predict this transition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1757.

Factors, Predicting Failure of Adjuvant Hormonotherapy of Breast Carcinoma. A Study in Tamoxifen Treated Patients.

Abstract Background: The latest clinical trials indicate better performance of aromatase inhibitors, compared to tamoxifen, in adjuvant hormonotherapy of breast carcinoma. The identification of molecular markers, predicting resistance to tamoxifen, could help to identify patients, which are most likely to benefit from aromatase inhibitors in up-front adjuvant hormonotherapy.Material and methods: Tissue microarrays were constructed from archival paraffin blocks of primary tumors of 179 patients with estrogen receptor positive operable breast carcinoma in stage I-III, subsequently treated with tamoxifen for five years or until relapse, with at least 7 years follow up available. The amplifications of Her-2 and cyclin D1 genes were evaluated by fluorescence in-situ hybridization. The level of progesterone receptor (PR) and Ki67 were estimated by immunohistochemistry.Results: 54 of above patients recurred during follow up. In univariate analysis of disease free survival, the presence of more than three nodal metastases (RR=4,5 p<0,001), grade 3 (RR=2,3 p=0,035), cyclin D1 (RR=3,06 p<0,001) and Her-2 (RR=3,06 p<0,001) amplifications were identified as significant risk factors, together with the negativity of PR (RR=2,1 p=0,013). In multivariate analysis, only clinical stage III (RR=2,6 p=0,003), cyclin D1 (RR=2,7 p=0,001) and Her-2 (RR=2,1 p=0,014) amplifications proved significant. In 77 patients who received adjuvant chemotherapy no statistically significant risk factor was identified. In multivariate analysis of 102 patients without adjuvant chemotherapy only stage III (RR=6,9 p=0,001) and Her-2 amplification (RR=4,5 p=0,001) were confirmed.Discussion: The advanced clinical stage, cyclin D1 and Her-2 gene amplifications represent factors, predicting the failure of adjuvant tamoxifen treatment, but their predictive value is much lower in patients receiving adjuvant chemotherapy. This fact indicates, they can reflect the common biological aggressiveness of tumor and need not to be tamoxifen specific. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6031.

Homozygous Mutations in ADAMTS10 and ADAMTS17 Cause Lenticular Myopia, Ectopia Lentis, Glaucoma, Spherophakia, and Short Stature

Weill-Marchesani syndrome (WMS) is a well-characterized disorder in which patients develop eye and skeletal abnormalities. Autosomal-recessive and autosomal-dominant forms of WMS are caused by mutations in ADAMTS10 and FBN1 genes, respectively. Here we report on 13 patients from seven unrelated families from the Arabian Peninsula. These patients have a constellation of features that fall within the WMS spectrum and follow an autosomal-recessive mode of inheritance. Individuals who came from two families and met the diagnostic criteria for WMS were each found to have a different homozygous missense mutation in ADAMTS10. Linkage analysis and direct sequencing of candidate genes in another two families and a sporadic case with phenotypes best described as WMS-like led to the identification of three homozygous mutations in the closely related ADAMTS17 gene. Our clinical and genetic findings suggest that ADAMTS17 plays a role in crystalline lens zonules and connective tissue formation and that mutations in ADAMTS17 are sufficient to produce some of the main features typically described in WMS.

Carcinoma Ex Spiradenoma/Cylindroma Confirmed by Immunohistochemical and Molecular Loss-of-Heterozygosity Profiling

We present a case of spiradenoma/cylindroma with admixed carcinoma of unknown origin, resolved using immunohistochemical and molecular loss-of-heterozygosity (LOH) profiling. The patient, a woman in her mid-70s, initially presented with separate mammary (ductal) carcinomas of the right and left breasts that were treated with radical mastectomies. For 9 years, the patient remained disease free until complaining of a slow-growing skin nodule on the lower back that was excised under clinical suspicion of metastatic mammary carcinoma. Histopathological exam revealed a benign eccrine spiradenoma/cylindroma and an intermixed carcinoma, with a differential diagnosis of either primary eccrine carcinoma or mammary carcinoma metastatic to the spiradenoma/cylindroma. Histological features and immunohistochemical staining favored eccrine carcinoma but not unequivocally; therefore, LOH profiles were performed on archival paraffin block tissue from the 3 neoplastic lesions (4 components). The mammary carcinomas showed disparate LOH at 5 of 7 (right breast) and 4 of 7 (left breast) informative genetic loci, establishing these carcinomas as separate primary neoplasms. Both the spiradenoma/cylindroma and eccrine carcinoma revealed no LOH at the tested loci, establishing the unknown carcinoma as an independent carcinoma arising within a spiradenoma/cylindroma. This neoplasm is referred to in the literature as carcinoma ex spiradenoma/cylindroma and spiradenocylindrocarcinoma.

