Identification of molecularly unique tumor-associated mesenchymal stromal cells in breast cancer patients.

The tumor microenvironment is a complex mixture of cell types that bi-directionally interact and influence tumor initiation, progression, recurrence, and patient survival. Mesenchymal stromal cells (MSCs) of the tumor microenvironment engage in crosstalk with cancer cells to mediate epigenetic control of gene expression. We identified CD90+ MSCs residing in the tumor microenvironment of patients with invasive breast cancer that exhibit a unique gene expression signature. Single-cell transcriptional analysis of these MSCs in tumor-associated stroma identified a distinct subpopulation characterized by increased expression of genes functionally related to extracellular matrix signaling. Blocking the TGFß pathway reveals that these cells directly contribute to cancer cell proliferation. Our findings provide novel insight into communication between breast cancer cells and MSCs that are consistent with an epithelial to mesenchymal transition and acquisition of competency for compromised control of proliferation, mobility, motility, and phenotype.

The Shared Core Resource as a Partner in Innovative Scientific Research: Illustration from an Academic Microscopy Imaging Center.

Core facilities have a ubiquitous and increasingly valuable presence at research institutions. Although many shared cores were originally created to provide routine services and access to complex and expensive instrumentation for the research community, they are frequently called upon by investigators to design protocols and procedures to help answer complex research questions. For instance, shared microscopy resources are evolving from providing access to and training on complex imaging instruments to developing detailed innovative protocols and experimental strategies, including sample preparation techniques, staining, complex imaging parameters, and high-level image analyses. These approaches require close intellectual collaboration between core staff and research investigators to formulate and coordinate plans for protocol development suited to the research question. Herein, we provide an example of such coordinated collaboration between a shared microscopy facility and a team of scientists and clinician-investigators to approach a complex multiprobe immunostaining, imaging, and image analysis project investigating the tumor microenvironment from human breast cancer samples. Our hope is that this example may be used to convey to institute administrators the critical importance of the intellectual contributions of the scientific staff in core facilities to research endeavors.

The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ.

Microenvironmental and molecular factors mediating the progression of Breast Ductal Carcinoma In Situ (DCIS) are not well understood, impeding the development of prevention strategies and the safe testing of treatment de-escalation. We addressed methodological barriers and characterized the mutational, transcriptional, histological, and microenvironmental landscape across 85 multiple microdissected regions from 39 cases. Most somatic alterations, including whole-genome duplications, were clonal, but genetic divergence increased with physical distance. Phenotypic and subtype heterogeneity was frequently associated with underlying genetic heterogeneity and regions with low-risk features preceded those with high-risk features according to the inferred phylogeny. B- and T-lymphocytes spatial analysis identified three immune states, including an epithelial excluded state located preferentially at DCIS regions, and characterized by histological and molecular features of immune escape, independently from molecular subtypes. Such breast pre-cancer atlas with uniquely integrated observations will help scope future expansion studies and build finer models of outcomes and progression risk.

Collagen Organization in Relation to Ductal Carcinoma In Situ Pathology and Outcomes.

BACKGROUND: There is widespread interest in discriminating indolent from aggressive ductal carcinoma in situ (DCIS). We sought to evaluate collagen organization in the DCIS tumor microenvironment in relation to pathologic characteristics and patient outcomes. METHODS: We retrieved fixed tissue specimens for 90 DCIS cases within the population-based Vermont DCIS Cohort. We imaged collagen fibers within 75 µm of the tumor/stromal boundary on hematoxylin and eosin-stained slides using multiphoton microscopy with second-harmonic generation. Automated software quantified collagen fiber length, width, straightness, density, alignment, and angle to the tumor/stroma boundary. Factor analysis identified linear combinations of collagen fiber features representing composite attributes of collagen organization. RESULTS: Multiple collagen features were associated with DCIS grade, necrosis pattern, or periductal fibrosis (P < 0.05). After adjusting for treatments and nuclear grade, risk of recurrence (defined as any second breast cancer diagnosis) was lower among cases with greater collagen fiber width [hazard ratio (HR), 0.57 per one standard deviation increase; 95% confidence interval (CI), 0.39-0.84] and fiber density (HR, 0.60; 95% CI, 0.42-0.85), whereas risk was elevated among DCIS cases with higher fiber straightness (HR, 1.47; 95% CI, 1.05-2.06) and distance to the nearest two fibers (HR, 1.47; 95% CI, 1.06-2.02). Fiber length, alignment, and fiber angle were not associated with recurrence (P > 0.05). Five composite factors were identified, accounting for 72.4% of the total variability among fibers; three were inversely associated with recurrence (HRs ranging from 0.60 to 0.67; P ≤ 0.01). CONCLUSIONS: Multiple aspects of collagen organization around DCIS lesions are associated with recurrence risk. IMPACT: Collagen organization should be considered in the development of prognostic DCIS biomarker signatures.

