Evangelia Razis

Potential value of PTEN in predicting cetuximab response in colorectal cancer: An exploratory study

BackgroundThe epidermal growth factor receptor (EGFR) is over-expressed in 70–75% of colorectal adenocarcinomas (CRC). The anti-EGFR monoclonal antibody cetuximab has been approved for the treatment of metastatic CRC, however tumor response to cetuximab has not been found to be associated with EGFR over-expression by immunohistochemistry (IHC). The aim of this study was to explore EGFR and the downstream effector phosphatase and tensin homologue deleted on chromosome 10 (PTEN) as potential predictors of response to cetuximab.MethodsCRC patients treated with cetuximab by the Hellenic Cooperative Oncology group, whose formalin-fixed paraffin-embedded tumor tissue was available, were included. Tissue was tested for EGFR and PTEN by IHC and fluorescence in situ hybridization (FISH).ResultsEighty-eight patients were identified and 72 were included based on the availability of tissue blocks with adequate material for analysis on them. All patients, except one, received cetuximab in combination with chemotherapy. Median follow-up was 53 months from diagnosis and 17 months from cetuximab initiation. At the time of the analysis 53% of the patients had died. Best response was complete response in one and partial response in 23 patients. In 16 patients disease stabilized. Lack of PTEN gene amplification was associated with more responses to cetuximab and longer time to progression (p = 0.042).ConclusionPTEN could be one of the molecular determinants of cetuximab response. Due to the heterogeneity of the population and the retrospective nature of the study, our results are hypothesis generating and should be approached with caution. Further prospective studies are needed to validate this finding.

Phase II Study of Neoadjuvant Imatinib in Glioblastoma: Evaluation of Clinical and Molecular Effects of the Treatment

Purpose: Phase I-II studies indicate that imatinib is active in glioblastoma multiforme. To better understand the molecular and clinical effects of imatinib in glioblastoma multiforme, we conducted a neoadjuvant study of imatinib with pretreatment and posttreatment biopsies. Experimental Design: Patients underwent a computerized tomography-guided biopsy of their brain tumors. If diagnosed with glioblastoma multiforme, they were immediately treated with 7 days of imatinib 400 mg orally twice daily followed by either definitive surgery or re-biopsy. Pretreatment and posttreatment tissue specimens were tested by immunohistochemistry for Ki67 and microvessel destiny, and posttreatment specimens were analyzed for the presence of intact imatinib in tissue. Furthermore, pretreatment and posttreatment pairs were analyzed by Western blotting for activation of platelet-derived growth factor receptor, epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase/AKT, and mitogen-activated protein kinase signaling pathways. Pharmacokinetic studies were also done. Results: Twenty patients were enrolled. Median survival was 6.2 months. Intact imatinib was detected in the posttreatment tissue specimens using mass spectrometry. There was no evidence of a drug effect on proliferation, as evidenced by a change in Ki67 expression. Biochemical evidence of response, as shown by decreased activation of AKT and mitogen-activated protein kinase or increased p27 level, was detected in 4 of 11 patients with evaluable, matched pre- and post-imatinib biopsies. Two patients showed high-level EGFR activation and homozygous EGFR mutations, whereas one patient had high-level platelet-derived growth factor receptor-B activation. Conclusions: Intact imatinib was detected in glioblastoma multiforme tissue. However, the histologic and immunoblotting evaluations suggest that glioblastoma multiforme proliferation and survival mechanisms are not substantially reduced by imatinib therapy in most patients. (Clin Cancer Res 2009;15(19):6258–66)

Sustained complete remission of metastatic dermatofibrosarcoma protuberans with imatinib mesylate.

Dermatofibrosarcoma protuberans (DFSP) is a soft tissue tumor which may recur locally and rarely causes metastases to vital organs. DFSPs have specific chromosomal abnormalities involving the platelet-derived growth factor &bgr;-chain locus (PDGFB) which may render these tumors responsive to targeted therapy with the tyrosine kinase inhibitor imatinib mesylate. A patient with locally recurrent and metastatic DFSP resistant to first-line chemotherapy was treated with imatinib mesylate 400 mg/day. The tumor was examined by a novel fluorescence in situ hybridization (FISH) method for specific rearrangements of the PDGFB locus. The patient was followed for response and toxicity by physical examination and imaging studies. FISH revealed PDGFB rearrangement indicative of multiplication of the PDGFB fusion locus within a ring chromosome. Physical examination showed response within the first month of treatment, and subsequent computed tomography and fluorodeoxyglycose positron emission tomography documented complete response to imatinib therapy. Our patient is now in sustained complete remission for 20 months with minimal toxicity. We conclude that sustained complete remission of metastatic DFSP with specific FISH abnormalities involving the PDGFB locus can be obtained with imatinib mesylate with minimal toxicity for the patient.