Clinical Significance of P16 in Langerhans Cell Histiocytosis

Background. The p16 protein has been identified as a tumor suppressor that functions by inhibiting the cyclin-dependent kinases CDK4 and CDK6. Deletions or point mutations in the p16 gene have been found in a number of human cancers, emphasizing its importance in regulating cell cycle progression. The authors have chosen to analyze P16 expression in biopsies from children with LCH and correlated it with clinical manifestation and outcome in a broad range of organs.Patients and Methods: Archival paraffin block were retrieved from children diagnosed with LCH and followed up at Asan medical center and Chungnam National University Hospital between 1998 and 2007. Pathological Langerhans cells were identified morphologically associated with the characteristic inflammatory type infiltrate and were confirmed by immunohistochemical staining with antibodies to CD1a, and S100. The disease was classified as single system defined as uni- or multifocal involvement of a single organ system or multi-system defined as involvement of multiple organ systems with or without organ dysfunction. The slides were stained with P16 antibody and semi-quantitatively evaluated using 5 grade systems, negative means absence of staining, ±, weakly positive, +, similar with lymphocytes around the LCs, 2+, stronger than lymphocystes, 3+, much stronger than lymphocytes.Results: The specimens from 57 patients (31 boys and 26 girls) were available for immunohistochemistry. Of these, 12 slides were not suitable because the quality of staining was not good for evaluation or the tumor cells are too difficult to be differentiated. The median age of 45 patients (24 boys and 21 girls) was 9.5 years, range 5 months to 22 years with a definite diagnosis of LCH based on CD1a positivity. The P16 protein was expressed in various degrees except one specimen. No significant difference was found between the level of P16 expression in lesions from patients with single-system, and multi-system disease. But higher tendency of relapse have shown in 1+, 2+ and 3+ groups compared with negative and weakly positive groups.Conclusions: This study has shown a diffuse expression of P16 protein in both single and multi-system lesions of LCH. P16 might be involved in the pathogenesis of LCH.

β‐catenin gene alterations in a variety of so‐called calcifying odontogenic cysts

Calcifying odontogenic cysts (COCs) evidence a variety of histopathological features, in addition to biological behaviors. Several systems have been proposed for the classification of COCs. However, the molecular changes underlying their development remain largely unknown. In an effort to elucidate the participation of beta-catenin gene mutation in COC development, we evaluated the genetic alterations and expression of beta-catenin in different COC subtypes with archival paraffin blocks. beta-catenin gene mutations were detected in all subtypes. Immunohistochemically, all cases of COCs evidenced cytoplasmic and nuclear beta-catenin expression. Beta-catenin gene mutations and beta-catenin overexpression have been identified as characteristics of COCs. Therefore, the constitutive activation of Tcf/Lef-dependent transcription appears to be intimately involved in their development. These findings indicate that aberrations of the Wnt signaling pathway may perform a crucial role in the development and differentiation status of the odontogenic epithelium in COCs via the deregulation of cell proliferation.

Frequency of the basal‐like phenotype in African breast cancer

Basal-like breast carcinoma has been recognized as a subtype with specific prognostic implications. However, there is a lack of reports about this category of breast tumors in African women. The aim of this study was to explore the basal-like phenotype in breast cancer patients in an African population, and a registry-based series was included from the well-defined Kyadondo County in Uganda (1.7 millions). We studied a total of 65 archival paraffin blocks of invasive breast cancer using antibodies against cytokeratin 5/6 and P-cadherin, and these markers were expressed in 34% of all cases and in 52% of ER (estrogen receptor)-negative tumors. All basal-like tumors were ER negative (p<0.0005) and PR (progesterone receptor) negative (p=0.002). Basal-like breast carcinomas were of a higher histologic grade (p=0.001), had high mitotic counts (p=0.002), and marked nuclear pleomorphism (p=0.002). P-cadherin-positive tumors had a high Ki-67 proliferative rate (p=0.039). In conclusion, the basal-like phenotype is frequent in this African series of breast cancer and is strongly associated with poor prognostic factors. Our findings might be significant in relation to clinical management of these patients, including novel targeted therapy.

