Pretreatment Tumor Biopsies Research Articles

Expression of excision repair cross-complementation group 1 protein predicts poor outcome in patients with small cell lung cancer

Alterations in apoptosis-related proteins and DNA damage repair proteins are associated with resistance to chemotherapy or radiotherapy, which is the most important cause of treatment failure in small cell lung cancer (SCLC). Pretreatment tumor biopsy specimens from 77 patients with SCLC (limited stage: 40, extensive stage: 37) were analyzed for p53, bcl-2, bax and ERCC1 expression by immunohistochemistry. All patients were treated with platinum-based doublets. The most commonly used regimen was etoposide/cisplatin (50 patients). In patients with limited stage SCLC, thoracic irradiation was performed either concurrently with chemotherapy or sequentially. High expression of p53, bcl-2, bax and ERCC1 was observed in 40 (52%), 72 (94%), 38 (49%) and 13 (17%) patients, respectively. High expression of ERCC1 was associated with poor OS (1-year, 23% vs. 53%; p=0.026). When grouped according to stage, a significant correlation between high expression of ERCC1 and poor outcome was observed only in patients with limited stage SCLC (p=0.017). High expression of p53, bcl-2 and bax was not correlated with patient outcome. Multivariate analysis showed that extensive stage (p=0.006) and male gender (p=0.009) were independent predictors of poor OS, while high expression of ERCC1 failed to reach statistical significance despite a trend (p=0.057). In limited stage patients, high expression of ERCC1 was an independent prognostic factor for poor OS (p=0.046), along with male gender (p=0.033). High expression of ERCC1 protein may be a useful predictor of poor outcome in SCLC patients treated with chemotherapy with or without radiotherapy, especially in limited stage SCLC.

Low Expression of Bax Predicts Poor Prognosis in Patients with Locally Advanced Esophageal Cancer Treated with Definitive Chemoradiotherapy

The present study evaluated the prognostic significance of apoptosis-related proteins, p53, Bcl-2, Bax, and galectin-3 in patients with locally advanced esophageal cancer treated with definitive chemoradiotherapy. A total of 63 patients with locally advanced esophageal cancer (squamous cell carcinoma: 62; adenocarcinoma: 1; stages II-IV) were treated with definitive chemoradiotherapy using 5-fluorouracil and cisplatin combined with radiotherapy. Pretreatment tumor biopsy specimens were analyzed for p53, Bcl-2, Bax, and galectin-3 expression by immunohistochemistry. High expression of Bax, p53, Bcl-2, and galectin-3 was observed in 67%, 47%, 24%, and 29% of patients, respectively. The median overall survival (OS) of total patients was 14 months with 16% of 3-year OS. High expression of p53, Bcl-2, and galectin-3 did not show correlation with clinicopathologic characteristics, including patient outcome. Low expression of Bax was significantly correlated with lack of clinical complete response (P=0.023). Low expression of Bax was also associated with poor OS (median, 8 months versus 16 months; P=0.0008) in univariate analysis. In multivariate analysis, low expression of Bax was the most significant independent predictor of poor OS (P=0.009), followed by low dose intensity of cisplatin and lack of clinical complete response. Low expression of Bax was significantly associated with the poor survival of patients with locally advanced esophageal cancer treated with chemoradiotherapy using 5-fluorouracil and cisplatin. Immunohistochemical staining for Bax with a pretreatment biopsy specimen might be useful to select the optimal treatment options for these patients.

Phase II Trial of Ixabepilone, an Epothilone B Analog, in Patients With Metastatic Breast Cancer Previously Untreated With Taxanes

Ixabepilone is an epothilone B analog that binds to microtubules and results in microtubule stabilization and mitotic arrest. Ixabepilone was evaluated for efficacy and safety in a phase II clinical trial for women with metastatic breast cancer. Patients were eligible if they had not previously received treatment with a taxane and had measurable metastatic breast cancer. Ixabepilone was administered at 6 mg/m(2)/d intravenously days 1 through 5 every 3 weeks until unacceptable toxicity or disease progression. Patients underwent pretreatment and post-treatment tumor biopsies, and tissues were analyzed for acetylated alpha-tubulin, tau-1, and p53 expression when possible. Twenty-three patients received 210 cycles with a median of eight cycles (range, two to 22 cycles) per patient. Thirteen patients (57%; exact 95% CI, 34.5% to 76.8%) had partial responses, six patients (26%) had stable disease, and four patients (17%) had progressive disease. Median time to progression and duration of response were 5.5 and 5.6 months, respectively. Four patients required dose reductions for neutropenia, neuropathy, or fatigue. Grade 3 or 4 toxicities included neutropenia (22%), fatigue (13%), anorexia (9%), and motor neuropathy (4%). Thirty-nine percent of patients experienced grade 1, 13% experienced grade 2, and none experienced grade 3/4 sensory neuropathy. The six patients with paired biopsies all had increases in tumor alpha-tubulin acetylation after treatment. Baseline or cycle 2 acetylated alpha-tubulin, tau-1, or p53 expression did not correlate with clinical response. Women with metastatic breast cancer previously untreated with taxanes have a meaningful durable response to single-agent ixabepilone therapy. Minimal hematologic toxicity and no grade 3 sensory neuropathy were noted.

