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Mohammed Z M A, Edwards J, Orange C, Mallon E, Doughty J C, McMillan D C & Going J J (2012) Histopathology 61, 283-292 Breast cancer outcomes by steroid hormone receptor status assessed visually and by computer image analysis Aims: To compare the assessment of steroid hormone receptor immunohistochemistry by eye and by computer-aided image analysis, and to examine their relationships with survival in breast cancer. Methods and results: Allred scores and weighted histoscores for oestrogen receptor (ER) and progesterone receptor (PR) immunohistochemistry were determined by eye (visual histoscore) for 459 primary invasive ductal breast carcinomas in triplicate tissue microarrays. Histoscores were also determined by computerized image analysis (automated histoscore). ER and PR status determined by these different methods were compared with each other and in their ability to predict survival over at least 142 months of follow-up. Allred and visual histoscore were highly associated for ER and PR (both P < 0.001). By univariate analysis, Allred score and visual histoscore for ER and PR were highly associated with recurrence-free and cancer-specific survival (both P < 0.001) in patients with invasive ductal breast cancer overall, in those who received tamoxifen, and in those with recurrence on tamoxifen. Visual and automated histoscores were in excellent agreement for ER and PR (both P < 0.001), and were equally effective in predicting recurrence and survival for patients with invasive breast cancer who received tamoxifen. Conclusions: Automated histoscore appears to be a valid alternative to visual histoscore or Allred score for determining ER and PR status. [ABSTRACT FROM AUTHOR]

Hypoxia inducible factor-1 alpha (HIF-1α) is known as an important transcription factor in endocrine tumours. It is elevated in hypoxic tumour microenvironment, increasing angiogenesis and enabling tumour cells to enter the circulation. We therefore hypothesised that patients with advanced prostate cancer disease have high tumoural HIF-1α xpression and worse disease specific survival. Aim of this study was to assess expression of HIF-1α in prostate cancer specimens taken before and after castrate resistance to address its cellular location and to examine if this is associated with clinicopathological features and clinical outcome of the particular prostate cancer cohort. 50 pairs of hormone naive and castrate resistant prostate cancer specimens were analysed employing tissue microarray technology. Immunohistochemistry was performed using an antibody to HIF-1α. HIF-1α expression was observed in both, cytoplasm and nucleus. Cytoplasmic HIF-1α expression correlated positively with metastases at diagnosis (p=0.005), whereas nuclear HIF-1α expression correlated with metastases at relapse (p=0.041). Cytoplasmic and nuclear HIF-1α expression did not change from hormone naive to hormone castrate resistant tumours. No significant association was observed in this study between tumoural HIF-1α expression, biochemical relapse and patient survival. HIF-1α was associated with the presence of metastases at time of diagnosis and time of relapse. HIF-1α is likely to play a role in progressive prostate cancer. [ABSTRACT FROM AUTHOR]

Aims To evaluate the relationships between immunohistochemical markers related to cellular senescence, cell proliferation and histological grade of epithelial dysplasia ( OD) of the oral cavity. In addition, the predictive value of these markers for progression of OD was assessed. Methods and results Retrospective immunohistochemical analyses were performed on 86 formalin-fixed paraffin-embedded specimens of OD and oral squamous cell carcinoma ( OSCC) for Ki67, phosphorylated histone H2 AX (γH2 AX), p53, p16, trimethyl-histone H3 (Lys9) (H3K9me3) and cyclin D1 (CycD1). Three separate areas representing the highest severity of OD on each slide were annotated digitally by two independent pathologists. Mean automated histoscores of the selected markers were generated and compared to that of age-matched healthy controls ( n = 24). Follow-up data of OD were retrieved and anonymized by a clinical team member and linked using unique participant identifiers. The median follow-up was 10.9 years (interquartile range: 10.1-11.5). Ki67 ( P < 0.0001), γH2 AX ( P = 0.03) and p53 ( P = 0.04) were increased significantly with higher histological grade of OD. γH2 AX ( P = 0.03), but not histological grade of OD ( P = 0.73), was associated prospectively with disease progression. Using the median histoscore for γH2 AX (median histoscore = 17) as a cut-off, histoscore ≥17 was associated with an increased risk of disease progression [hazard ratio ( HR) = 3.15, 95% confidence interval ( CI): 1.41-7.39, P = 0.0064]. Conclusions Although proliferation marker Ki67, DNA damage/checkpoint markers γH2 AX and p53 were increased in higher grade of OD, only γH2 AX was predictive of disease progression. These observations may reflect the role of DNA replicative stress in the transformation from OD to OSCC. Larger studies should evaluate whether γH2 AX can be used as a predictive marker of OD. [ABSTRACT FROM AUTHOR]

