Georg Lurje

FCGR2A and FCGR3A Polymorphisms Associated With Clinical Outcome of Epidermal Growth Factor Receptor–Expressing Metastatic Colorectal Cancer Patients Treated With Single-Agent Cetuximab

PURPOSE Cetuximab, a chimeric immunoglobulin G 1 (IgG1) anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), has shown efficacy in 10% of patients with metastatic colorectal cancer (CRC). Recent studies demonstrate antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the modes of action for rituximab and trastuzumab. Fragment c (Fc) portion of IgG1 mAb has shown to induce ADCC. Fragment c gamma receptors (FcgammaR) play an important role in initiating ADCC. Studies have shown that two IgG FcgammaR polymorphisms (FCGR2A-H131R and FCGR3A-V158F) independently predict response to rituximab in patients with follicular lymphoma. We tested the hypothesis of whether these two polymorphisms are associated with clinical outcome in metastatic CRC patients treated with single-agent cetuximab. PATIENTS AND METHODS Thirty-nine metastatic CRC patients were enrolled onto the ImClone0144 trial. Using an allele-specific polymerase chain reaction (PCR) -based method, gene polymorphisms of FCGA2A-H131R and FCGA3A-V158F were assessed from genomic DNA extracted from peripheral blood samples. RESULTS FCGR2A-H131R and FCGR3A-V158F polymorphisms were independently associated with progression-free survival (PFS; P = .037 and .055, respectively; log-rank test). Combined analysis of these two polymorphisms showed that patients with the favorable genotypes (FCGR2A, any histidine allele, and FCGR3A, any phenylalanine allele) showed a median PFS of 3.7 months (95% CI, 2.4 to 4.4 months), whereas patients with any two unfavorable genotypes (FCGR2A arginine/arginine or valine/valine) had a PFS of 1.1 months (95% CI, 1.0 to 1.4 months; P = .004; log-rank test). CONCLUSION Our preliminary data suggest that these two polymorphisms may be useful molecular markers to predict clinical outcome in metastatic CRC patients treated with cetuximab and that they may indicate a role of ADCC of cetuximab.

EGFR signaling and drug discovery.

Dysregulation of human epidermal growth factor receptor (ErbB/HER) pathways by over-expression or constitutive activation can promote tumor processes including angiogenesis and metastasis and is associated with poor prognosis in many human malignancies. In addition to cancer, ErbB signaling has also been implicated in cardiovascular and neurodegenerative diseases. Conversely, inhibition of ErbB pathways with targeted agents, such as monoclonal antibodies (MoAbs) or tyrosine kinase inhibitors (TKIs), blocks cell cycle progression, inhibits the production of pro-angiogenic factors and induces apoptosis in numerous in vitro and xenograft models. Accordingly, the ErbB receptor family with their most prominent members EGFR and HER-2 represents validated targets for anti-cancer therapy, and anti-ErbB MoAbs (cetuximab, panitumumab, and trastuzumab) and TKIs (gefitinib, erlotinib, and lapatinib) have now been approved for the treatment of advanced colorectal cancer, squamous cell carcinoma of the head and neck, advanced non-small-cell lung cancer, as well as pancreatic and breast cancer. Although results have been encouraging, more work remains to be done.

Interleukin‐8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models

Interleukin‐8 (IL‐8), a chemokine with a defining CXC amino acid motif, is known to possess tumorigenic and proangiogenic properties. Overexpression of IL‐8 has been detected in many human tumors, including colorectal cancer (CRC), and is associated with poor prognosis. The goal of our study was to determine the role of IL‐8 overexpression in CRC cells in vitro and in vivo. We stably transfected the IL‐8 cDNA into two human colon cancer cell lines, HCT116 and Caco2, and selected IL‐8‐secreting transfectants. Real‐time RT‐PCR confirmed that IL‐8 mRNA was overexpressed in IL‐8 transfectants with 45‐ to 85‐fold higher than parental cells. The IL‐8‐transfected clones secreted 19‐ to 28‐fold more IL‐8 protein than control and parental cells as detected by ELISA. The IL‐8 transfectants demonstrated increased cellular proliferation, cell migration and invasion based on functional assays. Growth inhibition studies showed that IL‐8 overexpression lead to a significant resistance to oxaliplatin (p < 0.0001). Inhibition of IL‐8 overexpression with small interfering RNA reversed the observed increases in tumorigenic functions and oxaliplatin resistance, suggesting that IL‐8 not only provides a proliferative advantage but also promotes the metastatic potential of colon cancer cells. Using a tumor xenograft model, IL‐8‐expressing cells formed significantly larger tumors than the control cells with increased microvessel density. Together, these findings indicate that overexpression of IL‐8 promotes tumor growth, metastasis, chemoresistance and angiogenesis, implying IL‐8 to be an important therapeutic target in CRC.

