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The accurate estimation of prognosis in patients with melanoma is of increasing importance with novel adjuvant therapies on the horizon. The current prediction of prognosis employs techniques involving sentinel lymph node biopsy, which carries an associated morbidity and is of little use in patients who develop direct distant metastases or direct in-transit metastases. New strategies or factors are therefore needed to improve the accuracy of determination of prognosis. nm23 is a putative metastasis suppressor; however, conflicting data exist as to its role in melanoma progression and its use as a potential prognostic marker. The purpose of this study was to use the technique of tissue microarray to study a cohort of melanoma patients with long-term follow-up data in order to ascertain its potential use as a prognostic marker. One hundred and twenty patients with primary cutaneous melanoma were included in the tissue microarray and a commercially available immunohistochemical marker for nm23 was used for protein detection. nm23 expression was strongly correlated with Clark's level (P

To establish the relationships between quantitative perfusion computed tomography (CT) parameters-specifically, primary tumor blood flow, blood volume, transit time, and permeability surface-area product-and immunohistologic markers of angiogenesis in colorectal cancer.After institutional review board approval and informed patient consent were obtained for this prospective study, 23 patients (11 men, 12 women; mean age, 68.4 years; age range, 34.8-87.1 years) with colorectal adenocarcinoma underwent a 65-second perfusion CT examination, and tumor blood flow, blood volume, mean transit time, and permeability surface-area product were determined. After surgery, resected specimens were sectioned and stained immunohistochemically to identify CD34 for quantification of microvessel density (MVD), to identify smooth muscle actin for assessment of pericyte coverage index, to identify vascular endothelial growth factor (VEGF), and to identify glucose transporter protein (GLUT-1). Perfusion CT measurements were correlated with MVD, pericyte coverage index, VEGF expression, and GLUT-1 expression by using Pearson or Spearman rank correlation analysis, with significance assigned at the 5% level.Mean blood flow, blood volume, transit time, and permeability surface-area product values were 72.1 mL/min/100 g of tissue +/- 28.4 (standard deviation), 6.2 mL/100 g of tissue +/- 1.4, 9.3 seconds +/- 3.9, and 13.9 mL/min/100 g of tissue +/- 3.2, respectively. Blood volume (r = 0.59, P = .002) and permeability surface-area product (r = 0.46, P = .03) correlated positively with MVD, but blood flow (r = 0.27, P = .22) and transit time (r = -0.18, P = .44) did not. There were no significant associations between any perfusion CT parameter and pericyte coverage index (ror= 0.36, P.05), VEGF score (rhoor= 0.30, Por= .15), or GLUT-1 score (rho0.21, Por= .33).Tumor permeability surface-area product and blood volume correlate positively with MVD and may reflect the microvascularity of colorectal tumors.

Delays in tissue fixation following tumour vascular clamping and extirpation may adversely affect subsequent protein and mRNA analysis. This study investigated the effect of surgically induced ischaemia in a xenograft model of a colorectal cancer on the expression of a range of prognostic, predictive, and hypoxic markers, with a particular emphasis on thymidylate synthase. Vascular occlusion of human tumour xenografts by D-shaped metal clamps permitted defined periods of tumour ischaemia. Alterations in protein expression were measured by immunohistochemistry and spectral imaging, and changes in mRNA were measured by reverse transcriptase–polymerase chain reaction. Thymidylate synthase expression decreased following vascular occlusion, and this correlated with cyclin A expression. A similar reduction in dihydropyrimidine dehydrogenase was also seen. There were significant changes in the expression of several hypoxic markers, with carbonic anhydrase-9 showing the greatest response. Gene transcriptional levels were also noted to change following tumour clamping. In this xenograft model, surgically induced tumour ischaemia considerably altered the gene expression profiles of several prognostic and hypoxic markers, suggesting that the degree of tumour ischaemia should be minimised prior to tissue fixation.

Quantification of protein expression in tissue sections stained by immunohistochemistry has traditionally involved visual grading techniques. However, if these results are to be used to predict tumor behavior and permit targeted therapy, there is a need for more accurate, objective, and reproducible methods. This study investigated the utility of spectral imaging as a method of quantifying thymidylate synthase protein expression in immunohistochemically stained sections of primary rectal cancer and normal rectal mucosa by comparing it with the current gold standard of manual visual grading. There was good correlation between estimates of thymidylate synthase stain intensity and area derived by spectral imaging and visual grading in both tumor and normal mucosal sections, suggesting that spectral imaging is a valid way of quantifying biologic sections stained by immunohistochemistry.