Introduction: Recently, we showed that the length of cardiac mucosa in healthy volunteers correlated with age and obesity. We have now examined the immunohistological characteristics of this expanded cardia to determine whether it may be due to columnar metaplasia of the distal oesophagus. Methods: We used the squamocolumnar junction (SCJ), antral and body biopsies from the 52 Helicobacter pylori-negative healthy volunteers who had participated in our earlier physiological study and did not have hiatus hernia, transsphincteric acid reflux, Barrett's oesophagus or intestinal metaplasia (IM) at cardia. The densities of inflammatory cells and reactive atypia were scored at squamous, cardiac and oxyntocardiac mucosa of SCJ, antrum and body. Slides were stained for caudal type homeobox 2 (CDX-2), villin, trefoil factor family 3 (TFF-3) and liver-intestine (LI)-cadherin, mucin MUC1, Muc-2 and Muc-5ac. In addition, biopsies from 15 Barrett's patients with/ without IM were stained and scored as comparison. Immunohistological characteristics were correlated with parameters of obesity and high-resolution pH metry recording. Results: Cardiac mucosa had a similar intensity of inflammatory infiltrate to non-IM Barrett's and greater than any of the other upper GI mucosae. The immunostaining pattern of cardiac mucosa most closely resembled non-IM Barrett's showing only slightly weaker CDX-2 immunostaining. In distal oesophageal squamous mucosa, expression of markers of columnar differentiation (TFF-3 and LI-cadherin) was apparent and these correlated with central obesity (correlation coefficient (CC)=0.604, p=0.001 and CC=0.462, p=0.002, respectively). In addition, expression of TFF-3 in distal oesophageal squamous mucosa correlated with proximal extension of gastric acidity within the region of the lower oesophageal sphincter (CC=-0.538, p=0.001). Conclusions: These findings are consistent with expansion of cardia in healthy volunteers occurring by squamo columnar metaplasia of distal oesophagus and aggravated by central obesity. This metaplastic origin of expanded cardia may be relevant to the substantial proportion of cardia adenocarcinomas unattributable to H. pylori or transsphincteric acid reflux. [ABSTRACT FROM AUTHOR]

Background: Lymphovascular invasion (LBVI) including lymphatic (LVI) and blood (BVI) vessel invasion is a critical step in cancer metastasis. In breast cancer, the optimal detection method of LBVI remains unclear. This research aimed to compare the prognostic value of different assessments of the LVI and BVI in patients with early breast cancer. Methods: The study cohort included 360 patients with a median follow-up of 168 months. LBVI on H&E sections (LBVIH&E) was reviewed centrally and blinded to the pathology report. Immunohistochemical staining for D2-40 and Factor VIII was performed to identify LVID2-40 and BVIFVIII. Results: LBVIH&E, LVID2-40 and BVIFVIII were present in 102 (28%), 127 (35%) and 59 (16%) patients respectively. In node-negative patients (206), LBVIH&E, LVID2-40 and BVIFVIII were present in 41 (20%), 53 (26%) and 21 (10%) respectively. In triple-negative patients (120), LBVIH&E, LVID2-40 and BVIFVIII were present in 35 (29%), 46 (38%) and 16 (13%) respectively. LBVIH&E was significantly associated with tumour recurrence in the whole cohort (P < 0.001), node-negative patients (P = 0.001) and triple-negative patients (P = 0.004). LVID2-40 and BVIFVIII were significantly associated with tumour recurrence in whole cohort, node-negative (all P < 0.001) and triple-negative patients (P = 0.002). In multivariate survival analysis, only LVID2-40 and BVIFVIII were independent predictors of cancer specific survival in the whole cohort (P = 0.023 and P < 0.001 respectively), node-negative patients (P =0.004 and P= 0.001 respectively) and triple-negative patients (P =0.014 and P= 0.001 respectively). Conclusion: Assessment of LVI and BVI by IHC using D2-40 and Factor VIII improves prediction of outcome in patients with node-negative and triple-negative breast cancer. [ABSTRACT FROM AUTHOR]