Polymorphisms and Clinical Outcome in Recurrent Ovarian Cancer Treated with Cyclophosphamide and Bevacizumab

Purpose: This study was designed to evaluate the associations between angiogenesis gene polymorphisms and clinical outcome in ovarian cancer patients treated with low-dose cyclophosphamide and bevacizumab. Experimental Design: Seventy recurrent/metastatic epithelial ovarian cancer patients were enrolled in a phase II clinical trial. Genomic DNA was available from 53 blood samples. Polymorphisms were analyzed using the PCR-RFLP protocol. A 5′ end 33P γATP-labeled PCR protocol was used to analyze dinucleotide repeats. Results: Patients genotyped A/A or A/T for the IL-8 T-251A gene polymorphism had a statistically significant lower response rate (19%; 0%) than those homozygous T/T (50%; P = 0.006, Fishers exact test). Patients carrying a minimum one C allele (C/C; C/T) of the CXCR2 C+785T polymorphism showed a median progression-free survival (PFS) of 7.4 months compared with the PFS of 3.7 months for those homozygous T/T (P = 0.026, log-rank test). Patients with the VEGF C+936T polymorphism C/T genotype had a longer median PFS of 11.8 months, compared with those with the C/C and T/T genotype, which had median PFS of 5.5 months and 3.2 months, respectively (P = 0.061, log-rank test). Patients carrying both AM 3′end alleles <14 CA repeats had the shortest median PFS of 3.4 months; patients with at least one allele >14 repeats or both alleles >14 repeats showed a median PFS of 6.4 months and 7.2 months, respectively (P = 0.008, log-rank test). Conclusion: Our data suggest that the IL-8 A-251T polymorphism may be a molecular predictor of response to bevacizumab-based chemotherapy. The CXCR2 C+785T, VEGF C+936T single nucleotide polymorphisms and the AM 3′ dinucleotide repeat polymorphisms may be molecular markers for PFS in ovarian cancer patients.

Polymorphisms in Cyclooxygenase-2 and Epidermal Growth Factor Receptor Are Associated with Progression-Free Survival Independent of K-ras in Metastatic Colorectal Cancer Patients Treated with Single-Agent Cetuximab

Purpose: Recently, an objective response rate of 12% was reported in a phase II study of cetuximab in patients with epidermal growth factor receptor (EGFR)-expressing metastatic colorectal cancer (mCRC) refractory to fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy (IMC-0144). In this large molecular correlates study, we tested whether K-ras mutation status and polymorphisms in genes involved in the EGFR-signaling pathway were associated with clinical outcome in IMC-0144. Experimental Design: We analyzed all available tissue samples from 130 of 346 mCRC patients enrolled in the IMC-0144 phase II clinical trial of cetuximab. Genomic DNA was extracted from formalin-fixed paraffin-embedded tumor tissues, and K-ras mutation status and the genotypes were analyzed using PCR-RFLP, direct DNA-sequencing, and 5′-end [γ-33P] ATP–labeled PCR-protocols. Results: The PFS of patients with cyclooxygenase-2 (COX-2) −765 G>C [C/C; risk ratio (RR), 0.31; 95% confidence interval (95% CI), 0.12-0.84; P = 0.032], COX-2 +8473 T>C (C/C; RR, 0.67; 95% CI, 0.40-1.13; P = 0.003), EGF +61 A>G (G/G; RR, 0.57; 95% CI, 0.34-0.95; P = 0.042), and EGFR +497 G>A (A/G; RR, 0.82; 95% CI, 0.56-1.20; P = 0.017) genotypes was significantly longer compared with those with other genotypes. In addition, patients whose tumors did not have K-ras mutations showed better RR, PFS, and overall survival than patients with K-ras mutations. In multivariable analysis, COX-2 +8473 T>C (adjusted P = 0.013) and EGFR +497 G>A (adjusted P = 0.010) remained significantly associated with progression-free survival, independent of skin rash toxicity, K-ras mutation status, and Eastern Cooperative Group performance status. Conclusions: Polymorphisms in COX-2 and EGFR may be useful independent molecular markers to predict clinical outcome in patients with mCRC treated with single-agent cetuximab, independent of skin rash toxicity, K-ras mutation, and Eastern Cooperative Oncology Group performance status.