A prospective study to evaluate the role of gene expression profiles (GEP) in predicting clinical outcome of patients (pts) receiving preoperative chemotherapy for oesophagogastric (OG) cancer

4501 Background: Whilst preoperative chemotherapy has demonstrated survival benefit for pts with potentially resectable OG cancer it is not possible to predict the benefit for an individual pt. This study was designed to prospectively correlate GEP with clinical outcome. Methods: Eligible pts were deemed to have resectable disease after staging CT, EUS, and laparoscopy as indicated & following discussion at the multidisciplinary team meeting. All pts received neoadjuvant platinum & fluoropyrimidine based chemotherapy & clinical data were entered prospectively onto a study specific database. GEP were produced from total RNA isolated from snap frozen pre treatment tumour biopsies obtained at baseline endoscopy. Labelled cDNA was hybridised versus a universal human reference using an in house c DNA array of 22,000 clones. Results: Of the pts with adequate follow up accrued between 2002–2005, 35 met the quality control measures for the arrays. Median age=66 yrs (47–83); male=32, female=3; tumour subsites: oesophagus=23, oesophago-gastric junction (OGJ)=12; adenocarcinoma=35; T stage: T 2=3, T3=30, T4=2; N stage: N0=12, N1=23; performance status 0=7, 1=28. Median follow up=938 days. Median overall survival (OS) = 570 days. Prognostic groups were designated according to the median OS (days) of the group: good > median and poor < median. Supervised hierarchical clustering of normalised data revealed significantly differentially expressed genes based on OS (p<0.01) with 2 distinct clusters: a poor outcome group: N= 17 (2yr OS 17.6%) [95% CI: 4.3–38.3], a good outcome group: N=18 (2 yr OS 55%) [95% CI: 30.5–74.8]. Of the differentially expressed genes, those involved in receptor tyrosine kinase signalling & cell growth were amongst the most significantly affected pathways. Conclusions: This novel technique using GEP in tumour biopsies has successfully identified groups of tumours with distinct gene expression profiles that correlate with survival. The approach warrants further validation in a larger cohort. It could facilitate the development of tailored treatment according to individual tumour biology in OG cancer. No significant financial relationships to disclose.

Low expression of bax predicts poor prognosis in patients with locally advanced esophageal cancer treated with definitive chemoradiotherapy

4597 Background: The present study evaluated the prognostic significance of apoptosis-related proteins, p53, bcl-2, bax, and galectin-3 in patients with locally advanced esophageal cancer treated with definitive chemoradiotherapy (CRT). Methods: Sixty-three patients with locally advanced esophageal cancer (stage II-IV) were treated with definitive CRT using 5-fluorouracil and cisplatin combined with radiotherapy. Pretreatment tumor biopsy specimens were analyzed for p53, bcl-2, bax, and galectin-3 expression by immunohistochemistry. Results: High expression of bax, p53, bcl-2, and galectin-3 was observed in 67%, 47%, 24%, and 29% of patients, respectively. The median overall survival (OS) of total patients was 14 months with 16% of 3-year OS. High expression of p53, bcl- 2, and galectin-3 did not demonstrate correlation with clinicopathologic characteristics, including patient outcome. Low expression of bax was significantly correlated with clinical complete response (p=0.023). Low expression of bax was also associated with poor OS (median, 8 months vs. 16 months; P=0.0008) in univariate analysis. In multivariate analysis, low expression of bax was the most significant independent predictor of poor OS (p=0.01) followed by clinical complete response and low radiation dose. Conclusions: Low expression of bax was significantly associated with the poor survival of patients with locally advanced esophageal cancer treated with CRT using 5-fluorouracil and cisplatin. Immunohistochemical staining for bax with a pretreatment biopsy specimen might be useful to select the optimal treatment options for these patients. No significant financial relationships to disclose.