Objective Colorectal cancer (CRC) is a major contributor to cancer mortality and morbidity. LIM kinase 2 (LIMK2) promotes tumour cell invasion and metastasis. The objectives of this study were to determine how LIMK2 expression is associated with CRC progression and patient outcome, and to use genetically modified Drosophila and mice to determine how LIMK2 deletion affects gastrointestinal stem cell regulation and tumour development. Design LIMK2 expression and activity were measured by immunostaining tumours from CRC-prone mice, human CRC cell lines and 650 human tumours. LIMK knockdown in Drosophila or Limk2 deletion in mice allowed for assessment of their contributions to gastrointestinal stem cell homeostasis and tumour development. Results LIMK2 expression was reduced in intestinal tumours of cancer-prone mice, as well as in human CRC cell lines and tumours. Reduced LIMK2 expression and substrate phosphorylation were associated with shorter patient survival. Genetic analysis in Drosophila midgut and intestinal epithelial cells isolated from genetically modified mice revealed a conserved role for LIMK2 in constraining gastrointestinal stem cell proliferation. Limk2 deletion increased colon tumour size in a colitisassociated colorectal mouse cancer model. Conclusions This study revealed that LIMK2 expression and activity progressively decrease with advancing stage, and supports the hypothesis that there is selective pressure for reduced LIMK2 expression in CRC to relieve negative constraints imposed upon gastrointestinal stem cells. [ABSTRACT FROM AUTHOR]

Purpose: The introduction of the bowel cancer screening programme has resulted in increasing numbers of patients being diagnosed with node-negative disease. Unfortunately, approximately 30 % will develop recurrence following surgery. Given the toxicity associated with adjuvant chemotherapy, it is important to identify high-risk patients who may benefit from adjuvant therapy. This study aims to identify which clinicopathological factors and genetic profiling markers predict outcome in node-negative disease. Methods: Forty-nine microsatellite stable (MSS) patients undergoing curative resection between 1991 and 1993 were included. Local immune response was assessed by Klintrup criteria and vascular invasion status assessed through Miller’s elastin staining. Comparative genomic hybridisation (CGH) on a range of loci provided data on allelic imbalance. Analysis of survival included clinicopathological and CGH data in a multivariate (Cox) model. Results: On binary logistical regression analysis, 4p deletion was independently associated with low Klintrup score (HR 0.16; 95 % CI (0.03–0.96); P = 0.045), venous invasion (HR 4.19; 95 % CI (1.08–16.29); P = 0.039) and higher Dukes’ stage (HR 6.43; 95 % CI (1.22–33.97); P = 0.028). Minimum follow-up was 109 months and there were 24 cancer deaths. On multivariate analysis, high Klintrup score (HR 0.33; 95 % CI (0.12–0.93); P = 0.036), 4p− (HR 4.01; 95 % CI (1.58–10.21); P = 0.004) and 5q− (HR 3.81; 95 % CI (1.54–9.47); P = 0.004) were significantly associated with survival. Conclusion: 4p−, 5q− and low Klintrup score were independently associated with poor cancer-specific survival in node-negative MSS colorectal cancer. Confirmatory work in a larger cohort is needed to determine whether these markers may be used to identify patients who may benefit from adjuvant chemotherapy. [ABSTRACT FROM AUTHOR]