Despite the use of sentinel node biopsy techniques, the search continues for other strategies to improve the accuracy of estimating prognosis in melanoma patients. Various biomarkers have previously been studied for use in this role, but none has yet achieved acceptance in routine practice. We have applied the novel technology of tissue microarray for the high throughput screening of a cohort of 120 primary cutaneous melanoma specimens for expression of the transmembrane glycoprotein CD44, splice variant 3 (v3), which has previously been implicated in tumor progression. A highly significant correlation between CD44v3 expression and Breslow thickness, Clark’s level and patient age was demonstrated (Spearman correlation p < 0.001). Regarding clinical outcome, CD44v3 expression was shown to be significantly associated with better outcome (v 2 5 7.2219, p 5 0.0072). Furthermore, subgroup analysis revealed a sequentially improved survival probability associated with the intensity of CD44v3 staining (v 2 5 12.5162, p 5 0.0058). Analysis in a Cox multivariate model, however, did not show CD44v3 to be independently predictive of prognosis. The implications of these findings are considered, and the use of CD44v3 as a potential prognostic marker or a target for therapeutic manipulation are discussed. ' 2005 Wiley-Liss, Inc.

Primary systemic therapy (PST) for operable breast cancer enables the identification of in vivo biological markers that predict response to treatment. A total of 118 patients with T2-4 N0-1 M0 primary breast cancer received six cycles of anthracycline-based PST. Clinical and radiological response was assessed before and after treatment using UICC criteria. A grading system to score pathological response was devised. Diagnostic biopsies and postchemotherapy surgical specimens were stained for oestrogen (ER) and progesterone (PgR) receptor, HER-2 and cell proliferation (Ki-67). Clinical, radiological and pathological response rates were 78, 72 and 38%, respectively. There was a strong correlation between ER and PgR staining (P0.0001). Higher Ki-67 proliferation indices were associated with PgR- tumours (median 28.3%, PgR+ 22.9%; P = 0.042). There was no relationship between HER-2 and other biological markers. No single pretreatment or postchemotherapy biological parameter predicted response by any modality of assessment. In all, 10 tumours changed hormone receptor classification after chemotherapy (three ER, seven PgR); HER-2 staining changed in nine cases. Median Ki-67 index was 24.9% before and 18.1% after treatment (P = 0.02); the median reduction in Ki-67 index after treatment was 21.2%. Tumours displaying75% reduction in Ki-67 after chemotherapy were more likely to achieve a pathological response (77.8 vs 26.7%, P = 0.004).

Background and purpose Hypoxia and proliferation are important determinants of radiation responsiveness; prospective measures of these before radiotherapy may enable individualisation of treatment schedules. Immunohistochemical techniques offer a potential means of achieving this in routine biopsy material. Material and methods Cellular hypoxia as measured by pimonidazole fixation and immunohistochemistry has been evaluated in a series of human bladder cancers with dual staining of sections for pimonidazole and either the vascular markers, CD31/34, or proliferation markers, Ki-67 or cyclin A. Twenty one tumour specimens were examined suitable for the double staining technique. Results The median hypoxic fraction was 9% (range 0–38). Seven tumours did not stain for pimonidazole and 11 exhibited necrosis. The mean vascular density ranged from 16.7 to 160.6 vessels per mm 2 . The median hot spot count was 30 (range 16–43). There was a statistically significant increase in vessel density in hypoxic compared to oxic regions measured by both vessel density ( P =0.02) and hot spot count ( P =0.004). Proliferation indices decreased from oxic to hypoxic areas close to blood vessels. Conclusions We have demonstrated that bladder cancer exhibits a range of hypoxia, proliferation and vascular density which may be used to form the basis for patient selection for hypoxia modification, accelerated radiotherapy and vascular targeting agents.