Predictive and Pharmacodynamic Biomarker Studies in Tumor and Skin Tissue Samples of Patients With Recurrent or Metastatic Squamous Cell Carcinoma of the Head and Neck Treated With Erlotinib

PURPOSE Pharmacodynamic tissue studies were conducted on a phase I/II trial of erlotinib and cisplatin in patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). Levels of epidermal growth factor receptor (EGFR), downstream signaling components, and markers of angiogenesis and apoptosis were evaluated to determine the relationship between correlative end points and clinical outcomes. PATIENTS AND METHODS Pretreatment and during-treatment tumor and skin biopsies, and archival tumor specimens were evaluated for EGFR, phosphorylated (p) -EGFR, extracellular signal-regulated kinase (ERK), p-ERK, Akt, p-Akt, Ki67, p27, p-nuclear factor kappa B (NFkappaB), p-signal transducer and activator of transcription 3 (STAT3), and EGFR gene copy number. Results On 37 archival samples, response to therapy was evident in two of four (50%) patients with high EGFR gene copy number tumors and in four of 27 (15%) patients with low gene copy number tumors. On nine paired tumor biopsies, elevated pretreatment levels of p27 and p-STAT3 predicted for prolonged time to progression (TTP) and overall survival (OS; P < or = .03). With treatment, a decrease in p-EGFR, p-NFkappaB, and p27 correlated with increased TTP, OS, or both TTP and OS, respectively (P < or = .04). Multidimensional scaling (MDS) models revealed clustering profiles of tumor markers by immunofluorescence could predict response. On 32 paired skin biopsies, suppression of p-EGFR with therapy correlated with increased OS (P = .045). CONCLUSION High EGFR gene copy in tumor specimens may predict which patients have an increased likelihood of response to erlotinib, and decreased p-EGFR level in skin biopsies during therapy may represent a potential surrogate marker for improved clinical outcome. MDS represents a novel way to evaluate the relationships between molecular markers and clinical outcome. Additional biomarker studies with larger sample sizes are required to elucidate HNSCC patients who may benefit from this targeted therapy.

Relationship of estrogen and progesterone receptors to clinical outcome in metastatic endometrial carcinoma: A Gynecologic Oncology Group Study

Introduction The goal of this study was to explore the relationship between the expression of hormone receptors in metastatic endometrial tumors and clinical response to daily tamoxifen citrate and intermittent weekly medroxyprogesterone acetate. Study design Patients with measurable recurrent or advanced endometrial cancer were enrolled on a clinical trial, Gynecologic Oncology Group Study 119. A pretreatment tumor biopsy was obtained and subjected to immunohistochemical analyses. Estrogen receptor-α (ER-α) and progesterone receptor (PR) were assessed on frozen tissues, and PR isoforms A and B were detected on fixed tissues. The receptors were scored using a semi-quantitative HSCORE, with a cut off greater than 75 considered positive. Results Of the 60 eligible patients, 45 had evaluable tissues for all receptors. For ER, 40% of the cases were positive; for PR, 45% were positive. The sub-cellular distribution of PRA was exclusively nuclear, and 16% of the tumors demonstrated positive staining. PRB was nuclear and cytoplasmic, with 22% of the tumors staining for nuclear PRB and 36% of the tumors staining for cytoplasmic PRB. ER and PR from frozen tissues and PRA and cytoplasmic PRB from fixed tissues significantly decreased with increasing tumor grade. The co-expression of ER-α with PR from the frozen tissues ( r = 0.68, p < 0.001) and PRA ( r = 0.58, p < 0.001) from the fixed tissues was statistically significant. The ER HSCORE was related to both response and overall survival; there was no statistically significant correlation of PR with clinical response in this small number of patients. Conclusion ER-α measured in metastatic endometrial carcinoma tissue prior to hormonal therapy was statistically significantly related to clinical response to daily tamoxifen and intermittent medroxyprogesterone acetate.

First Study of the Safety, Tolerability, and Pharmacokinetics of CP-724,714 in Patients with Advanced Malignant Solid HER2-Expressing Tumors

To test the tolerability, safety, and recommended phase II dose of CP-724,714, a reversible, highly selective, oral HER2 tyrosine kinase inhibitor in patients with advanced solid tumor malignancies that express HER2. A phase I trial evaluated escalating doses of CP-724,714, administered daily in 21-day cycles. Pharmacokinetics/pharmacodynamics were evaluated in serial blood samples and in pretreatment and posttreatment tumor and skin biopsies. Thirty female patients [median age, 51 years (range, 37-71); median performance status, 1 (range, 0-1)] received CP-724,714 at four dose levels: 250 mg once daily (4 patients), 250 mg twice daily (15 patients), 250 mg thrice daily (6 patients), and 400 mg twice daily (5 patients). Dosing at 400 mg twice daily and 250 mg thrice daily was not feasible due to reversible, cholestatic liver dysfunction. Treatment-related adverse events were nausea (58%), asthenia (23%), hyperbilirubinemia (27%), elevated transaminases (30%), and skin rash (30%); neither diarrhea nor cardiomyopathy was observed. No objective responses were observed in 28 evaluable patients; 8 (29%) patients had stable disease. Twenty-seven (96%) patients received prior trastuzumab and were heavily pretreated (median prior chemotherapy, 6; range, 1-11). Systemic exposure exceeded the in vivo efficacy threshold required in preclinical studies. Dose-limiting toxicities included hyperbilirubinemia, elevated alanine aminotransferase, thrombocytopenia and pulmonary embolus. Although the protocol-specified maximum tolerated dose of CP-724,714 was 250 mg thrice daily, the recommended phase II dose was 250 mg twice daily due to excessive late-cycle hepatotoxicity. Despite extensive prior treatment, 29% of patients had stable disease. A phase II trial has been initiated in patients with breast cancer.