Background Several well-established tumour prognostic factors are used to guide the clinical management of patients with breast cancer. Lymphovascular invasion and angiogenesis have also been reported to have some promise as prognostic factors. The aim of the present study was to examine the prognostic value of tumour lymphovascular invasion and microvessel density compared with that of established prognostic factors in invasive ductal breast cancer. Methods In addition to hormone receptor status and Ki-67 proliferative activity, lymphovascular invasion and microvessel density and their relationship with survival were examined in patients with invasive ductal breast cancer. Full sections and tissue microarrays (n = 384 patients) were utilised to assess these factors and were scored by appropriate methods. Results On univariate analysis tumour size (P < 0.05), lymph node involvement (P < 0.01), lymphovascular invasion (P < 0.05), microvessel density (P < 0.05) and local- regional treatment (P < 0.01) were associated with poorer survival in ER negative tumours. On multivariate analysis in ER negative tumours lymph node involvement (P < 0.01) and localregional treatment (P < 0.05) were independently associated with poorer cancer-specific survival. On univariate analysis tumour grade (P < 0.05), lymph node involvement (P < 0.001), HER-2 (P < 0.05), Ki-67 (P < 0.01) and lymphovascular invasion (P < 0.001) were associated with poorer survival in ER positive tumours. On multivariate analysis lymph node involvement (P < 0.001), Ki-67 (P < 0.001) and lymphovascular invasion (P < 0.05) were independently associated with poorer cancer-specific survival in ER positive tumours. Conclusion Lymphovascular invasion but not microvessel density was independently associated with poorer survival in patients with ER positive but not ER negative invasive ductal breast cancer. [ABSTRACT FROM AUTHOR]

We previously reported that AR phosphorylation at serine 213 was associated with poor outcome and may contribute to prostate cancer development and progression. This study investigates if specific AR phosphorylation sites have differing roles in the progression of hormone naïve prostate cancer (HNPC) to castrate resistant disease (CRPC). A panel of phosphospecific antibodies were employed to study AR phosphorylation in 84 matched HNPC and CRPC tumours. Immunohistochemistry measured Androgen receptor expression phosphorylated at serine residues 94 (pAR94), 308 (pAR308), 650(pAR650) and 791 (pAR791). No correlations with clinical parameters were observed for pAR94 or pAR650 in HNPC or CRPC tumours. In contrast to our previous observation with serine 213, high pAR308 is significantly associated with a longer time to disease specific death (p = 0.011) and high pAR791 expression significantly associated with a longer time to disease recurrence (p = 0.018) in HNPC tumours and longer time to death from disease recurrence (p = 0.040) in CRPC tumours. This observation in CRPC tumours was attenuated in high apoptotic tumours (p = 0.022) and low proliferating tumours (p = 0.004). These results demonstrate that understanding the differing roles of AR phosphorylation is necessary before this can be exploited as a target for castrate resistant prostate cancer. [ABSTRACT FROM AUTHOR]