Long non-coding RNA chromogenic in situ hybridisation signal pattern correlation with breast tumour pathology.

AIM: Long non-coding RNAs (lncRNAs) are potential biomarkers for breast cancer risk stratification. LncRNA expression has been investigated primarily by RNA sequencing, quantitative reverse transcription PCR or microarray techniques. In this study, six breast cancer-implicated lncRNAs were investigated by chromogenic in situ hybridisation (CISH). METHODS: Invasive breast carcinoma (IBC), ductal carcinoma in situ (DCIS) and normal adjacent (NA) breast tissues from 52 patients were screened by CISH. Staining was graded by modified Allred scoring. RESULTS: HOTAIR, H19 and KCNQ1OT1 had significantly higher expression levels in IBC and DCIS than NA (p<0.05), and HOTAIR and H19 were expressed more strongly in IBC than in DCIS tissues (p<0.05). HOTAIR and KCNQ101T were expressed in tumour cells; H19 and MEG3 were expressed in stromal microenvironment cells; MALAT1 was expressed in all cells strongly and ZFAS1 was negative or weakly expressed in all specimens. CONCLUSION: These data corroborate the involvement of three lncRNAs (HOTAIR, H19 and KCNQ1OT1) in breast tumourigenesis and support lncRNA CISH as a potential clinical assay. Importantly, CISH allows identification of the tissue compartment expressing lncRNA.

Antisecretory medication is associated with decreased Helicobacter pylori detection in gastric marginal zone lymphoma.

Helicobacter pylori status influences the prognosis and management of gastric extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), so accurate determination of H pylori status is of clinical importance. The low rate of histologic H pylori positivity among gastric MALT lymphoma cases at our institution prompted investigation for possible causes. A case series of 24 patients as having gastric MALT lymphoma (with no diffuse large B-cell component) in a tertiary care setting between 1997 and 2010 was identified, and clinical records were reviewed. Immunohistochemical staining for H pylori and BCL10 was performed. This study received institutional review board approval (protocol number M13-033). Thirty-nine percent of cases (9/23) were H pylori positive by histology, and 4 additional patients had positive serologic results; overall, 57% of cases (13/23) were positive for H pylori. Treatment with antisecretory medications was associated with a lower likelihood of histologic positivity (13% among treated patients vs 75% among untreated; P = .04). Nuclear localization of BCL10 was seen in 2 cases and was not associated with H pylori status. Antisecretory medications decrease the likelihood of histologic detection of H pylori in gastric MALT lymphoma cases. Incorporation of results of serologic or other testing is needed to ensure correct classification with respect to H pylori status.

Human papillomavirus type distribution in invasive cervical cancers from Madhya Pradesh: implications for vaccination programs in central India.

Invasive cervical carcinoma (ICC) remains a leading cause of female mortality in India. There is a paucity of data about human papillomavirus (HPV) type distribution among ICCs from Central India. Formalin-fixed, paraffin-embedded ICC specimens (n=270 patients) from Madhya Pradesh state were screened for HPV by GP5/6 primer-based polymerase chain reaction followed by cycle sequencing and NCBI BLAST search. HPV was detected in 93.3% of the tumors. Thirteen types were detected: HPV16 (73.7%), HPV18 (11.9%), HPV45 (2.2%), HPV66 (1.5%), HPV35 (1.1%), and HPV56 (0.7%). HPV31, 51, 58, 59, 67, 82 and JEB2 were each seen in 1 case (0.4%). HPV16 was more common among women less than 40 yr than 40 yr or older (P=0.006), and HPV18 was more common in women aged 40 yr or older (P=0.013). Squamous cell carcinomas were more frequently HPV-positive than adenocarcinomas (P=0.01). HPV18 was more common in adenocarcinoma than in squamous cell carcinoma (P=0.02). These data contribute to the pan-India profile of HPV-type relationship to ICC and show the potential of current vaccines to greatly relieve (by up to 85.6%) the ICC burden in Central India. The findings are also suggestive that next-generation HPV vaccines might be designed on a regional basis rather than from compounded global data.