Purpose: Identification of factors that assist prediction of tumor response to radiotherapy may aid in refining treatment strategies and improving outcome. Possible association of molecular marker expression profiles with locoregional control of head and neck squamous cell carcinoma was investigated in a randomized trial of conventional versus continuous hyperfractionated accelerated radiotherapy (CHART). Experimental Design: Tumor material was obtained from 402 patients. Immunohistochemistry was used to assess Ki-67, CD31, p53, Bcl-2, and cyclin D1 expression. A hierarchical clustering algorithm with a Bayesian information criterion was used to group tumors with similar marker expression; resulting expression profiles were then compared in terms of their difference in outcome after CHART and conventionally fractionated radiotherapy. Results: Molecular marker profile was an independent prognostic factor for locoregional control. This was confirmed in multivariate analysis, including clinical variables such as tumor and nodal status, primary site, histological grade, age, and gender (P < 0.001 and P = 0.006 for local and nodal relapse, respectively). In particular, Bcl-2-positive tumors responded significantly better than average in both arms of the trial. Tumors negative for p53- and Bcl-2, with high and randomly patterned Ki-67 expression, responded worse than average with no benefit from CHART. Tumors with similarly negative p53 and Bcl-2, but low Ki-67 staining, with an organized pattern, benefit significantly from CHART schedule. Conclusions: This study demonstrates the potential of molecular profiles to predict radiotherapy response of head and neck squamous cell carcinoma and for treatment stratification. Distinct expression profiles correlate with three distinct clinical phenotypes, including good locoregional control, poor locoregional control, and an outcome strongly dependent upon fractionation schedule.

The application of a cost-effective spectral imager to spatially segmenting absorptive and fluorescent chemical probes on the basis of their spectral characteristics has been demonstrated. The imager comprises a computer-controlled spectrally selective element that allows random access to a bandwidth of 15 nm between 400 and 700 nm. Further, the use of linear un-mixing of the spectral response of a sample at a single pixel has been facilitated using non-negative least squares fitting. Examples are given showing the separation of dye distributions, such as immunohistochemical markers for tumour hypoxia, from multiply stained thin tissue sections, imaged by trans-illumination microscopy. A quantitative study is also presented that shows a correlation between staining intensity and normal versus tumour tissue, and the advantage of reducing the amount of data captured for a particular study is also demonstrated. An example of the application to fluorescence microscopy is also given, showing the separation of green fluorescent protein, Cy3 and Cy5 at a single pixel. The system has been validated against samples of known optical density and of known overlapping combinations of coloured filters. These results confirm the ability of this technique to separate spectral responses that cannot be resolved with conventional colour imaging.

Cyclin A expression was studied in a series of 65 squamous cell carcinomas of the head and neck (HNSCC) and compared with known markers of proliferation, iododeoxyuridine (IdUrd) and Ki-67, to assess whether aberrant expression was prevalent. Patients had previously been administered IdUrd to study cell kinetics in relation to outcome of radiotherapy. The data showed that all three parameters were highly correlated although the absolute values were different. The median labelling indices (LI) for IdUrd, cyclin A and Ki-67 were 10.7, 17.1 and 30.8% respectively, reflecting the known pattern of differential cell cycle expression. However, there were a significant number of cases in which an unexpected relationship between cyclin A and either IdUrd or Ki-67 was present. Some of these were attributable to overexpression but others indicated underexpression. Although the greater variability and range of cyclin A expression, coupled with its more closely associated role in cell cycle regulation, might suggest that it may be a more informative marker for cell proliferation than Ki-67, the aberrant expression seen in over one third of cases would indicate that caution should be exercised in interpreting cyclin A as a surrogate marker of proliferation in HNSCC. © 2000 Cancer Research Campaign

This study investigates the proliferation characteristics of 81 primary basal cell carcinomas (BCC) using detection of the Ki-67 antigen by immunohistochemistry. The tumours were classified into distinct sub-types based on their histological growth pattern and differentiation status. The mean Ki-67 growth fraction was 0.293 and this was found to vary between the different growth patterns, with morpheic, infiltrating and superficial tumours showing the highest levels of proliferation at 0.373, 0.351 and 0.335, respectively; the nodular and micronodular growth patterns were significantly lower at 0.248 and 0.232, respectively. No overall association was seen between proliferation and differentiation status although certain histological growth patterns such as nodular showed a greater propensity to differentiate. Proliferation was related to tumour size, with larger lesions exhibiting higher growth fractions although this may have also been related to tumour subtype as infiltrating and morpheic tumours tended to present with larger tumour diameters. The spatial distribution of proliferating cells by Ki-67 labelling was not related to tumour subtype, differentiation or growth fraction. These studies have shown BCC to possess proliferative characteristics akin to other solid tumours commonly regarded as more rapidly dividing. There was an association between growth fraction and tumour subtype consistent with higher proliferation in the lesions considered to be more aggressive.