Gender Disparities in Metastatic Colorectal Cancer Survival

Purpose: Previous studies have shown that estrogen prevents colon cancer in postmenopausal women, indicating a role in colorectal cancer carcinogenesis and tumor progression. We investigated the interactions between sex, age, ethnicity, and year of diagnosis on overall survival (OS) in patients with metastatic colorectal cancer (MCRC). Experimental Design: We screened 52,882 patients with MCRC from 1988 to 2004, using the Surveillance Epidemiology and End Results registry. Age at diagnosis, sex, ethnicity, tumor location, year of diagnosis, OS, and cancer-specific survival were evaluated using Cox proportional hazards model. The models were adjusted for marital status, tumor site, tumor differentiation, and treatment with radiation and/or surgery. Results: We observed that younger women (18-44 years old) with MCRC lived longer than younger men (17 months versus 14; P < 0.0001, log-rank test). In contrast, older women (55 years and older) had significantly worse OS than older men (7 months versus 9; P < 0.0001, log-rank test). In multivariate analysis, we found that gender discrepancies have widened in recent years; young women diagnosed after 2000 have improved cancer-specific survival, compared to men (hazard ratio, 0.778; 95% confidence interval, 0.669-0.904), but those diagnosed before 2000 benefit less (hazard ratio, 0.931; 95% confidence interval, 0.821-1.056). Conclusion: As one of the largest data sets analyzed to establish that younger women with MCRC survive longer than younger men, hormonal status not only seems to play an important role in the development and pathogenesis of colorectal cancer but also may be of prognostic significance. These data warrant further studies to determine the role of estrogen in colorectal cancer. (Clin Cancer Res 2009;15(20):6391–7)

Survival of metastatic gastric cancer: Significance of age, sex and race/ethnicity

BACKGROUND Despite the success of modern chemotherapy in the treatment of large bowel cancers, patients with metastatic gastric cancer continue to have a dismal outcome. Identifying predictive and prognostic markers is an important step to improving current treatment approaches and extending survival. METHODS Extracting data from the US NCIs Surveillance, Epidemiology, and End Results (SEER) registries, we compared overall survival for patients with metastatic gastric cancer by gender, age, and ethnicity using Cox proportional hazards models. 13,840 patients (≥ 18 years) were identified from 1988-2004. Males and females were categorized by age grouping and ethnicity. RESULTS 19% of Hispanic patients were diagnosed < 45 years of age as compared to 5.5% of Caucasians. Caucasian patients and men were more likely to be diagnosed with tumors in the gastric cardia (P<0.001). In our survival analysis, we found that women had a lower risk of dying as compared to men (P<0.001). Overall survival diminished with age (P<0.001). The median overall survival was 6 months in patients of ≤ 44 years old as compared to 3 months in patients 75 years and older. Gender differences in overall survival significantly varied by race and tumor grade/differentiation (P for interaction = 0.003 and 0.005, respectively). CONCLUSION This is the largest study of metastatic gastric cancer patients from the SEER registry to show that age, gender, and tumor location are significant independent prognostic factors for overall survival in patients with metastatic gastric cancer.