Biomarkers of benefit from cetuximab-based therapy in metastatic colorectal cancer: interaction of EGFR ligand expression with RAS/RAF, PIK3CA genotypes

BackgroundMore than half of patients with KRAS-wild type advanced colorectal cancer (CRC) fail anti-EGFR monoclonal antibodies. We studied EGFR-axis messenger RNA (mRNA) expression and RAS, RAF, PIK3CA mutations in order to identify additional biomarkers of cetuximab efficacy.MethodsPreviously genotyped (KRAS, NRAS, BRAF, PIK3CA mutations) formalin-fixed paraffin-embedded tumour biopsies of 226 cetuximab-treated CRC patients (1st to 3rd line therapy) were assessed for mRNA expression of epidermal growth factor receptor (EGFR) and its ligands EGF, Transofrming Growth Factor-a (TGFA), Amphiregulin (AREG) and Epiregulin (EREG) with real time quantitative PCR. Mutations were detected in 72 (31.9%) tumours for KRAS, in 6 (2.65%) for BRAF, in 7 (3.1%) for NRAS and in 37 (16.4%) for PIK3CA.ResultsOnly PIK3CA mutations occasionally coexisted with other gene mutations. In univariate analysis, prognostic significance for survival ( from metastases until death) was seen for BRAF mutations (Hazard Ratio HR 8.1, 95% CI 3.4-19), codon 12-only KRAS mutations (HR 1.62, 95% CI 1.1-2.4), high AREG mRNA expression only in KRAS wild type CRC (HR 0.47, 95% CI 0.3-0.7) and high EREG mRNA expression irrespective of KRAS mutation status (HR 0.45, 95% CI 0.28-0.7). EREG tumoural mRNA expression was significantly associated with a 2.26-fold increased likelihood of objective response to cetuximab therapy (RECIST 1.1). In multivariate analysis, favourable predictive factors were high AREG mRNA in KRAS wild type tumours, high EREG mRNA, low Ephrin A2 receptor mRNA. Cetuximab-treated patients with AREG-low KRAS wild type CRC fared very poorly, their survival being similar to KRAS mutant CRC. Patients with KRAS codon 13 or other non-codon 12 mutations had a median survival (30 months, 95% CI 20–35) similar to that of patients with KRAS wild-type (median survival 29 months, 95% CI 25–35), in contrast to patients with KRAS codon 12 mutations who fared worse (median survival 19 months, 95% CI 15–26).ConclusionsBRAF and codon 12 KRAS mutations predict for adverse outcome of CRC patients receiving cetuximab. AREG mRNA reflects EGFR signalling in KRAS wild type tumours, predicting for cetuximab efficacy when high and failure when low. EREG may have a prognostic role independent of KRAS mutation.

Greek BRCA1 and BRCA2 mutation spectrum: two BRCA1 mutations account for half the carriers found among high-risk breast/ovarian cancer patients

Abstract127 Greek breast/ovarian cancer families were screened for germline BRCA1/2 mutations by dHPLC followed by direct sequencing. Our results indicated 16 and 5 breast/ovarian cancer families bearing deleterious mutations in the BRCA1 and BRCA2 genes, respectively. Two novel BRCA2 germline mutations (G4X and 3783del10) are reported here for the first time. Subsequent compilation of our present findings with previously reported mutation data reveals that in a total of 287 Greek breast/ovarian cancer families, 46 and 13 carry a deleterious mutation in BRCA1 and BRCA2, respectively. It should be noted that two BRCA1 mutations, 5382insC and G1738R, both located in exon 20, account for 46% of the families found to carry a mutation. Based on our mutation analysis results, we propose here a hierarchical, cost-effective BRCA1/2 mutation screening protocol for individuals of Greek ethnic origin. The suggested protocol can impact on the clinical management of breast-ovarian cancer families on a national healthcare system level.

Paclitaxel and carboplatin as first-line chemotherapy combined with gefitinib (IRESSA) in patients with advanced breast cancer: a phase I/II study conducted by the Hellenic Cooperative Oncology Group.