PURPOSE: To assess the relationship between helical dynamic contrast material-enhanced (DCE) computed tomographic (CT) parameters and immunohistochemical markers of hypoxia in patients with operable non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: After institutional review board approval was obtained, 20 prospective patients who were suspected of having NSCLC underwent whole-tumor DCE CT with kinetic modeling (Patlak analysis) 24 hours before scheduled surgery. Flow-extraction product (in milliliters per 100 milliliters per minute) and blood volume (in milliliters per 100 milliliters) were derived. After surgery, matched whole-tumor sections were stained for exogenous and endogenous markers of hypoxia (pimonidazole infused intravenously 24 hours before surgery, immediately after DCE CT; glucose transporter protein). Correlation between DCE CT parameters and immunohistochemical markers was assessed by using the Spearman rank correlation. DCE CT parameters and immunohistochemical markers were also compared according to pathologic subtype, grade, stage, and nodal status by using the Mann-Whitney test. P values less than .05 indicated a statistically significant difference. RESULT: Fourteen patients with confirmed primary NSCLC underwent resection. There were negative correlations between blood volume and pimonidazole staining (r = -0.48, P = .004), and between flow-extraction product and glucose transporter protein expression (r = -0.50, P = .002). Flow-extraction product was significantly higher in adenocarcinomas than in squamous cell tumors (17.73 vs 11.46; P = .043). Glucose transporter protein expression was significantly lower for adenocarcinomas than for squamous tumors (14.07 vs 33.03; P < .001) and in node negative than in node positive tumors (15.63 vs 23.85; P = .005). CONCLUSION: Blood volume and flow-extraction product derived at DCE CT correlated negatively with pimonidazole and glucose transporter protein expression, indicating the potential of these CT parameters as imaging biomarkers of hypoxia.

The goal of targeted therapy has driven a search for markers of prognosis and response to adjuvant therapy. The surgical resection of a solid tumour induces tissue ischaemia and acidosis, both potent mediators of gene expression. This study investigated the impact of colorectal cancer (CRC) surgery on prognostic and predictive marker levels. Tumour expression of thymidylate synthase, thymidine phosphorylase, cyclin A, vascular endothelial growth factor (VEGF), carbonic anhydrase-9, hypoxia inducible factor-1alpha, and glucose transporter-1 (GLUT-1) proteins was determined before and after rectal cancer surgery. Spectral imaging of tissue sections stained by immunohistochemistry provided quantitative data. Surgery altered thymidylate synthase protein expression (P=0.02), and this correlated with the change in the proliferation marker cyclin A. The expression of hypoxia inducible factor-1alpha, VEGF, and GLUT-1 proteins was also different following surgery. Colorectal cancer surgery significantly impacts on intratumoral gene expression, suggesting archival specimens may not accurately reflect in situ marker levels. Although rectal cancer was the studied model, the results may be applicable to any solid tumour undergoing extirpation in which molecular markers have been proposed to guide patient therapy.

Purpose: To investigate the ability of blood oxygen level–dependent (BOLD) MRI to depict clinically significant prostate tumor hypoxia. Methods and Materials: Thirty-three patients with prostate carcinoma undergoing radical prostatectomy were studied preoperatively, using gradient echo sequences without and with contrast medium enhancement, to map relative tissue oxygenation according to relaxivity rates and relative blood volume (rBV). Pimonidazole was administered preoperatively, and whole-mount sections of selected tumor-bearing slices were stained for pimonidazole fixation and tumor and nontumor localization. Histologic and imaging parameters were independently mapped onto patient prostate outlines. Using 5-mm grids, 861 nontumor grid locations were compared with 237 tumor grids (with >50% tumor per location) using contingency table analysis with respect to the ability of imaging to predict pimonidazole staining. Results: Twenty patients completed the imaging and histologic protocols. Pimonidazole staining was found in 33% of nontumor and in 70% of tumor grids. The sensitivity of the MR relaxivity parameter R 2 * in depicting tumor hypoxia was high (88%), improving with the addition of low rBV information (95%) without changing specificity (36% and 29%, respectively). High R 2 * increased the positive predictive value for hypoxia by 6% (70% to 76%); conversely, low R 2 * decreased the likelihood of hypoxia being present by 26% (70% to 44%) and by 41% (71% to 30%) when combined with rBV information. Conclusion: R 2 * maps from BOLD-MRI have high sensitivity but low specificity for defining intraprostatic tumor hypoxia. This together with the negative predictive value of 70% when combined with blood volume information makes BOLD-MRI a potential noninvasive technique for mapping prostatic tumor hypoxia.