Intratumoral DNA content heterogeneity in breast carcinomas demonstrated by core punch tissue sampling and flow cytometry.

Further to advancements in instrumentation and fluorescent dye technologies, there has been a resurgence of interest in the flow cytometric assay of formalin-fixed, paraffin-embedded specimens. Here we present a novel, simple and effective alternative to whole block sectioning that allows selective multisampling of tissues within a specimen block and the investigation of intratumoral heterogeneity. Formalin-fixed, paraffin-embedded breast carcinoma specimens were core-punched using 1.0 mm diameter needles and assayed by flow cytometry using a modified Hedley method. Intratumoral heterogeneity for DNA index and per cent S-phase fraction was detected in 10 of 23 (44%) and 11 of 23 (47%) specimens respectively. Macro-level genomic heterogeneity is common in breast cancer even within a single surgical specimen block. Studies investigating the relationship of DNA content heterogeneity to other markers of genomic instability such as mutations, deletions, insertions and translocations are warranted.

HPV E6/E7 RNA in situ hybridization signal patterns as biomarkers of three-tier cervical intraepithelial neoplasia grade.

Cervical lesion grading is critical for effective patient management. A three-tier classification (cervical intraepithelial neoplasia [CIN] grade 1, 2 or 3) based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification, CAP-ASCCP LAST consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV E6/E7 and p16 mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic in situ hybridization (CISH). Specimens were also screened by immunohistochemistry for p16INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay's high sensitivity for HPV support the use of HPV E6/E7 RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when 'LSIL vs. HSIL' assignment is equivocal.

Evidence that alpha-9 human papillomavirus infections are a major etiologic factor for oropharyngeal carcinoma in black South Africans.

Human papillomavirus (HPV) infection, most commonly genotype 16 of the alpha-9 family, is implicated in the etiology of a subset of oropharyngeal squamous cell carcinomas (OPSC) worldwide. Data are scarce regarding OPSC in South Africans, and three prior studies suggest no significant etiologic role for HPV. We aimed to investigate for evidence of HPV etiology in OPSCs from black South Africans by polymerase chain reaction (PCR) methodologies with determination of HPV subtype by sequencing, in situ hybridization (ISH), and p16INK4a immunohistochemistry (IHC), as a surrogate marker for an HPV-driven tumor. It was hypothesized that HPV-driven tumors would be positive by PCR plus IHC and/or ISH whereas OPSCs with HPV background infections (HPV-passenger) would be positive by PCR alone. Formalin-fixed, paraffin embedded tissues from 51 OPSCs collected between 2005 and 2010 from 41 patients were analyzed for HPV by GP5?6? PCR (targeting the HPV L1 region), pU-1M/pU- 2R PCR (targeting the HPV E6/E7 region) and HPV-31 specific PCR (targeting the E5 region), chromogenic ISH, and p16INK4a IHC. All cases positive by PCR were subject to sequencing to determine HPV genotype. The patient mean age was 58.0 years and 88 % were male. Of the 51 evaluable tumors, 48 (94.1 %) were positive for HPV DNA by PCR: 25 (49.1 %) met criteria for an HPV-driven tumor, 23 (45.1 %) for HPV-passenger, and 3 (5.9 %) were HPV unrelated. Sequencing of the PCR-positive cases revealed the following genotypes: combined HPV-16 and 31 (41.7 %), HPV-31 (25.0 %), HPV-16 (22.9 %), combined HPV-16 and 18 (6.3 %), and a single case each of HPV 18 and HPV 33. Studies via ISH were negative in all cases. In accordance with worldwide trends but contrary to prior South African data, HPV likely plays an etiologic role in a significant subset (at least 49.1 %) of OPSC in black South Africans. We found that the alpha-9 HPV family, particularly HPV-16 and 31 either in combination or separately, to predominate in our sample tumors. The use of multiple PCR primers increased sensitivity of viral detection, and a HPV-31 specific primer confirmed the presence of this genotype in many samples. Further studies including HPV E6/E7 mRNA assays are needed to better elucidate the pathogenic role of HPV in black South African OPSCs.