Distinctive Expression Pattern of ErbB Family Receptors Signifies an Aggressive Variant of Bladder Cancer

Expression of various members of the ErbB family (epidermal growth factor receptor/ErbB-1, ErbB-2, ErbB-3 and ErbB-4) is associated with disease stage and survival in patients with urothelial carcinoma. We examined the correlation of ErbB family receptor expression with the progression of urothelial carcinoma and survival. A urothelial carcinoma tissue array was constructed from 248 archival paraffin blocks and quality control studies were ascertained. The tissue microarray was stained for epidermal growth factor receptor, ErbB-2, ErbB-3 and ErbB-4, and analyzed using an automated reader. Patient data included grade, stage, growth pattern, recurrence and survival. Kaplan-Meier estimates of 5-year overall and recurrence-free survival were 58% and 27%, respectively. Patients with high grade, invasive or nonpapillary disease had a worse prognosis than patients with low grade, superficial or papillary disease (p <0.0001). High epidermal growth factor receptor or low ErbB-4 expression was associated with nonpapillary, high grade and invasive tumors as well as with significantly shorter recurrence-free and overall survival (p <0.002, 0.028 and 0.047, respectively). Levels of ErbB-2 and ErbB-3 expression were not associated with overall or recurrence-free survival. The expression profiles of ErbB-4 and epidermal growth factor receptor are prognostic in urothelial carcinoma. They may help in selecting patients at high risk with bladder cancer for more aggressive therapy.

166 INVITED PKB-FoxO

Expression of various members of the ErbB family (epidermal growth factor receptor/ErbB-1, ErbB-2, ErbB-3 and ErbB-4) is associated with disease stage and survival in patients with urothelial carcinoma. We examined the correlation of ErbB family receptor expression with the progression of urothelial carcinoma and survival.A urothelial carcinoma tissue array was constructed from 248 archival paraffin blocks and quality control studies were ascertained. The tissue microarray was stained for epidermal growth factor receptor, ErbB-2, ErbB-3 and ErbB-4, and analyzed using an automated reader. Patient data included grade, stage, growth pattern, recurrence and survival.Kaplan-Meier estimates of 5-year overall and recurrence-free survival were 58% and 27%, respectively. Patients with high grade, invasive or nonpapillary disease had a worse prognosis than patients with low grade, superficial or papillary disease (p <0.0001). High epidermal growth factor receptor or low ErbB-4 expression was associated with nonpapillary, high grade and invasive tumors as well as with significantly shorter recurrence-free and overall survival (p <0.002, 0.028 and 0.047, respectively). Levels of ErbB-2 and ErbB-3 expression were not associated with overall or recurrence-free survival.The expression profiles of ErbB-4 and epidermal growth factor receptor are prognostic in urothelial carcinoma. They may help in selecting patients at high risk with bladder cancer for more aggressive therapy.

A new estrogen receptor on ER-negative breast cancer?

10604 Background: Premenopausal women and BRCA-1 mutation carriers have estrogen receptor-negative (ER-) breast cancers that paradoxically seem responsive to hormonal deprivation such as oophorectomy. GPR30 is a G protein-coupled receptor that is activated not only by estradiol but also tamoxifen and fulvestrant. The prevalence and significance of GPR30 in breast cancer are unknown. We hypothesized that GPR30 may be present in ER-negative tumors and may promote growth of ER-negative breast cancer in premenopausal patients. Methods: An immunohistochemical staining protocol for rabbit polyclonal GPR30 antibody (Novus Biological) was developed initially on the known GPR30-positive breast cancer cell line MCF-7 and the GPR30-negative breast cancer cell line MDA-MB-231 and later on a panel of human breast tumors from archived paraffin blocks. After an automated protocol was optimized, under an IRB-approved protocol all incoming breast cancer specimens were stained for GPR30 and reviewed by a pathologist blinded to the ER/PR status. Significance of associations was tested by chi square analysis. Results: Twenty-seven breast cancer tumors were studied. The mean age of the patients was 53 (range 20–87). All of 10 triple negative (ER-,PR-, HER2 neu-) and 2 ER-/Her2 neu+ tumors stained positive for GPR30. Only 4 of 11 ER+/Her2neu - tumors stained positive for GPR30 (p=0.004). The mean age of triple negative patients was 43.7 yr, while the mean age of ER+/Her2 neu- was 63.2 (p=0.02). Three out of 4 ER+/HER2 neu+ tumors stained positive for GPR30. Four patients were African Americans, 2 had triple negative tumors and 2 ER+/Her2+, all 4 were positive for GPR30. Conclusions: GPR30 appears to be frequently expressed in ER-negative breast cancer and may represent an adverse factor. ER+ tumors with good prognostic features (PR+, HER2 neu-) were mostly negative for GPR30, while high-risk tumors were positive for GRP30. Premenopausal status and African-American race are known risk factors for lower survival. In this study, the African-American patients had high-risk tumors that were also positive for GPR30. A longer- range study is needed to test the association of GPR30 expression with recurrence and death. Future studies to assess the function of GPR30 in breast cancer cells may yield prognostic and therapeutic applications. No significant financial relationships to disclose.