Introduction: Evidence for the role of inflammation in benign prostatic hyperplasia (BPH) is conflicting. Establishing the prognostic significance of local and systemic inflammation and tissue necrosis scoring systems in BPH may elucidate the potential of inflammatory pathways as a target of therapeutic intervention in these patients. Patients and Methods: Consecutive patients with histological BPH diagnosed between 1996 and 2005 were identified. Systemic inflammation was assessed by the modified Glasgow prognostic score (mGPS), local inflammation by the Klintrup-Makinen criteria and tissue necrosis was evaluated by an extent-based classification. Results: In 392 BPH patients, there was a trend for increased local inflammation and tissue necrosis to be associated with shorter time to failure of pre-operative medical treatment of BPH (p = 0.096 and 0.088, respectively). High modified Glasgow prognostic score was associated with older age (p = 0.002) and higher levels of deprivation (measured by the Scottish Index of Multiple Deprivation) (p = 0.021). Conclusions: The prognostic use of established scoring systems of systemic and local inflammation and tissue necrosis in BPH requires further investigation. It remains unclear as to whether targeting inflammation in BPH has therapeutic potential. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Introduction: Inflammation is postulated to link obesity and benign prostatic hyperplasia (BPH). The role of inflammation and the prognostic significance of body mass index (BMI) was investigated In BPH patients. Subjects and Methods: Consecutive patients with histological BPH were identified from 1996 to 2005. Systemic inflammation was assessed by modified Glasgow Prognostic Score (mGPS) and local in-flammation by Klintrup-Makinen criteria. Results: In 392 pa-tients, BMI was associated with cardiovascular disease (p = 0.033), type 2 diabetes mellltus (p = 4.45 x 10"8), aspirin us-age (p = 0.018) and failure of surgical treatment (p = 0.001). mGPS and Klintrup-Makinen scores were not associated with clinical variables or outcome measures. On multivariate analysis BMI was an Independent predictor of time to failure of surgical management of BPH, HR 1.56 (95% CI 1.11-2.19), p = 0.010. Conclusions: The mGPS and Klintrup-Makinen scores were not associated with BMI in BPH patients. High BMI is associated with failure of surgical management of BPH. Preoperative weight loss should be strongly encour-aged in these patients. [ABSTRACT FROM AUTHOR]

Aims: To compare visual and computerized image analysis of HER2 immunohistochemistry (IHC) with fluorescence in-situ hybridization (FISH) for HER2 status, and to examine the relationships with outcome in patients with primary operable invasive ductal breast cancer. Methods and results: Tissue microarrays for 431 breast cancer patients were used to compare different approaches to the assessment of HER2 status. The cores were scored visually and with the Slidepath Tissue IA system, using the NICE-approved scoring system for the HercepTest, as well as by FISH. The agreement between visual and image analysis of HER2 IHC was excellent [interclass correlation coefficient (ICCC) = 0.95, rs = 0.90, r = 0.91, k = 0.81, and P < 0.001]. The agreement of HER2 FISH with visual and image analysis of HER2 IHC was also excellent (ICCC = 0.95 and ICCC = 0.92, respectively). Univariate survival analysis showed equivalent associations of visual and image analysis of HER2 and HER2 FISH with both recurrence-free survival (all P < 0.01) and cancer-specific survival (all P < 0.05) in patients with invasive ductal breast cancer. Conclusions: Computerized image analysis of HER2 IHC gives results comparable to those obtained with visual assessment, with possible advantages in diagnostic pathology. [ABSTRACT FROM AUTHOR]

The cytokine TGF-beta acts as a tumor suppressor in normal epithelial cells and during the early stages of tumorigenesis. During malignant progression, cancer cells can switch their response to TGF-beta and use this cytokine as a potent oncogenic factor; however, the mechanistic basis for this is poorly understood. Here we demonstrate that downregulation of disabled homolog 2 (DAB2) gene expression via promoter methylation frequently occurs in human squamous cell carcinomas (SCCs) and acts as an independent predictor of metastasis and poor prognosis. Retrospective microarray analysis in an independent data set indicated that low levels of DAB2 and high levels of TGFB2 expression correlate with poor prognosis. Immunohistochemistry, reexpression, genetic knockout, and RNAi silencing studies demonstrated that downregulation of DAB2 expression modulated the TGF-beta/Smad pathway. Simultaneously, DAB2 downregulation abrogated TGF-beta tumor suppressor function, while enabling TGF-beta tumor-promoting activities. Downregulation of DAB2 blocked TGF-beta-mediated inhibition of cell proliferation and migration and enabled TGF-beta to promote cell motility, anchorage-independent growth, and tumor growth in vivo. Our data indicate that DAB2 acts as a tumor suppressor by dictating tumor cell TGF-beta responses, identify a biomarker for SCC progression, and suggest a means to stratify patients with advanced SCC who may benefit clinically from anti-TGF-beta therapies. [ABSTRACT FROM AUTHOR]