Thymidylate synthase expression in oral squamous cell carcinoma predicts response to S-1

The purpose of this research was to evaluate the predictive value of expression of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), or orotate phosphoribosyltransferase (OPRT) genes for response to S-1. Twenty-five patients with oral squamous cell carcinoma (OSCC) received S-1 80 mg/m2/day. Pretreatment tumor biopsies were analyzed for TS, DPD, TP or OPRT mRNA expression by real-time reverse transcription-PCR. TS protein expression was evaluated by immunohistochemistry using a polyclonal TS antibody. Twenty-five patients were evaluable for response and gene expression. Six of the 25 (24%) achieved complete remission and 4 of the 25 (16%) had a partial response. Median TS/beta-actin was 2.51 (range 0.98-7.07). Median TS/beta-actin was 1.26 in responding patients and 3.43 in non-responders (P=0.0001). Ten of 11 patients with TS/beta-actin <1.80 and 0 of 15 with higher values responded (P<0.0001). Overall survival was 29.7 months in patients with TS/beta-actin <1.80 and 41.7 months in patients with higher values (P=0.0013). No correlations were seen between expression of DPD, TP or OPRT mRNA and response or survival. Weak TS staining was seen in 6 of 25 tumors evaluable for immunohistochemistry, including 5 responders. All 4 of the patients with both weak staining and TS/beta-actin <1.80 responded. High TS mRNA expression predicts non-response to S-1. On the other hand, high levels of DPD or TP mRNA and low levels of OPRT mRNA are not associated with S-1 resistance. TS mRNA expression is considered to be a useful prognostic factor in OSCC patients with S-1 single-agent therapy.

Pilot study examining tumor expression of RAD51 and clinical outcomes in human head cancers

Radiation therapy and chemotherapy are commonly used treatments for head and neck cancer. RAD51 is a highly conserved DNA repair protein that serves a central function in the homologous recombination pathway. High levels of RAD51 protein expression have been reported in number of human cancer cell lines, and studies suggest that RAD51 overexpression can increase cellular resistance to radiation and some chemotherapeutic drugs. In this study, RAD51 protein levels were quantified by immunohistochemistry in tumor samples from twelve head and neck cancer patients who received identical treatment with induction chemotherapy (paclitaxel and carboplatinum) followed by radiation therapy given concurrently with additional chemotherapy (paclitaxel, fluorouracil, hydroxyurea). Patients with high RAD51 protein levels in their pre-treatment tumor biopsies demonstrated poorer cancer-specific survival rates than patients with lower RAD51 levels (33.3% vs. 88.9% at 2 years; p=0.025). In addition, within a subgroup of patients with normal tumor cell p53 expression, there was a non-significant trend toward better induction chemotherapy response rates observed in the tumors with lower RAD51 protein levels. These results suggest that tumor cell RAD51 expression levels may influence the outcome of patients with head and neck cancer treated with chemotherapy and radiotherapy.

Mcl-1, Vascular Endothelial Growth Factor-R2, and 14-3-3σ Expression Might Predict Primary Response against Radiotherapy and Chemotherapy in Patients with Locally Advanced Squamous Cell Carcinomas of the Head and Neck

This study was done to explore whether the expression of a selected set of proteins could predict primary response to radiotherapy or concomitant radiotherapy and chemotherapy in patients with advanced head and neck cancer. Forty-three pretreatment tumor biopsies were taken during diagnostic panendoscopy and examined for Mcl-1, vascular endothelial growth factor (VEGF)-R2, CD9, and 14-3-3sigma expression by immunohistochemistry. Forty-three patients underwent primary radiotherapy, of which, 29 patients received concomitant chemotherapy (low dose daily cisplatin, mitomycin C bolus). The primary end-point was locoregional tumor control 6 months after completion of radiotherapy. Mcl-1, VEGF-R2, CD9, and 14-3-3sigma expression were correlated with patients' primary response to radiotherapy and chemotherapy and with established clinicopathologic variables. Thirty complete and 13 partial responses were observed in our patient group. High expression levels of Mcl-1 (P=0.021), VEGF-R2 (P=0.032), and 14-3-3sigma (P=0.013), but not of CD9, in tumor biopsies was correlated with complete response. Overexpression of at least two of the three aforementioned proteins in pretreatment biopsies predicted-with a likelihood of 80%-whether a patient would achieve complete response to radiotherapy and chemotherapy. However, if only one of these proteins is overexpressed, there is a likelihood of 84.6% that this patient would not completely respond to therapy. Determining the expression levels of Mcl-1, VEGF-R2, and 14-3-3sigma may be helpful in predicting the early clinical response in head and neck tumor patients receiving primary radiotherapy and chemotherapy and may further allow a pretherapeutic selection of patients.