Background The aim of this study was to investigate the use of magnetic resonance imaging (MRI) for non-invasive measurement of rectal cancer angiogenesis and hypoxia. Methods Fifteen patients with rectal adenocarcinoma underwent preoperative dynamic contrast-enhanced (DCE) and blood oxygenation level-dependent (BOLD) MRI. Microvessel density (CD31 level), and expression of vascular endothelial growth factor (VEGF) and carbonic anhydrase (CA) 9 were measured immunohistochemically in histological tumour sections from 12 patients. Serum VEGF levels were also measured in 14 patients. Correlations between quantitative imaging indices and immunohistochemical variables were examined. Results There was good correlation between circulating VEGF and CD31 expression (rs = 0·88, P < 0·001). CD31 expression did not correlate with any dynamic MRI parameter, except transfer constant, with which it correlated inversely (rs = −0·65, P = 0·022). Tissue and circulating VEGF levels did not correlate, and neither correlated with any tumour DCE MRI parameter. No relationship was seen between BOLD MRI and CA-9 expression. Conclusion The negative correlation between transfer constant (reflecting tumour blood flow and microvessel permeability) with CD31 expression is paradoxical. DCE MRI methods for assessing tissue vascularity correlate poorly with histological markers of angiogenesis and hypoxia, suggesting that DCE MRI does not simply reflect static histological vascular properties in patients with rectal cancer.

This study investigated the influence of pre-treatment proliferation characteristics, assessed by Ki-67 staining, in patients treated in the CHART trial of accelerated radiotherapy in head and neck cancer. Histological material from 402 patients was collected and stained for the presence and pattern of Ki-67 staining. Locoregional control and overall survival were the main clinical endpoints. Increasing Ki-67 positivity was associated with decreasing differentiation (P < 0.001) and increasing N-stage (P < 0.004). Increasing N-stage was also associated with the progression of proliferation pattern from marginal to random (P < 0.001). Using a multivariate model, a trend was seen towards a greater benefit from CHART in the lower Ki-67 tumours (P = 0.08); this became significant by pooling the low and intermediate Ki-67 groups in comparison with the high Ki-67 group (P = 0.032). Tumours with marginal proliferation pattern showed a lower hazard ratio with CHART versus conventional for locoregional control (P = 0.005). The data presented in this study do not support that a high pre-treatment Ki-67 is associated with a therapeutic benefit from accelerated radiotherapy.

Introduction Biological markers that reliably predict clinical or pathological response to primary systemic therapy early during a course of chemotherapy may have considerable clinical potential. This study evaluated changes in Ki-67 labeling index and apoptotic index (AI) before, during, and after neoadjuvant anthracycline chemotherapy. Methods Twenty-seven patients receiving neoadjuvant FEC (5-fluorouracil, epirubicin, and cyclophosphamide) chemotherapy for operable breast cancer underwent repeat core biopsy after 21 days of treatment. Tissue from pre-treatment biopsy, day 21 and surgery was analysed for Ki-67 index and AI. Results The objective clinical response rate was 56%. Eight patients (31%) achieved a pathological response by histological criteria; two patients had a near-complete pathological response. A reduction in Ki-67 index was observed in 68% of patients at day 21 and 72% at surgery; Ki-67 index increased between day 21 and surgery in 54%. AI decreased in 50% of tumours by day 21, increased in 45% and was unchanged in one patient; 56% demonstrated rebound increases in AI by the time of surgery. Neither pre-treatment nor post-chemotherapy median Ki-67 index nor median AI at all three time points or relative changes at day 21 and surgery differed significantly between clinical or pathological responders and non-responders. Clinical responders had lower median Ki-67 indices at day 21 (11.4% versus 27.0%, p = 0.02) and significantly greater percentage reductions in Ki-67 at day 21 than did non-responders (-50.6% versus -5.3%, p = 0.04). The median day-21 Ki-67 was higher in pathological responders (30.3% versus 14.1%, p = 0.046). A trend toward increased AI at day 21 in pathological responders was observed (5.30 versus 1.68, p = 0.12). Increased day-21 AI was a statistically significant predictor of pathological response (p = 0.049). A strong trend for predicting pathological response was seen with higher Ki-67 indices at day 21 and AI at surgery (p = 0.06 and 0.06, respectively). Conclusion The clinical utility of early changes in biological marker expression during chemotherapy remains unclear. Until further prospectively validated evidence confirming the reliability of predictive markers is available, clinical decision-making should not be based upon individual biological tumour marker profiles.