Significant variations in nuclear structure occur between and within Gleason grading patterns 3, 4, and 5 determined by digital image analysis

Alterations in nuclei structure and DNA content captured from Gleason grading patterns 3, 4 and 5 of radical prostatectomy (RP) cases were determined by a computer-assisted microscope. Quantitative Nuclear Morphometry (QNM) profiles were created to evaluate variability in nuclear structure within each of these grades. A tissue microarray (TMA) was constructed using RP cases and the prostate cancer (PCa) TMA cores prepared from 20 GG-3, 9 GG-4, 10 GG-5 patterns, and 20 benign cancer-adjacent cases from RP archival paraffin blocks. Feulgen-stained nuclei were captured from 0.6 mm spots using the AutoCyte system. Pools of 1100 nuclei captured from each test group were used to calculate Multivariate Logistic Regression (MLR) models that generated predictive indices and predictive probabilities (PP) to make comparisons between and within each set of pooled nuclei. A single QNM profiles yielded areas of receiver operator characteristic curves (ROC) that distinguished differences among benign cancer-adjacent nuclei and GG-3 (ROC-AUC = 0.78); GG-4 (ROC-AUC = 0.86) and GG-5 (ROC-AUC = 0.88) with accuracies of 73%, 78% and 80% respectively. Applying PP plots generated from MLR models of GG 3, 4, and 5 nuclei clearly demonstrated marked heterogeneity within each of these three GG patterns. QNM signatures illustrate alterations in nuclei structure, based upon nuclear morphometry within each of these three GG patterns, and might signify potential variations in PCa disease risk of progression outcomes. In the future a modified system of Gleason grading that considers not only glandular architecture but also quantitative nuclear grade may ensure accuracy in prognosis.

Comparing Vasculogenic Mimicry With Endothelial Cell-lined Vessels: Techniques for 3D Reconstruction and Quantitative Analysis of Tissue Components from Archival Paraffin Blocks

We previously described techniques to generate 3-dimensional reconstructions of the tumor microcirculation using immunofluorescence histochemistry and laser scanning confocal microscopy on serial sections from archival formalin-fixed, paraffin-embedded tissues. By aligning sequential z-stacks in an immersive visualization environment (ImmersaDesk), the need to insert fiduciary markers into tissue was eliminated. In this study, we developed methods to stitch overlapping confocal z-series together to extend the surface area of interest well beyond that captured by the confocal microscope objective and developed methods to quantify the distribution of markers of interest in 3 dimensions. These techniques were applied to the problem of comparing the surface area of nonendothelial cell-lined, laminin-rich looping vasculogenic mimicry (VM) patterns that are known to transmit fluid, with the surface area of endothelial cell-lined vessels in metastatic uveal melanoma to the liver in 3 dimensions. After labeling sections with antibodies to CD34 and laminin, the surface area of VM patterns to vessels was calculated by segmenting out structures that labeled with laminin but not with CD34 from those structures labeling with CD34, or CD34 and laminin. In metastatic uveal melanoma tissues featuring colocalization of high microvascular density [66.4 microvessels adjusted for 0.313 mm2 area (range 56.7 to 72.7)] and VM patterning, the surface area of VM patterns was 11.6-fold greater (range 10.8 to 14.1) than the surface provided by CD34-positive vessels. These methods may be extended to visualize and quantify molecular markers in 3 dimensions in a variety of pathologic entities from archival paraffin-embedded tissues.