Predicting the Future From Trials of the Past: Epidermal Growth Factor Receptor Expression and Outcome of Fractionated Radiation Therapy Trials

The epidermal growth factor receptor (EGFR; HER 1; ErbB1) has been at the center of an explosion of translational research over the past 20 years. The EGFR is expressed or overexpressed in a large number of malignancies, including non–small-cell lung (NSCLC), head and neck, esophageal, gastric, colorectal, breast, prostate, bladder, renal, pancreatic, and ovarian cancers. EGFR expression is often found in association with increased expression of its ligands, most notably epidermal growth factor (EGF), tumor growth factor-alpha, or amphiregulin. EGFR activation and signaling affects many aspects of cell biology relevant to malignant transformation, including cell cycle progression, migration and invasion, differentiation, and cell survival. The antiapoptotic effects of EGFR activation may be particularly relevant to the assessment of treatment outcomes in patients afflicted with EGFR expressing tumors have been ascribed to regulation of Bcl-2 family members. In this issue, Bentzen et al examine EGFR expression status in 304 patients with available pretreatment tumor biopsy material among 918 patients randomized to Continuous Hyperfractionated Accelerated Radiotherapy (CHART) versus conventionally fractionated radiotherapy. The EGFR index was defined as the proportion of tumor cells with EGFR membrane staining. Significant benefit in locoregional tumor control from CHART was seen in patients with head and neck squamous cell carcinoma with high EGFR expression (2P .010) but not in patients with low EGFR expression (2P .85). By contrast, EGFR expression status was not linked to patient survival or rate of distant metastases. Bentzen et al used immunohistochemical analysis to assess EGFR expression levels, which raises some technical concerns. First, manual methods of immunohistochemical scoring of antigen expression as used in this report are fraught with variability. This variability may explain the lack of association of EGFR expression with other biomarkers evaluated in this study (Ki-67 index, Ki-67 pattern, p53 index, p53 intensity, bcl-2 expression, or cyclin D1 index). Another concern centers on the retrospective analysis of archived tissue stored for different periods of time. The CHART head and neck phase III trial accrued patients over 5 years from March 1990 to April 1995. During this extended period, oxidation of antigens/DNA/RNA may have occurred. A recent editorial in the Journal of Clinical Oncology by Meropol refers to work by Atkins et al in support of the notion that extended storage of tissue sections over time affects the sensitivity of EGFR immunostaining. However, despite these concerns, the study by Bentzen et al provides an interesting new perspective on intratumoral EGFR expression as a variable with obvious relevance to the design of fractionated radiotherapy. Several reports have illuminated molecular mechanisms leading to enhanced expression of the EGFR in epithelial malignancies. In addition to gene amplification, sequence analysis has revealed polymorphisms of intron 1 of the EGFR gene characterized by (CA)n dinucleotide repeats of different lengths that regulate transcription rates of the EGFR in cell lines and in tumor tissues in patients. The length of the (CA)n dinucleotide repeat tract is inversely correlated with the transcriptional activity of the EGFR gene. Further investigation of EGFR sequences expressed by tumor cells has also revealed alterations that primarily affect the activation state of the receptor rather than the expression levels. For example, in patients with colorectal cancer, a polymorphic variant of the EGFR gene (HER-1 R497K) has JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 23 NUMBER 24 AUGUST 2

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

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. 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. 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. 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.