Sentinel lymph node biopsy is the most accurate technique for establishing melanoma patient prognosis but is associated with morbidity and is of little value in those tumors that first metastasize to sites other than the regional nodal basin. Noninvasive methods of establishing prognosis are therefore desirable. We have used the novel technology of tissue microarray to study a cohort of 120 patients with melanoma with long-term follow-up data for the expression of P-cadherin. The tissue microarray was successfully constructed and stained. Loss of P-cadherin expression was found to be significantly correlated with outcome (log rank χ2 = 10.1336, P = 0.0015). The results suggest a fundamental role for P-cadherin in melanoma progression and identify it as a potential prognostic marker and a possible target for therapeutic manipulation.

Purpose: To investigate the presence of hypoxia in human prostate carcinoma by using pimonidazole immunohistochemical labeling in radical prostatectomy specimens. Methods and Materials: Forty-three patients (median age, 69 years; range, 49–83 years) with localized prostate adenocarcinoma received 0.5 gm/m 2 i.v. pimonidazole 16–24 h before radical prostatectomy. Hypoxia was detected with a monoclonal antibody directed against pimonidazole and scored in formalin-fixed, paraffin-embedded sections. Median and maximal vessel counts were measured with CD34. Results: Thirty-seven patients completed the study. Pimonidazole binding was present in prostate carcinomas in 34 of 37 patients (92%) and in benign prostatic hyperplasia in 35 of 37 patients (95%). A positive correlation of 3+ pimonidazole binding with Gleason score was demonstrated (Spearman's rank, p = 0.044). Vascularity scores did not correlate with hypoxic status or clinical prognostic parameters. Conclusion: Prostate carcinoma and benign prostatic hyperplasia have significant areas of hypoxia; greater hypoxia scores are seen with more aggressive prostate cancer. It is postulated that a hypoxic microenvironment within the prostate might be responsible for the promotion of secondary genetic alterations and angiogenic stimulation, leading to malignant progression, a more aggressive cell phenotype, and greater radioresistance. Modification of radiation regimens to specifically target hypoxia might improve local tumor control.

The prognostic significance of a panel of molecular biomarkers in head and neck squamous cell carcinoma (HNSCC) for first failure site (primary (T), nodal (N) or distant (M)) was analysed in 309 patients randomised to continuous hyperfractionated accelerated radiotherapy (CHART) vs. conventionally fractionated radiotherapy. Multivariate competing risks analysis was performed using an accelerated failure-time model. First-order interactions between each marker and trial arm were also tested. Bcl2-positivity increased the time to T- and N-failures, increasing cyclin D I score decreased the time to N-failures. A random proliferative pattern and low Ki-67 decreased the time to M-failures. A high CD31 score was associated with a significantly longer time to T-failure after CHART, but not after conventional fractionation. Risks of T-, N- and M-failures could be estimated for individual patients. Competing risks analysis of failure sites allows the rational selection of patients for more aggressive loco-regional or systemic therapy. (C) 2004 Elsevier Ltd. All rights reserved.