Combined p16INK4a and human papillomavirus testing improves the prediction of cervical intraepithelial neoplasia (CIN II-III) in Thai patients with low-grade cytological abnormalities.

Thailand is in the process of developing a national cervical screening program. This study examined p16INK4a staining and HPV prevalence in abnormal cervical samples with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL), to evaluate the efficacy of combined HPV and p16INK4a detection to predict CIN II-III. Totals of 125 ASCUS and 87 LSIL cases were re-evaluated by Pap test and cervical cells of ASCUS and LSIL cases were prepared on slides for p16INK4a detection by immunocytochemistry. HPV genotyping of DNA extracts was performed by GP5+/6+ PCR and reverse line blot hybridization. Histopathologic tests were performed to identify cervical lesion. Total of 212 cases were diagnosed to normal (20), ASCUS (112), LSIL (78) and HSIL (2). HPV was detected in ASCUS (49/112, 43.8%), LSIL (60/78, 76.9%) and HSIL (2/2, 100%) cases. The majority of HPV positive samples typed for high-risk HPV. 55.7% (107/192) of abnormal cases (ASCUS, LSIL and HSIL) were positive p16INK4a. For the 111 HPV DNA positive cases, 34 of 49 (69.4%) ASCUS cases and 49 of 60 (81.7%) LSIL cases were p16INK4a positive. 140 biopsies were taken and histological classified: CIN negative (65 cases), CIN I (56 cases) and CIN II-III (19 cases). HPV DNA detection predicted CIN II-III with sensitivity and specificity of 84% and 49%, whereas p16INK4a staining showed higher sensitivity (89.5%) and specificity (56.2%). The prediction of CIN II-III was significantly better by combination of positive HPV DNA and p16INK4a with 93.8% sensitivity and 59.2% specificity. Detection of HPV DNA combined with p16INK4a in cervical cells can predict CIN II-III and may improve the screening diagnosis of Thai women at risk for CIN II-III or cancer.

Sarcomatoid renal cell carcinoma is an example of epithelial--mesenchymal transition.

BACKGROUND: Sarcomatoid renal cell carcinomas (SRCC) are composed of two cell populations, a sarcomatous component (SC) and a carcinomatous component (CC). SRCC are particularly aggressive and often present at an advanced stage at diagnosis. Epithelial-mesenchymal transition (EMT) has been proposed as a mechanism for the development of SC from CC. AIMS AND METHODS: E- to N-cadherin switching, localisation of ß-catenin, and expression of Snail and secreted protein acidic and rich in cysteine (SPARC) (markers of EMT) were studied to determine whether SRCC is an example of EMT. Expression of these markers was analysed by immunohistochemistry on 21 cases of SRCC that had both SC and CC and scored according to intensity and extent. RESULTS: E-cadherin expression was decreased in SC (Wilcoxon signed-rank test, p=0.0004) while N-cadherin expression was high in both components (p=0.46). Membranous ß-catenin expression was decreased in SC (p<0.0001) while cytoplasmic expression was increased (p=0.0002). Snail and SPARC had higher expression in SC (p=0.002 and p<0.0001, respectively). When the scores were dichotomised into low and high expression levels, the results using McNemar's test substantiated the above results. CONCLUSIONS: E- to N-cadherin switching, dissociation of ß-catenin from the membrane, and increased expression of Snail and SPARC in SC indicate that SRCC is an example of EMT. High expression of N-cadherin and Snail in CC suggest early involvement in initiating EMT. Once EMT is established, loss of E-cadherin, release of ß-catenin into the cytoplasm, and expression of SPARC correspond with mesenchymal phenotypic expression.

Screening for human papillomavirus in basaloid squamous carcinoma: utility of p16(INK4a), CISH, and PCR.

This study compares p16( INK4a) immunohistochemistry (IHC), HPV chromogenic in situ hybridization (ISH), and HPV polymerase chain reaction (PCR) genotyping for detection of HPV infection in basaloid squamous carcinoma (BSCC). A retrospective histopathological analysis of 40 BSCC from a single institution was carried out. p16 IHC, HPV DNA extraction and ISH, and HPV PCR genotyping were performed, and there was excellent agreement between all 3 methods of HPV detection. Analysis of variance yielded no significant differences between the results of the 3 tests ( P = .354) and Pearson product-moment correlation coefficients calculated for each pair of tests demonstrated direct correlation (r = .61 for PCR and IHC, r = .61 for PCR and ISH, and r = 1.00 for ISH and IHC). This supports the use of p16(INK4a) IHC as an initial screening tool for HPV infection in BSCC, while definitive evidence of HPV DNA can be sought subsequently with PCR or CISH.