The Cyclin-Dependent Kinase Inhibitor UCN-01 Plus Cisplatin in Advanced Solid Tumors: A California Cancer Consortium Phase I Pharmacokinetic and Molecular Correlative Trial

UCN-01 (7-hydroxy-staurosporine) is a novel antineoplastic agent targeting cyclin-dependent kinases, which shows potent in vitro and in vivo activity against a broad range of tumor types. Our group has previously shown that UCN-01 potentiates the apoptotic response of agents such as cisplatin in vitro by preventing sequence-specific abrogation of G2 arrest caused by DNA-damaging chemotherapies. This National Cancer Institute-sponsored phase I trial was designed to determine the safety, maximum tolerated dose, and pharmacokinetics of escalating doses of cisplatin in combination with UCN-01 in patients with advanced malignant solid tumors, as well as to do molecular correlative studies on tumor specimens. Cisplatin was infused over 1 hour before UCN-01 (45 mg/m2/d) given as a 72-hour continuous infusion. Escalation of cisplatin was planned through five dose levels at 20, 30, 45, 60, and 75 mg/m2. Ten patients were accrued. Accrual was halted at dose level 2 (cisplatin, 30 mg/m2) due to dose-limiting toxicities consisting of grade 5 sepsis with respiratory failure associated with grade 3 creatinine (one patient) and grade 3 atrial fibrillation (one patient). Plasma and salivary pharmacokinetics of UCN-01 were unaffected by cisplatin. Pretreatment and posttreatment tumor biopsies showed that UCN-01 was active against a key molecular target, the checkpoint kinase Chk1. This phase I trial failed to achieve targeted therapeutic dose levels of cisplatin when combined with prolonged infusion UCN-01. However, because preclinical data indicate that UCN-01 potentiates response to platinum, further studies with alternative dose schedules of the combination, or with other platinum analogues, are warranted.

Association of activated transcription factor NF-kappaB with chemo-radiotherapy resistance and poor outcome in esophageal adenocarcinoma

4029 Background: The incidence of esophageal/gastroesophageal junction adenocarcinoma (E/GEJAC) is rapidly rising in Western countries. Chemo-radiotherapy (CT-XRT) followed by surgery has been used for localized E/GEJAC. However, while 30% of patients achieve a pathologic complete response (pCR), most patients present with radioresistant residual, highly aggressive tumor. Thus there is a need to identify and target new molecular pathways associated with cancer resistance. Through microarray gene expression profiles we identified differentially expressed transcription factor NF-kappa B (NF-κB) associated signaling pathway in CT-XRT non-responders group. To validate our findings, we examined activated NF-κB protein expression, CT-XRT response and clinical outcome. Methods: Pre-treatment tumor biopsies and post-treatment resected residual tumor were obtained from patients enrolled in a phase II trial of pre-operative induction chemotherapy and CT-XRT for potentially resectable E/GEJAC. Activated (p65 nuclear localization) NF-κB protein expression was evaluated by immunohistochemistry and correlated with pCR and clinical outcome. Results: 31 patients, clinically staged II and III, were included in the analysis. Activated NF-κB was significantly associated with presence of residual tumor in the resected esophagectomy specimen [NF-κB positive in 1/9 (11%) pCR vs 12/22 (54.5%) non-pCR; P=.044]. Activated NF-κB expression was sustained in pre- and post-treatment tumor in 6 and de novo acquired in the remaining 6 of 12 NF-kB positive non-pCR group. Independent of clinical stage, tumor histology and lymph nodes invasion, activated NF-κB in the post-treatment tumor was associated with increased frequencies of perineural/connective/vascular invasion [NF-κB positive 10/13 (77%) vs NF-κB negative 0/7; P=.003], and distant metastasis [NF-κB positive 5/13 (38.5%) vs NF-κB negative 0/7; P=.08]. Conclusions: Our results suggest that activated NF-κB is associated with poor response to CT/CT-XRT and worse outcome in E/GEJAC, and thus may serve as a potential molecular target to improve treatment efficacy. No significant financial relationships to disclose.

The prognostic value of the hypoxia markers CA IX and GLUT 1 and the cytokines VEGF and IL 6 in head and neck squamous cell carcinoma treated by radiotherapy ± chemotherapy

BackgroundSeveral parameters of the tumor microenvironment, such as hypoxia, inflammation and angiogenesis, play a critical role in tumor aggressiveness and treatment response. A major question remains if these markers can be used to stratify patients to certain treatment protocols.The purpose of this study was to investigate the inter-relationship and the prognostic significance of several biological and clinicopathological parameters in patients with head and neck squamous cell carcinoma (HNSCC) treated by radiotherapy ± chemotherapy.MethodsWe used two subgroups of a retrospective series for which CT-determined tumoral perfusion correlated with local control. In the first subgroup (n = 67), immunohistochemistry for carbonic anhydrase IX (CA IX) and glucose transporter-1 (GLUT-1) was performed on the pretreatment tumor biopsy. In the second subgroup (n = 34), enzyme linked immunosorbent assay (ELISA) was used to determine pretreatment levels of the cytokines vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) in serum. Correlation was investigated between tumoral perfusion and each of these biological markers, as well as between the markers mutually. The prognostic value of these microenvironmental parameters was also evaluated.ResultsFor CA IX and GLUT-1, the combined assessment of patients with both markers expressed above the median showed an independent correlation with local control (p = 0.02) and disease-free survival (p = 0.04) with a trend for regional control (p = 0.06).In the second subgroup, IL-6 pretreatment serum level above the median was the only independent predictor of local control (p = 0.009), disease-free survival (p = 0.02) and overall survival (p = 0.005).ConclusionTo our knowledge, we are the first to report a link in HNSCC between IL-6 pretreatment serum levels and radioresistance in vivo. This link is supported by the strong prognostic association of pretreatment IL-6 with local control, known to be the most important parameter to judge radiotherapy responses.Furthermore, the combined assessment of CA IX and GLUT-1 correlated independently with prognosis. This is a valuable indication that a combined approach is important in the investigation of prognostic markers.

