Paul I. Richman

Epidermal Growth Factor Receptor Expression in Pretreatment Biopsies From Head and Neck Squamous Cell Carcinoma As a Predictive Factor for a Benefit From Accelerated Radiation Therapy in a Randomized Controlled Trial

PURPOSE Accelerated repopulation is a main reason for locoregional failure after fractionated radiotherapy for head and neck squamous cell carcinoma (HNSCC). Epidermal growth factor receptor (EGFR) is a key controller of cellular proliferation in HNSCC, which stimulated the current study to look for a direct link between EGFR status and a possible clinical advantage of accelerated radiotherapy. PATIENTS AND METHODS Immunohistochemical staining for EGFR was performed in 304 patients with available pretreatment tumor biopsy material among 918 patients randomized to receive continuous hyperfractionated accelerated radiotherapy versus conventionally fractionated radiotherapy. The EGFR index was estimated as the proportion of tumor cells with EGFR membrane staining. RESULTS Significant benefit in locoregional tumor control from continuous hyperfractionated accelerated radiotherapy was seen in patients with HNSCC with high EGFR expression (2P = .010) but not in those with low EGFR expression (2P = .85). EGFR status had no significant effect on survival or rate of distant metastases. The EGFR index was significantly associated with histologic grade and microvessel density. There was moderate support for an association between EGFR status and subsite within the head and neck region but no significant association with Ki-67 index, Ki-67 pattern, p53 index, p53 intensity, bcl-2 expression, or cyclin D1 index. CONCLUSION This study indicates a key role for the EGFR receptor in determining the proliferative cellular response to fractionated radiotherapy in HNSCC. It also shows that we can select the dose-fractionation regime that has the greatest chance of benefiting the patient. These results also encourage further development of EGFR targeting combined with fractionated radiotherapy in HNSCC.

Evaluation of ER, PgR, HER-2 and Ki-67 as predictors of response to neoadjuvant anthracycline chemotherapy for operable breast cancer

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 (P<0.0001). 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 displaying >75% reduction in Ki-67 after chemotherapy were more likely to achieve a pathological response (77.8 vs 26.7%, P=0.004).

Molecular marker profiles predict locoregional control of head and neck squamous cell carcinoma in a randomized trial of continuous hyperfractionated accelerated radiotherapy

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.

p53 status of head and neck cancer : relation to biological characteristics and outcome of radiotherapy

p53 status was investigated in 99 patients with squamous cell carcinoma of the head and neck region uniformly treated with accelerated radiotherapy and in whom tumour cell proliferation and DNA aneuploidy were assessed using bromodeoxyuridine (BrdUrd) incorporation and flow cytometry (FCM). Seventy-six percent of tumours were immunohistochemically positive for p53 protein, but heterogeneity was noticed both in the percentage of cells positive for p53 and in their level of expression. However, tumours which were either essentially all positive or all negative or showed sporadic positivity for p53 protein showed no differences in their level of aneuploidy, proliferation rate, tissue organisation or outcome with radiotherapy. There was a trend for those p53-positive tumours with the strongest expression to have more DNA aneuploidy and deregulation of proliferation organisation than weaker expressors; but there were no differences in proliferation rate or outcome of radiotherapy. These studies suggest that p53 protein stabilisation as assessed by immunohistochemistry does not have any major relationship with the biological characteristics and outcome of squamous cell cancer treated by accelerated radiotherapy.

Evaluation of Ki-67 proliferation and apoptotic index before, during and after neoadjuvant chemotherapy for primary breast cancer

IntroductionBiological 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.MethodsTwenty-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.ResultsThe 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).ConclusionThe 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.

bcl-2 expression in head and neck cancer: an enigmatic prognostic marker

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.

An analysis of p16 protein expression in sporadic malignant melanoma.

Inactivation of the p16 tumour suppressor gene has been reported frequently in melanoma cell lines, and mutations have been detected in familial melanoma kindreds. The aim of this study was to assess the role of p16 inactivation in melanocytic progression by measuring the level of p16 protein in a range of sporadic, benign and malignant melanocytic lesions. Using dual parameter flow cytometry, p16 protein expression was measured in 30 benign melanocytic naevi, 38 primary and 51 metastatic melanomas. A high level of p16 expression was demonstrated in benign melanocytic naevi (96% median nuclear positivity), with a significant reduction in primary melanomas (69%, P< 0.001). The median nuclear positivity of primary melanomas was significantly higher (P<0.03) than the level of expression in metastatic lesions (median positivity 37%). A progressive loss of p16 expression was demonstrated from benign melanocytic naevi through to primary and metastatic lesions. These data suggest that loss of p16 protein expression is not only associated with the early transformation of benign lesions, but also with the later stages of malignant progression.

Measurement of invasive potential provides an accurate prognostic marker for giant cell tumour of tendon sheath

Giant cell tumours of tendon sheath vary from solitary nodules to a multinodular variety that exhibits local infiltration. Recent advances in molecular oncology have defined a gene, nm23, expressed in normal cells which is responsible for inhibiting infiltration. The aim of this study was to investigate the expression of nm23 in a series of 52 giant cell tumours using immunohistochemistry and to assess its prognostic potential, nm23 expression was absent in 21% of tumours and this was associated with a highly significant risk of local recurrence (P<0.0001). Multivariate analysis of outcome showed nm23 expression to be more reliable than other clinicopathological parameters for predicting outcome. This immunohistochemieal test for nm23 is easily performed on standard paraffin sections and is recommended as an accurate prognostic marker for giant cell tumours of tendon sheath.

Validation of tissue microarray for the immunohistochemical profiling of melanoma.

Tissue microarray technology allows high throughput profiling of cancer specimens by immunohistochemical staining. Protein expression varies throughout tumour specimens resulting in heterogeneous staining patterns, which has led to doubts as to the accuracy of tissue microarray. In an effort to validate the use of tissue microarray for melanoma immunohistochemical investigation, a study was conducted comparing the concordance of MCAM staining between whole tumour specimens and tissue microarray core biopsies. Data on full tissue sections were compared with the results of one to four 0.6 mm core biopsies per tumour on a tissue array. It was revealed that concordance of tissue array core biopsies in quadruplicate compared with full-section analysis for the expression and intensity of expression of MCAM.

Cellular proliferation characteristics of basal cell carcinoma: relationship to clinical subtype and histopathology

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.