SummaryPaclitaxel (TaxolR) and carboplatin are an effective combination regimen for treating advanced breast cancer. Gefitinib (IRESSA) is the first epidermal growth factor receptor tyrosine kinase inhibitor to be approved for cancer treatment. This multicenter phase II trial treated 68 patients with advanced breast cancer with paclitaxel (175 mg/m2 over 3 h) and 3-weekly carboplatin (area under the curve of 6) for six cycles, and 250 mg/day gefitinib orally. Median age was 57 (range 35–77) years, patients had performance status 0 (69.1%), 1 (27.9%) 2 (2.9%), 82.4% of patients had visceral metastases and 63.2% had received adjuvant chemotherapy. Forty-eight (70.6%) patients completed six cycles of chemotherapy and 20 (29.4%) patients discontinued treatment (seven [10.3%] due to disease progression, seven [10.3%] due to toxicity, five [7.4%] withdrew consent and one [1.5%] died after the first cycle). Sixty-three (92.7%) patients were evaluable for response; nine (13.2%) had complete responses, 30 (44.1%) had partial responses, 21 (30.9%) had stable disease and three (4.4%) had disease progression. Grade 3/4 adverse events in ≥5% of patients except of alopecia, included neutropenia (17.7%), anemia (10.3%), diarrhea (7.4%), thrombocytopenia (5.9%) and peripheral neuropathy (5.9%). Of those tumor biopsies available for immunohistochemical analysis (n=60), 5.0% were positive and 35.0% negative for expression of all HER-family receptors. Comparable numbers of tumor biopsies were nuclear p27kipl positive and negative (39.7 and 42.7%, respectively), with the majority (72.1%) negative for cytoplasmic p27kipl. The observed efficacy data in this study were similar to those reported for the combination of paclitaxel and carboplatin alone.

Improved outcome of high-risk early HER2 positive breast cancer with high CXCL13-CXCR5 messenger RNA expression.

UNLABELLED The CXCL13-CXCR5 is a chemokine axis that is activated in some breast cancers. A total of 321 tissue blocks from a group of patients who received adjuvant, dose-dense chemotherapy for high-risk early breast cancer were examined. Activation of this axis was found to be associated with determinants of poor prognosis but also with improved outcome in the human epidermal growth factor receptor 2 overexpressing subpopulation. BACKGROUND Chemokines are important in cell migration and are thought to play a key role in metastasis. We explored the prognostic significance of C-X-C ligand-motif (CXCL) 12, CXCL13, and receptor (CXCR) 5 on disease-free survival (DFS) and overall survival (OS) in early breast cancer. METHODS A total of 595 patients with high risk, [corrected] early breast cancer were treated in a 2-arm trial (HE10/97) with dose-dense sequential epirubicin followed by cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) with or without paclitaxel. RNA was extracted from 321 formalin-fixed paraffin-embedded primary tumor tissue samples and quantitative reverse-transcriptase polymerase chain reaction was used to assess messenger RNA (mRNA) expression of CXCL12, CXCL13, and CXCR5; estrogen receptor; progesterone receptor (PgR); microtubule-associated protein tau and human epidermal growth factor receptor 2 (HER2). RESULTS CXCL13 and CXCR5 were found to be negatively associated with estrogen receptor and microtubule-associated protein tau mRNA expression and with dense lymphocytic infiltration, and were positively associated with nuclear grade. Only CXCL13 was positively associated with HER2. Multivariate analysis revealed an association between high CXCL13 mRNA expression and improved DFS (hazard ratio [HR] 0.48 [95% CI, 0.25-0.90]; Wald, P = .023) but not OS; whereas high CXCL12 expression was significantly associated with increased OS (HR 0.53 [95% CI, 0.33-0.85]; Wald, P = .009). In the HER2 mRNA overexpressing subgroup, high CXCL13 mRNA expression was associated with improved DFS (P < .001), whereas high CXCR5 was associated with increased DFS and OS (P = .004 and P = .049, respectively). CONCLUSIONS The CXCL13-CXCR5 axis is associated with classic determinants of poor prognosis, such as high grade, hormone receptor negativity, and axillary node involvement. Interestingly, this chemokine axis seems to be strongly associated with improved outcome in patients with HER2(+) disease.

Triple negative breast cancer: proposals for a pragmatic definition and implications for patient management and trial design.