Objectives: Real-time monitoring of disease status would be beneficial for timely decision making in the treatment of urothelial cancer (UC), and may accelerate the evaluation of clinical trials. Use of cell free tumor DNA (cftDNA) as a biomarker in liquid biopsy is minimally invasive and its successful use has been reported in various cancer types, including UC. The objective of this study was to evaluate the use of digital droplet PCR (ddPCR)-based assays to monitor UC after treatment.Method and Materials: Blood, urine and matching formalin fixed, paraffin embedded diagnostic specimens were collected from 20 patients diagnosed with stage T1 (n = 2) and T2/T3 (n = 18) disease. SNaPshot assays, Sanger sequencing and whole exome sequencing were used to identify tumor-specific mutations, and somatic mutation status was confirmed using patient-matched DNAs extracted from buffy coats and peripheral blood mononucleocytes. The ddPCR assays of the tumor-specific mutations were used to detect the fractional abundance of cftDNA in plasma and urine.Results: SNaPshot and Sanger sequencing identified point mutations in 70% of the patients that were assayable by ddPCR. Cases of remission and relapse monitored by assays for PIK3CA E542K and TP53 Y163C mutations in plasma and urine concurred with clinical observations up to 48 months from the start of chemotherapy. A new ddPCR assay for the telomerase reverse transcriptase (TERT) promoter (-124) mutation was developed. The TERT assay was able to detect mutations in cases below the limit of detection by SNaPshot. Whole exome sequencing identified a novel mutation, CNTNAP4 G727*. A ddPCR assay designed to detect this mutation was able to distinguish mutant from wild-type alleles.Conclusions: The study demonstrated that ddPCR assays could be used to detect cftDNA in liquid biopsy monitoring of the post-therapy disease status in patients with UC. Overall, 70% of the patients in our study harbored mutations that were assayable by ddPCR. [ABSTRACT FROM AUTHOR]

Background: Lymphovascular invasion (LBVI) including lymphatic (LVI) and blood (BVI) vessel invasion is a critical step in cancer metastasis. In breast cancer, the optimal detection method of LBVI remains unclear. This research aimed to compare the prognostic value of different assessments of the LVI and BVI in patients with early breast cancer.Methods: The study cohort included 360 patients with a median follow-up of 168 months. LBVI on H&E sections (LBVIH&E) was reviewed centrally and blinded to the pathology report. Immunohistochemical staining for D2-40 and Factor VIII was performed to identify LVID2-40 and BVIFVIII.Results: LBVIH&E, LVID2-40 and BVIFVIII were present in 102 (28%), 127 (35%) and 59 (16%) patients respectively. In node-negative patients (206), LBVIH&E, LVID2-40 and BVIFVIII were present in 41 (20%), 53 (26%) and 21 (10%) respectively. In triple-negative patients (120), LBVIH&E, LVID2-40 and BVIFVIII were present in 35 (29%), 46 (38%) and 16 (13%) respectively. LBVIH&E was significantly associated with tumour recurrence in the whole cohort (P < 0.001), node-negative patients (P = 0.001) and triple-negative patients (P = 0.004). LVID2-40 and BVIFVIII were significantly associated with tumour recurrence in whole cohort, node-negative (all P < 0.001) and triple-negative patients (P = 0.002). In multivariate survival analysis, only LVID2-40 and BVIFVIII were independent predictors of cancer specific survival in the whole cohort (P = 0.023 and P < 0.001 respectively), node-negative patients (P = 0.004 and P = 0.001 respectively) and triple-negative patients (P = 0.014 and P = 0.001 respectively).Conclusion: Assessment of LVI and BVI by IHC using D2-40 and Factor VIII improves prediction of outcome in patients with node-negative and triple-negative breast cancer. [ABSTRACT FROM AUTHOR]