Preradiotherapy Hemoglobin Level but not Microvessel Density Predicts Locoregional Control and Survival in Laryngeal Cancer Treated with Primary Radical Radiotherapy

To evaluate the roles of preradiotherapy hemoglobin level and microvessel density (MVD) as predictive factors for tumor control and survival in patients with laryngeal cancer treated with primary radiotherapy. Two hundred and fourteen patients with stage I-IV laryngeal cancer were included in the analysis. Patients were treated with once daily fractionated radiotherapy over 6.5 weeks or twice daily fractionated radiotherapy over 4.5 weeks up to total doses of 62 to 68 Gy. Preradiotherapy hemoglobin levels were obtained from patient journals, and pretreatment tumor biopsies were stained with CD34 antibody for the counting of microvessels. The prognostic implication of preradiotherapy hemoglobin level and MVD on tumor control and survival was tested. Five-year locoregional control probability was 88.9% for patients with preradiotherapy hemoglobin levels >137.5 g/L (median) and 64.4% for patients with preradiotherapy hemoglobin levels <137.5 g/L (P = 0.01). The corresponding figures for disease-free survival were 87.8 and 62.8% (P = 0.007), respectively, and for overall survival 58.1 and 40.3% (P < 0.001), respectively. In multivariate analysis, tumor stage and preradiotherapy hemoglobin level were significant prognostic factors for locoregional control and disease-free survival, whereas tumor stage, preradiotherapy hemoglobin-level, gender, and age were significant prognostic factors for overall survival. No correlation was found between MVD and tumor control and survival. Preradiotherapy hemoglobin level, but not MVD, predicts locoregional control and survival in patients with laryngeal cancer treated with radiotherapy.

Epidermal Growth Factor Receptor Tyrosine Kinase Inhibition Represses Cyclin D1 in Aerodigestive Tract Cancers

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are active in cancer therapy. Mechanisms engaged during these clinical responses need to be determined. We reported previously that epidermal growth factor stimulation markedly increased cyclin D1 protein expression in human bronchial epithelial (HBE) cells, and this was opposed by chemoprevention with all-trans-retinoic acid. The current study sought to determine whether the EGFR TKI erlotinib repressed cyclin D1 protein expression in immortalized HBE cells, lung cancer cell lines, and clinical aerodigestive tract cancers. The BEAS-2B immortalized HBE cell line was exposed to varying concentrations of erlotinib, and effects on proliferation, cell cycle distribution, G1 cyclin expression, and cyclin D1 reporter activity were measured. Non-small-cell lung cancer cell lines were also evaluated for changes in proliferation and cyclin protein expression after erlotinib treatments. A proof of principle clinical trial was conducted. During this study, patients underwent a 9-day course of erlotinib treatment. Pretreatment and posttreatment tumor biopsies were obtained, and changes in candidate biomarkers were determined by immunostaining. Plasma pharmacokinetics and tumor tissue erlotinib concentrations were measured. Erlotinib, at clinically achievable dosages, repressed BEAS-2B cell growth, triggered G1 arrest, and preferentially reduced cyclin D1 protein expression and transcriptional activation. Erlotinib also preferentially repressed proliferation and cyclin D1 protein expression in responsive, but not resistant, non-small-cell lung cancer cell lines. This occurred in the presence of wild-type EGFR sequence at exons 18, 19, and 21. Five patients were enrolled onto an erlotinib proof of principle clinical trial, and four cases were evaluable. Pharmacokinetic studies established therapeutic erlotinib plasma levels in all patients, but tissue levels exceeding 2 micromol/L were detected in only two cases. Notably, these cases had pathological evidence of response (necrosis) in posttreatment biopsies as compared with pretreatment biopsies. In these cases, marked repression of cyclin D1 and the proliferation marker Ki-67 was detected by immunohistochemical assays. Cases without pathological response to erlotinib did not exhibit changes in cyclin D1 or Ki-67 immunohistochemical expression and had much lower erlotinib tissue levels than did responding cases. Taken together, these in vitro and in vivo findings provide direct evidence for repression of cyclin D1 protein as a surrogate marker of response in aerodigestive tract cancers to erlotinib treatment. These findings also provide a rationale for combining an EGFR TKI with an agent that would cooperatively repress cyclin D1 expression in clinical trials for aerodigestive tract cancer therapy or chemoprevention.