Thymidylate synthase gene variations: predictive and prognostic markers

Since its introduction more than 50 years ago by Heidelberger et al., the fluoropyrimidine 5-fluorouracil (5-FU) has remained the mainstay of therapeutic regimens used in the treatment of colorectal cancer and other human malignancies, with single-agent response rates of 20% to 25% in advanced disease stage. Pharmacogenomics has emerged as a useful tool to address interindividual gene variations by analyzing the interplay of host and tumor genotype and drug efficacy and toxicity. Having a reliable panel of prognostic and predictive markers will be critical in selecting an individualized and tailored chemotherapy regimen based on the particular tumor and host genotype. Although conflicting results have been reported, higher thymidylate synthase (TS) protein and mRNA expression levels in tumors have generally been associated with poor clinical outcome in patients treated with 5-FU–based chemotherapy regimens. However, the cause of the variability in TS expression still remains not fully understood, although several germ-line polymorphisms seem to affect the expression of TS, some of which have been found to have an effect on prognosis and the probability of response to 5-FU–based chemotherapy. This review will provide an update on pharmacogenomic studies of TS that were aimed at elucidating their role as prognostic and predictive markers. [Mol Cancer Ther 2009;8(5):1000–7]

Thymidylate Synthase Haplotype is Associated with Tumor Recurrence in Stage II and Stage III Colon Cancer Patients

Background Tumor recurrence after curative resection is a major problem in the management of colon cancer therapy. Identifying molecular markers for tumor recurrence is critical for successfully selecting patients who are more likely to benefit from adjuvant chemotherapy. We analyzed the value of thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms as a prognostic marker in stage II and stage III colon cancer patients treated with 5-fluorouracil-based adjuvant chemotherapy. Methods Between 1987 and 2007, blood samples were obtained from 197 patients with stage II or stage III colon cancer at medical facilities at the University of Southern California. DNA was extracted from peripheral blood, and the genotypes were analyzed using PCR–restriction fragment length polymorphism technique. Results Patients harboring the TS 3RG/+6-bp haplotype were at greatest risk to develop tumor recurrence [relative risk (RR): 2.25; 95% confidence interval (CI): 1.04–4.85; adjusted P value=0.032]. TS enhancer region 3RG alone (RR: 3.48 years; 95% CI: 1.61–7.54; adjusted P value=0.013) or in combination with TS 1494del6 bp (RR: 3.41 years; 95% CI: 1.33–8.75; adjusted P value=0.044) proved to be adverse prognostic markers in both univariate and multivariable analysis. Conclusion ‘High-expression’ variants of TS 2R/3R repeat, TS enhancer region 3R G/C, TS 1494del6 bp, and TS haplotype analysis might help to identify stage II and stage III colon cancer patients who are at great risk of developing tumor recurrence, and also those who are more likely to benefit from 5-fluorouracil-based adjuvant chemotherapy. Larger, independent, prospective studies are, however, needed to confirm and validate our preliminary findings.

Gender-Related Survival Differences Associated with EGFR Polymorphisms in Metastatic Colon Cancer

Evidence is accumulating supporting gender-related differences in the development of colonic carcinomas. Sex steroid hormone receptors are expressed in the colon and interact with epidermal growth factor receptor (EGFR), a gene widely expressed in colonic tissue. Increased EGFR expression is linked with poor prognosis in colon cancer. Within the EGFR gene there are two functional polymorphisms of interest: a polymorphism located at codon 497 (HER-1 R497K) and a dinucleotide (CA)(n) repeat polymorphism located within intron 1. These germ-line polymorphisms of EGFR were analyzed in genomic DNA from 318 metastatic colon cancer patients, 177 males and 141 females, collected from 1992 to 2003. Gender-related survival differences were associated with the HER-1 R497K polymorphism (P(interaction) = 0.003). Females with the HER-1 497 Arg/Arg variant had better overall survival (OS) when compared with the Lys/Lys and/or Lys/Arg variants. In males the opposite was true. The EGFR dinucleotide (CA)(n) repeat also trended with a gender-related OS difference (P(interaction) = 0.11). Females with both short <20 (CA)(n) repeat alleles had better OS than those with any long >or=20 (CA)(n) repeats. In males the opposite was true. Combination analysis of the two polymorphisms taken together also revealed the same gender-related survival difference (P(interaction) = 0.002). These associations were observed using multivariable analysis. The two polymorphisms were not in linkage disequilibrium and are independent of one another. This study supports the role of functional EGFR polymorphisms as independent prognostic markers in metastatic colon cancer. As a prognostic factor, these variants had opposite prognostic implications based on gender.