Cyclin D1 Protein Tissue Detection in Laryngeal Cancer

Cyclin D1 (CCND1) is a set of periodic regulatory proteins that is believed to govern cell cycle transit from G1 into S phase. Overexpression of CCND1 leads to abnormal cellular proliferation which underlies processes of tumorigenesis; CCND1 can thus function as a cooperative oncogene in cell transformation. In the present study we investigate the immunohistochemical expression of CCND1 in a well-documented series of 58 laryngeal squamous cell carcinomas (LSCC) and search for statistical associations between CCND1 index and various clinicopathological parameters including several immunomarkers’ expression as well as patients’ disease-free survival. Tissue sections from archival paraffin blocks were stained using the avidin-biotin-peroxidase complex method; the H-295 rabbit polyclonal antibody was applied at dilution of 1:150. The percentage of CCND1 immunoreactive tumor cells for each tumor was counted by an image analysis system. CCND1 staining was confined to cell nuclei and, in the examined samples, ranged from undetectable (i.e. 0% of tumor cells, n = 6) to the majority of tumor cells (i.e. 89% of tumor cells) with mean value: 15.73%. In tumor adjacent, non invasive lesions, strong CCND1 staining was noticed in areas with cellular atypia. In cases with nodal metastases, no change in CCND1 expression in the nodal metastases compared with the primary tumors was observed. p53 protein accumulation in malignant cells was positively linked with CCND1 index (Mann-Whitney U: 205.5, p = 0.034). CCND1 expression appears to be an early event in processes of tumorigenesis and tumor progression in some LSCC. Apart from p53 protein accumulation, CCND1 immunohistochemical expression does not seem to correlate with nodal metastasis, disease recurrence or any other clinicopathological prognostic indicator.

Bacterial Etiology for Chronic Villitis is Not Supported by Polymerase Chain Reaction for 16S rRNA DNA

Chronic villitis is characterized by chorionic villi infiltrated by lymphocytes, histiocytes, and sometimes plasma cells. In a small percentage of cases, an infectious agent can be demonstrated within areas of chronic villitis. However, the pathogenesis of most lesions is idiopathic. Chronic villitis may represent the direct spread of chronic endometrial infection by bacterial organisms that are particularly problematic for culture. To test this hypothesis, polymerase chain reaction (PCR) using primers for the universal bacterial 16S rRNA DNA was performed on DNA extracted from areas of chronic villitis selected from placentas in the Yale Pathology database. Specific areas of chronic villitis were first confirmed by examination of sections stained with hematoxylin and eosin and then removed from archived paraffin blocks. Control tissue spiked with known bacterial counts was also prepared to test the sensitivity of the experiment. All tissue was deparaffinized, dehydrated, and digested with proteinase K. DNA extraction was performed with the Gentra Puregene kit. PCR was done using primers p11 and p13 for the 16S rRNA DNA. The 233-bp amplified target product was identified by agarose gel electrophoresis. Nineteen specimens with multifocal chronic villitis without confinement to anchoring villi were studied. None of the chronic villitis specimens had a demonstrable product using the PCR primers for 16S rRNA DNA, despite adequate DNA in the samples and controls. The assay was sensitive down to approximately 1500 bacteria per specimen. In conclusion, these data do not support a bacterial etiology for chronic villitis.

Okkulte lymphatische Tumorzelldisseminierung beim Pankreaskarzinom

The aim of this study was to determine the frequency and effect on prognosis of occult tumor cells in regional lymph nodes judged to be tumor-free in conventional histopathology of pancreatic cancer patients. Among 115 patients who underwent pancreatic resection for pancreatic (n=84) or distal common bile duct malignancy (n=12) or carcinoma of the papilla (n=19), 48 (42%) were staged pN0. Archival paraffin blocks of 271 resected regional lymph nodes of 41 pN0 patients were reevaluated for occult tumor cells using monoclonal antibody Ber-EP4. Cases with or without isolated tumor cells were compared regarding the distribution of various clinicopathological factors. Of 41 pN0 patients, 16 (39%) exhibited single Ber-Ep4 immunoreactive cells or small cell clusters in at least one lymph node. The occurrence of occult tumor cells was not dependent on other clinicopathological factors such as pT stage, grading, or curative resection. However, those cells were encountered more frequently in common bile duct carcinomas (100%) than in pancreatic (36%) or papilla (20%) carcinomas (P=0.009). Occult tumor cells impaired prognosis significantly in uni- and multivariate analyses (estimated 5-year survival 53% for pN0((i-)) vs 10% for pN0((i+)) and 9% for pN1/N2; P=0.0047). Occult tumor cells are frequent in apparently tumor-free lymph nodes of pancreatic cancer patients and often overlooked in conventional histopathology. They are encountered even in limited stages of disease and they impair prognosis, which is comparable to that of patients with true lymphatic metastases. Occult tumor cells in lymph nodes of pancreatic cancer patients could be used to stratify adjuvant therapy.