In trials in triple negative breast cancer (TNBC), oestrogen and progesterone receptor negativity should be defined as < 1% positive cells. Negativity is a ratio of <2 between Her2 gene copy number and centromere of chromosome 17 or a copy number of 4 or less. In routine practice, immunohistochemistry is acceptable given stringent quality assurance. Triple negativity emerging after neoadjuvant treatment differs from primary TN and such patients should not enter TNBC trials. Patients relapsing with TN metastases should be eligible even if their primary was positive. Rare TN subtypes such as apocrine, adenoid-cystic and low-grade metaplastic tumours should be excluded. TN and basal-like (BL) signatures overlap but are not equivalent. Since the significance of basal cytokeratin or EGFR overexpression is not known and we lack validated assays, these features should not be used to subclassify TN tumours. Tissue collection in trials is mandatory so the effect on outcome of different tumour phenotypes and BRCA mutation can be explored. No prospective studies have established that TN tumours have particular sensitivity or resistance to any specific chemotherapy agent or radiation. TNBC patients should be treated according to tumour and clinical characteristics.

Paclitaxel and Gemcitabine, As First-Line Chemotherapy, Combined with Trastuzumab in Patients with Advanced Breast Cancer: A Phase II Study Conducted by the Hellenic Cooperative Oncology Group (HeCOG)

Purpose: Advanced breast cancer (ABC) is an incurable disease. Standard first-line treatment for patients with HER-2/neu overexpressing tumors includes the combination of the humanized monoclonal antibody trastuzumab with chemotherapy, mainly paclitaxel. This combination is the first to demonstrate a survival advantage in this group of patients. To improve on these results, we investigated a triplet, paclitaxel-gemcitabine-trastuzumab (TGH), in a phase II study. Patients and Methods: Patients with ABC were accrued to the study. Treatment consisted of paclitaxel 80 mg/m2/week, gemcitabine 1000 mg/m2 every 2 weeks, and trastuzumab 4 mg/kg loading dose and then 2 mg/kg/week. Patients were treated on study for a total of 12 weeks. Response evaluation was performed at the end of the 12 weeks. Continuation of treatment beyond the 12 weeks was left to the discretion of the investigator. Primary study endpoint was response. Toxicity assessment and survival were secondary endpoints. Results: Between November 2000 and May 2002, 40 patients were accrued and 32 patients completed all 12 weeks of therapy. One patient died of septic shock during therapy. Grade III and IV neutropenia was seen in 12.5% of cases each. Grade III anemia was seen in two patients, and grade III and IV thrombocytopenia in three and two patients, respectively. Both paclitaxel and gemcitabine were delivered at 86% of the planned dose intensity. Six patients achieved a complete response (CR) and 15 a partial response for an overall response rate of 52.5%. An additional 25% demonstrated stable disease and 20% progressive disease. Median duration of response was 14 months. All six patients who achieved CR are still in CR for 6 to 19 months. After a median follow up of 12.2 months, 19 patients have progressed and 7 have died. Median time to progression is 13.7 months, whereas median survival has not been reached. Conclusion: TGH is a well-tolerated and effective regimen for the first-line treatment of ABC. Randomized comparison between paclitaxel, trastuzumab, and triplets are warranted.

Dose-dense sequential chemotherapy with epirubicin and paclitaxel versus the combination, as first-line chemotherapy, in advanced breast cancer: a randomized study conducted by the Hellenic Cooperative Oncology Group.

PURPOSE To compare the efficacy of two different schedules of epirubicin and paclitaxel, as first-line chemotherapy, in patients with advanced breast cancer (ABC). PATIENTS AND METHODS From October 1997 until May 1999, 183 eligible patients with ABC entered the study. Chemotherapy in group A (93 patients) consisted of four cycles of epirubicin at a dose of 110 mg/m(2) followed by four cycles of paclitaxel at a dose of 225 mg/m(2) in a 3-hour infusion. All cycles were repeated every 2 weeks with granulocyte colony-stimulating factor support. The therapeutic regimen in group B (90 patients) consisted of epirubicin (80 mg/m(2)) immediately followed by paclitaxel (175 mg/m(2) in a 3-hour infusion) every 3 weeks for six cycles. RESULTS In total, 79 patients (85%) in group A and 72 patients (80%) in group B completed treatment. The median relative dose-intensity of epirubicin was 0.96 in both groups, and that of paclitaxel was 0.96 and 0.97 in groups A and B, respectively. The complete response rate was higher in group A (21.5% v 9% P =.02). Nevertheless, there was no significant difference in the overall response rate between the two groups (55% v 42%, P =.10). Severe neutropenia was more frequently observed with concurrent treatment. After a median follow-up of 16.5 months, median time to progression was 10 months in group A and 8.5 months in group B (P =.27), and median survival was 21.5 and 20 months, respectively (P =.17). CONCLUSION The present study failed to demonstrate a significant difference in overall response rate between dose-dense sequential administration of epirubicin and paclitaxel compared with the combination of the two drugs given on the same day, even though the sequential treatment resulted in a significantly higher complete response rate.