Evaluation of epidermal growth factor receptor (EGFR) as a predictive marker in patients with non-small-cell lung cancer (NSCLC) receiving first-line gefitinib combined with platinum-based chemotherapy

7013 Background: In two Phase III trials (INTACT 1 & 2), gefitinib (‘Iressa’, ZD1839) in combination with first-line platinum-based chemotherapy regimens failed to show benefit in terms of survival when compared with chemotherapy alone in patients (pts) with NSCLC. The primary objective of this analysis was to evaluate if EGFR staining was predictive of survival. Methods: Pretreatment tumor biopsies were assessed by immunohistochemistry for EGFR using the DakoCytomation pharmDx assay™. The percentage of tumor cells was assessed using 4 levels of intensity: no staining, 0; weak, 1+; moderate, 2+; strong, 3+; as well as the presence of complete membrane staining. 516 pts (INTACT 1/2, 219/297) with tumor samples fully evaluable for EGFR were analyzed in the per protocol population of the combined trials. The analysis was stratified by trial and performed independently for pts randomized to placebo or gefitinib. A restricted backwards elimination Cox regression analysis was conducted to identify independent EGFR factors. Variables found to be significant (p<0.1) were also tested for treatment interaction to determine if they served as predictive factors. Results: The demographics of the analysis population were representative of the overall population in each trial as were common disease characteristics. The analysis showed that two EGFR-based variables are independent strong prognostic factors for both placebo- and gefitinib-treated patients. However, no interactions with treatment (ie no value for predicting benefit of gefitinib treatment) were indicated for either of these two variables (p=0.8688 and 0.4967) or for any other EGFR-based variables. Conclusion: Given the overall trial results for INTACT 1 & 2 showing no survival benefit between gefitinib and placebo, it was highly unlikely that EGFR variables would identify a subset of pts who receive survival benefit. The data presented support this hypothesis. 'Iressa' is a trademark of the AstraZeneca group of companies 'PharmDx assay' is a trademark of DakoCytomation Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca Pharmaceuticals; DakoCytomation AstraZeneca; DakoCytomation; Genentech AstraZeneca AstraZeneca AstraZeneca Pharmaceuticals

Evaluation of epidermal growth factor receptor (EGFR) as a predictive marker in patients with non-small-cell lung cancer (NSCLC) receiving first-line gefitinib combined with platinum-based chemotherapy

7013 Background: In two Phase III trials (INTACT 1 & 2), gefitinib (‘Iressa’, ZD1839) in combination with first-line platinum-based chemotherapy regimens failed to show benefit in terms of survival when compared with chemotherapy alone in patients (pts) with NSCLC. The primary objective of this analysis was to evaluate if EGFR staining was predictive of survival. Methods: Pretreatment tumor biopsies were assessed by immunohistochemistry for EGFR using the DakoCytomation pharmDx assay™. The percentage of tumor cells was assessed using 4 levels of intensity: no staining, 0; weak, 1+; moderate, 2+; strong, 3+; as well as the presence of complete membrane staining. 516 pts (INTACT 1/2, 219/297) with tumor samples fully evaluable for EGFR were analyzed in the per protocol population of the combined trials. The analysis was stratified by trial and performed independently for pts randomized to placebo or gefitinib. A restricted backwards elimination Cox regression analysis was conducted to identify independent EGFR factors. Variables found to be significant (p<0.1) were also tested for treatment interaction to determine if they served as predictive factors. Results: The demographics of the analysis population were representative of the overall population in each trial as were common disease characteristics. The analysis showed that two EGFR-based variables are independent strong prognostic factors for both placebo- and gefitinib-treated patients. However, no interactions with treatment (ie no value for predicting benefit of gefitinib treatment) were indicated for either of these two variables (p=0.8688 and 0.4967) or for any other EGFR-based variables. Conclusion: Given the overall trial results for INTACT 1 & 2 showing no survival benefit between gefitinib and placebo, it was highly unlikely that EGFR variables would identify a subset of pts who receive survival benefit. The data presented support this hypothesis. 'Iressa' is a trademark of the AstraZeneca group of companies 'PharmDx assay' is a trademark of DakoCytomation Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca Pharmaceuticals; DakoCytomation AstraZeneca; DakoCytomation; Genentech AstraZeneca AstraZeneca AstraZeneca Pharmaceuticals