Purpose To investigate the localization and distribution of cytochrome P450 CYP1B1 protein expression in patients diagnosed with prostate carcinoma compared to those with bladder carcinoma. To validate CYP1B1 as a molecular target for the development of selective cancer therapeutics for use in combination with radiation. Methods and materials Prostatectomy specimens ( n = 33) of moderate Gleason grade (3 + 3 and 3 + 4) were analyzed immunohistochemically for CYP1B1 protein expression using a specific monoclonal antibody for the enzyme. The intensity of CYP1B1 staining was assessed both semiquantitatively using visual scoring and quantitatively by spectral imaging microscopy using reference spectra and compared with bladder carcinoma ( n = 22). Results CYP1B1 protein expression was present in 75% of prostate carcinomas ( n = 27) compared to 100% of bladder carcinomas ( n = 22). In both cases, CYP1B1 protein expression was heterogeneous and localized in the cytoplasm of the tumor cells but absent from the surrounding stromal tissue. CYP1B1 was also detected in premalignant prostatic intraepithelial neoplasia ( n = 2, 100%), as well as noncancerous tissues, including benign prostatic hyperplasia ( n = 27, 82%), metaplastic prostatic urothelium ( n = 8, 100%), and hyperplastic prostatic urothelium ( n = 14, 100%). Higher CYP1B1 protein expression in bladder vs. prostate carcinoma was confirmed by their corresponding average normalized absorbances (± standard deviation), measured as 1.40 ± 0.44 and 0.55 ± 0.09, respectively. Overall CYP1B1 staining intensity in prostate carcinoma was similar to that in prostatic intraepithelial neoplasia, benign prostatic hyperplasia, and hyper-/metaplastic urothelial tissue. No CYP1B1 was detected in normal prostate tissue. Conclusions CYP1B1 is overexpressed in prostate carcinoma at a high frequency and is also detectable in the associated premalignant and hyperplastic tissue, implicating a possible link with malignant progression and CYP1B1 as a suitable target for therapy. Spectral imaging microscopy has highlighted differences in CYP1B1 protein expression between different cancers.

Purpose: The role of bcl-2 overexpression in cancer presents a paradox. In some tumor types, it is associated with favorable outcome, whereas in others the reverse is true. The purpose of this study was to explore the influence of bcl-2 in a large series of head and neck cancer patients treated in the CHART randomized trial. Methods and Materials: Histologic material was obtained from 400 patients; bcl-2 expression was assessed by immunohistochemistry as either positive or negative cytoplasmic staining. Results: Positivity of bcl-2 was recorded in 12.8% (9.5–16.5%, 95% confidence limits) of tumors. There were significant differences in positive tumors within different sites with nasopharynx showing the highest incidence (46.2%). A multivariate logistic regression analysis showed that bcl-2 was strongly associated with histologic dedifferentiation, as well as increasing N stage and female gender. In univariate analyses, bcl-2 positive patients had a lower locoregional relapse rate (RR 0.57, p = 0.02) and improved survival (RR 0.49, p = 0.004) compared to bcl-2 negative patients; this became more significant in multivariate analysis. Conclusion: These data demonstrate that bcl-2 overexpression is a marker of what is considered to be more advanced and aggressive disease yet it is associated with a more favorable outcome irrespective of the treatment schedule.

Microbeam radiation therapy (MRT) using high doses of synchrotron X-rays can destroy tumours in animal models whilst causing little damage to normal tissues. Determining the spatial distribution of radiation doses delivered during MRT at a microscopic scale is a major challenge. Film and semiconductor dosimetry as well as Monte Carlo methods struggle to provide accurate estimates of dose profiles and peak-to-valley dose ratios at the position of the targeted and traversed tissues whose biological responses determine treatment outcome. The purpose of this study was to utilise (Gamma)-H2AX immunostaining as a biodosimetric tool that enables in situ biological dose mapping within an irradiated tissue to provide direct biological evidence for the scale of the radiation burden to 'spared' tissue regions between MRT tracks. (Gamma)-H2AX analysis allowed microbeams to be traced and DNA damage foci to be quantified in valleys between beams following MRT treatment of fibroblast cultures and murine skin where foci yields per unit dose were approximately five-fold lower than in fibroblast cultures. Foci levels in cells located in valleys were compared with calibration curves using known broadbeam synchrotron X-ray doses to generate spatial dose profiles and calculate peak-to-valley dose ratios of 30-40 for cell cultures and approximately 60 for murine skin, consistent with the range obtained with conventional dosimetry methods. This biological dose mapping approach could find several applications both in optimising MRT or other radiotherapeutic treatments and in estimating localised doses following accidental radiation exposure using skin punch biopsies.