Cervical cancer screening in north east Thailand using the visual inspection with acetic acid (VIA) test and its relationship to high-risk human papillomavirus (HR-HPV) status.

AIM: This study investigated the utility of visual inspection with acetic acid (VIA) as a method for cervical cancer screening in Thailand and examined the relationship of VIA to high-risk human papillomavirus (HR-HPV) status. METHODS: Cervical cells were collected from 160 patients receiving a Pap smear. VIA was performed on the cervix of the patients by application of 5% acetic acid. HPV screening of DNA extracted from cytology samples was performed by PCR using the GP5+/6+ primer system followed by reverse line blot hybridization genotyping. RESULTS: The majority (96.9%) of the patients were diagnosed with normal or inflammatory cytologic changes. 32.8% of normal cytology and 42.0% of inflammation cases showed positive acetowhite staining. 3.1%, 38.1% and 42.5% of subjects were positive for an abnormal Pap test, VIA test, and HPV DNA, respectively. VIA demonstrated 50% sensitivity and 66.7% specificity for abnormal histology with PPV and NPV values of 50% and 66.7%, respectively, whereas HPV DNA test showed 100% sensitivity. HPV16 was the most common (54.4%) and HR-HPV was detected in 36.3% of all cases. 48.5% of HR-HPV positive and 36.8% of HR-HPV negative cervices stained with acetowhite following the VIA test. CONCLUSION: The VIA test is a simple method for cervical cancer screening; however, a significant proportion of patients with normal or inflammatory cytology were positive by this test. Further, HR-HPV in women without acetowhite staining was demonstrated. Therefore, some form of HR-HPV detection test may be required for combination with cervical cell screening even in low-resource nations.

Multinucleated giant cells' incidence, immune markers, and significance: a study of 172 cases of papillary thyroid carcinoma.

Multinucleated giant cells (MGCs) are often detected in cases of papillary thyroid carcinoma (PTC). Their origin and significance, however, has not been established. One possibility is that they form in response to injury induced by fine needle aspiration biopsy (FNAB). Other hypotheses are that the chemically-altered colloid produced by PTC induces MGCs to act as colloidophages, or else MGCs are a non-specific immune response ingesting neoplastic follicle cells. We assigned 172 cases of PTC a semi-quantitative score for MGCs. Cases with "many" MGCs were immunohistochemically stained for AEI/AEIII, CD68, and CD163 to assess for epithelial vs histiocytic differentiation, and for thyroglobulin and TTF-1 to assess for MGC ingestion of colloid or thyroid follicle cells respectively. Overall, we identified MGCs in 100/172 (58.1%) PTC specimens; in 45 (26.2%), "many" MGCs were found, while in 55 (31.9%) MGCs were "few." The mean sizes of PTC in cases with many as opposed to rare/no MGCs was 2.50 cm vs 1.8 [P = 0.003]. The cases of PTC with many MGCs had higher multifocality (26/45 vs 51/127 [P = 0.06]), extrathyroidal extension (21/45 vs 36/127 [P = 0.03]), and recurrence (8/45 vs 9/127 [P = 0.08]), than did cases with rare or no MGCs. The majority of patients both with and without numerous MGCs had previous histories of FNA or hemilobectomy: 40/45 and 99/127 respectively (P = 0.062). The majority of MGCs were positive for CD68 (45/45), CD163 (44/45), thyroglobulin (34/45) and negative for AEI/AEIII (44/45) and TTF-1 (44/45). These results indicate that MGCs in PTC are of histiocytic origin. Cases of PTC with many MGCs have a significantly greater likelihood of extrathyroidal extension and greater tumor size than cases with few/no MGCs. MGCs appear to be functioning largely as colloidophages.

Valves of the deep venous system: an overlooked risk factor.