Comparative study of CD34, α‐SMA and h‐caldesmon expression in the stroma of gynaecomastia and male breast carcinoma

To address the fibroblastic/myofibroblastic nature of stroma in gynaecomastia and in male breast carcinoma, the expression of CD34, alpha-smooth muscle actin (SMA) and h-caldesmon in the stromal cells was investigated by immunohistochemistry. Representative archival paraffin blocks were collected from male patients with gynaecomastia (32 cases) and mammary carcinoma (24 cases) between 1984 and 2004 and CD34, alpha-SMA and h-caldesmon were assessed immunohistochemically using a streptavidin-biotin method. Thirty cases of gynaecomastia showed a CD34+, alpha-SMA- and h-caldesmon- immunophenotype with different CD34 staining intensity in the various histological subtypes. Positivity for alpha-SMA and negativity for CD34 and h-caldesmon was found in a case of florid gynaecomastia relating to reactive fibrosis due to previous surgical intervention. Acquisition of alpha-SMA expression by stromal fibroblasts but absence of CD34 staining was identified in 22 cases of male breast carcinoma. The immunophenotype of periductal connective tissue stroma in gynaecomastia appears to parallel the phenotype of normal breast stroma. In male breast carcinoma the stromal cell immunophenotype is similar to that of its female counterpart showing myofibroblastic differentiation. However alpha-SMA+ and CD34- are not specific to malignancy because such findings are also encountered in reactive fibrosis.

Tissue microarrays: Potential in the Indian subcontinent

Tissue microarrays (TMAs) are a means of combining hundreds of specimens of tissue on to a single slide for analysis simultaneously. The evolution of this technology to validate the results of cDNA microarrays has impacted tremendously in accurately identifying prognostic indicators significant in determining survival demographics for patients. TMAs can be generated from archival paraffin blocks, combined with sophisticated image analysis software for reading TMA immunohistochemistry, and a staggering amount of useful information can be generated in terms of the biomarkers useful in predicting patient outcome. There is a wide range of uses for the TMA technology including profiling of specific proteins in cancerous tissues and non-cancerous tissues. Given the wide variety of tissue resources available in India, investment in a dedicated TMA facility will be of immense use in the research arena in India. This review article discusses the basics of TMA construction, design, the software available for the analysis of this technology and its relevance to Indian scientists. A potential workflow structure for setting up a TMA facility is also included.

Antimony concentrations in nodal tissue can confirm sentinel node identity

The sentinel node biopsy procedure is a highly accurate method of staging patients with cutaneous melanoma and the tumor-harboring status of sentinel nodes is the most important prognostic factor. For the procedure to provide accurate prognostic information, however, it is essential that ‘true’ sentinel nodes are removed and examined thoroughly. A technique to confirm sentinel node identity may reduce the false-negative rate of the procedure. We have found that antimony (originating from the antimony sulfide colloid used for preoperative lymphoscintigraphy in our institution) can be measured in tissue sections of sentinel nodes using inductively coupled plasma mass spectrometry. The aims of this study were to determine whether antimony concentrations can be used to confirm that removed sentinel nodes are ‘true’ sentinel nodes and to differentiate sentinel nodes from nonsentinel nodes. In all, 24 patients who had both a tumor-positive sentinel node and a tumor-negative nonsentinel node removed from one regional node field during the same operation, were identified. Tissue sections (50 μm) thick were cut from archival paraffin blocks of each of the sentinel nodes and nonsentinel nodes. Antimony concentrations in the tissue sections were measured using inductively coupled plasma mass spectrometry. The median and mean concentrations of antimony in parts per billion were 0.526 and 1.198, respectively (range 0.020–7.596) in the sentinel nodes, and 0.043 and 0.123 (range 0–0.800) in the nonsentinel nodes (P=0.004). In four of the 24 pairs, both the presumed sentinel nodes and the nonsentinel nodes had very low antimony levels (less than 0.18 parts per billion), suggesting that nodes designated as sentinel nodes may not have been ‘true’ sentinel nodes. It is concluded that determination of antimony concentrations within sentinel nodes using the highly sensitive method of inductively coupled plasma mass spectrometry can confirm the identity of sentinel nodes and validate the sentinel node technique.