Deep venous valves are frequent sites of deep venous thrombosis initiation. However, the possible contribution of the valvular sinus endothelium has received little attention in studies of thrombosis risk. We hypothesized that the endothelium of valve sinus differs from that of vein lumen with up-regulation of anticoagulant and down-regulation of procoagulant activities in response to the local environment. In pursuit of this hypothesis, we quantified endothelial protein C receptor (EPCR), thrombomodulin (TM), and von Willebrand factor (VWF) by immunofluorescence in great saphenous veins harvested at cardiac bypass surgery. We found significantly increased expression of EPCR and TM in the valvular sinus endothelium as opposed to the vein lumenal endothelium, and the opposite pattern with VWF (paired t test for TM and EPCR, each P < .001; for VWF, P = .01). These data support our hypothesis and suggest that variation in valvular sinus thromboresistance may be an important factor in venous thrombogenesis.

Immunohistochemical analysis of ER, PR, Her2 and CK5/6 in infiltrative breast carcinomas in Indian patients.

AIM: Breast cancer is biologically a heterogeneous disease. Patients with the same diagnostic profile have markedly different clinical outcomes. Gene expression studies identified distinct breast cancer subtypes that differ in prognosis. Aim is to identify the immunohistochemical subtypes of breast carcinoma and correlate the results with pathological features associated with adverse prognosis in our study population. METHOD: We included 107 consecutive cases of invasive breast carcinoma and sub classified using immunohistochemical staining for ER, PR, Her2, and CK5/6 into the following subtypes: luminal A, luminal B, basal-like, Her2(+) and unclassified. Associations between tumor subtypes and tumor characteristics were examined. RESULTS: The proportion of each subtype in our patient population was: luminal A 37.4%, luminal B 11.1%, Her2(+) 29% and basal-like 7.5%. The following variables were significantly associated with IHC breast cancer subtypes: patient age (p<0.05), overall histopathology grade (p<0.001), nuclear grade (p<0.005) and mitotic index (p<0.001). Her2(+) and basal like subtypes were associated with poor differentiation (p<0.01), higher nuclear grade (p<0.05) and high mitotic index (p<0.05). CONCLUSIONS: Our data show a higher proportion of patients in the study population undergo total mastectomy and harbor poorly differentiated, node positive tumors than reported. There was also a relatively high percentage of the Her2(+) subtype (29%).

The polymerase chain reaction and pathology practice.

Pathology practice is increasingly augmented with molecular tests for improved diagnostics and patient management. The polymerase chain reaction (PCR) is foremost amongst these techniques. This review explains the principles of PCR and the methodological factors that contribute to a successful assay. Key PCR technique variations, such as reverse transcription (RT)-PCR and quantitative real-time (q) PCR, are described and an overview is provided of how PCR products are analysed. The review includes examples of PCR usage in clinical practice for the detection of infectious and genetic diseases, for tumour diagnostics and in molecular forensic applications such as specimen identity confirmation.

Smoking increases the risk of high-grade vaginal intraepithelial neoplasia in women with oncogenic human papillomavirus.

OBJECTIVES: In a large retrospective study, the association of smoking with human papillomavirus (HPV) genotype and vaginal intraepithelial neoplasia (VAIN) grade was analyzed. METHODS: A SNOMED search was performed for vaginal biopsy or resection specimens diagnosed as VAIN over an 11-year period. The diagnosis of VAIN grade was confirmed by histological review. HPV genotype was determined by GP5+/6+ PCR and dot blot hybridization with type-specific oligonucleotide probes. Smoking history was obtained by chart review. Statistical analysis was performed using the chi-square test. RESULTS: We identified specimens from 111 patients (age range 15-84); 64% (n=71) were diagnosed with high-grade VAIN (HGVAIN) and 36% (n=40) with low-grade VAIN (LGVAIN). High-risk (HR) HPV genotypes were identified in 83% of specimens (n=92), other types in 17% (n=19). Twenty-one different HPV genotypes were detected in total. Smoking history was available for 81% (n=90). Forty-one percent (n=37) had a positive smoking history. There was no significant difference in infection with HR vs. other types (p=0.92) among smokers when compared to non-smokers. In patients with HR HPV genotypes, smokers were at an increased risk for HGVAIN lesions when compared to patients who had never smoked (83% vs. 59%, p=0.02). CONCLUSIONS: These data indicate an increased risk for HGVAIN in HR HPV positive women who smoke compared to HR HPV positive non-smokers.