Abstract: The identification of genomic rearrangements in breast/ovarian cancer families has widened the mutational spectrum of the BRCA1 gene, increasing the number of patients who can benefit from molecular screening. More than 60 different BRCA1 genomic rearrangements with mapped breakpoints have been reported up to date, in all exons of the gene. The proportion of BRCA1 mutations due to genomic rearrangements varies from 8 to 27% in different populations, probably due to both ethnic diversity and the technical approach employed. In order to estimate the contribution of BRCA1 genomic rearrangements to hereditary breast/ovarian cancer (HBOC) predisposition in Greek families, probands from 95 families with breast/ovarian history but negative for point mutations or small insertions/deletions in BRCA1 and BRCA2 genes, were screened using Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF). Two large deletions of 4.2 and 4.4 kb were identified in exons 20 and 24 respectively. Additional screening, using diagnostic primers for the above deletions in exons 20 and 24, performed on another 86 probands from families with breast/ovarian cancer history and 210 cases of sporadic breast/ovarian cancer resulted in the identification of two more large genomic rearrangements. One, identified in a familial case, identical to the previous exon 24 deletion and a second, identified in a case reported as sporadic, 3.2 kb deletion involving exon 20 and reported elsewhere in another Greek patient. Three out of four genomic rearrangements described in this study were detected in patients who had developed both breast and ovarian cancer; thus suggesting a correlation between the specific phenotype and the high probability of detecting inherited rearrangements in BRCA1.

Abstract: BACKGROUND: The purpose of this study was to evaluate the effect of adjuvant treatment with tamoxifen on the CK-19 mRNA+ cells in patients with early-stage breast cancer. PATIENTS AND METHODS: CK-19 mRNA+ cells were prospectively and longitudinally detected using a specific real-time PCR assay for CK-19 mRNA in 119 patients with estrogen and/or progesterone receptor-positive tumors during the period of tamoxifen administration. RESULTS: Twenty-two (18.5%) patients had detectable CK-19 mRNA+ cells after the completion of adjuvant chemotherapy and in 15 (68.2%) of them adjuvant tamoxifen could not eliminate these cells (persistently positive). In 68 (57.1%) patients, no CK-19 mRNA+ cells could be detected throughout the follow-up period (persistently negative). Seven (46.7%) of the 15 persistently positive and six (8.8%) of the 68 persistently negative patients developed disease recurrence (P = 0.00026). Persistency of CK-19 mRNA+ cells was associated with a significantly lower median disease-free interval (P = 0.0001) and overall survival (P = 0.0005). Multivariate analysis revealed that the detection of CK-19 mRNA+ cells during the administration of tamoxifen was associated with an increased risk of relapse [hazard ratio (HR) = 22.318, P = 0.00006] and death (HR = 13.954, P < 0.00001). CONCLUSIONS: The detection of CK-19 mRNA+ cells throughout the period of adjuvant tamoxifen treatment is an independent poor prognostic factor in patients with early breast cancer.

Abstract: PURPOSE: To examine the prognostic value of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTCs) in early-stage breast cancer patients focusing on clinically relevant subgroups based on estrogen receptor (ER) and HER2 expression. PATIENTS AND METHODS: CK-19 mRNA-positive CTCs were detected by real-time reverse transcriptase polymerase chain reaction in the blood of 444 consecutive, stage I-III, breast cancer patients before initiation of adjuvant chemotherapy. The association between detection of CK-19 mRNA-positive CTCs and clinical outcome was analyzed for patients with ER-positive, ER-negative, triple-negative, HER2-positive, and ER-positive/HER2-negative tumors. RESULTS: CK-19 mRNA-positive CTCs were detected in 181 (40.8%) of 444 patients; 109 (41.9%) of 260 patients with ER-positive tumors; 71 (40.6%) of 175 patients with ER-negative tumors; 27 (35%) of 77 patients with triple-negative tumors; 35 (39.8%) of 88 patients with HER2-positive tumors; and 82 (44.1%) of 186 patients with ER-positive/HER2-negative tumors. After a median follow-up of 53.5 months, patients with CK-19 mRNA-positive CTCs experienced reduced disease-free survival (DFS; P < .001) and overall survival (OS; P < .001); this was mainly observed in patients with ER-negative (P < .001 and P < .001, respectively) but not ER-positive tumors (P = .172 and P = .425, respectively) and in patients with triple-negative (P = .008 and P = .001, respectively) and HER2-positive (P = .023 and P = .040, respectively) but not ER-positive/HER2-negative tumors (P = .210 and P = .578, respectively). In multivariate analysis, the interaction between CK-19 mRNA-positive CTCs and ER status was the strongest independent prognostic factor for reduced DFS (hazard ratio [HR], 3.808; 95% CI, 2.415 to 6.003; P < .001) and OS (HR, 4.172; 95% CI, 2.477 to 9.161; P < .001). CONCLUSION: Detection of CK-19 mRNA-positive CTCs before adjuvant chemotherapy predicts poor clinical outcome mainly in patients with ER-negative, triple-negative, and HER2-positive early-stage breast cancer.

Abstract: PURPOSE: The aim of this study was to evaluate the clinical relevance of the simultaneous detection of cytokeratin (CK)-19 messenger RNA (mRNA)- and HER2 mRNA-positive cells in peripheral blood of women with early-stage breast cancer. PATIENTS AND METHODS: CK-19 mRNA- and HER2 mRNA-positive cells were detected using a real-time and a nested reverse-transcriptase polymerase chain reaction assay, respectively, in a cohort of 185 women with early-stage breast cancer before the initiation of any adjuvant systemic treatment. Detection of CK-19 mRNA- and HER2 mRNA-positive cells in the peripheral blood was correlated with clinical outcome. RESULTS: Overall, 63 of the 185 patients (34%) had detectable CK-19 mRNA-positive cells, and 33 (52.3%) also had detectable HER2 mRNA-positive cells. Patients with CK-19/HER2 mRNA-negative cells showed a trend toward longer disease-free survival (DFS) compared with patients with CK-19 mRNA-positive/HER2 mRNA-negative cells (P = .054) and had longer DFS than patients with CK-19/HER2 mRNA-positive cells (P < .001). Similarly, overall survival (OS) was higher in patients with CK-19/HER2 mRNA-negative cells compared with patients with CK-19 mRNA-positive/HER2 mRNA-negative cells (P = .039) or CK-19/HER2 mRNA-positive cells (P < .001). Patients with CK-19/HER2 mRNA-positive cells had shorter DFS but not OS compared with patients with CK-19 mRNA-positive/HER2 mRNA-negative cells. In multivariate analysis, the simultaneous detection of CK-19 mRNA- and HER2 mRNA-positive cells was independently associated with early relapse. CONCLUSION: The simultaneous detection of CK-19 mRNA- and HER2 mRNA-positive cells in peripheral blood predicts poor clinical outcome for women with early-stage breast cancer.

Abstract: PURPOSE: To evaluate the predictive and prognostic value of peripheral blood cytokeratin-19 (CK-19) mRNA-positive cells in axillary lymph node-negative breast cancer patients. PATIENTS AND METHODS: Peripheral blood was obtained from 167 node-negative breast cancer patients before the initiation of any systemic adjuvant therapy, and was analyzed for the presence of CK-19 mRNA-positive cells using a real time polymerase chain reaction assay. The association with known prognostic factors and the effect of CK-19 mRNA-positive cells on patients' prognosis was investigated. RESULTS: CK-19 mRNA-positive cells were detected in the blood of 36 (21.6%) of the 167 patients. There was no correlation between the detection of CK-19 mRNA-positive cells in the peripheral blood and the various known pathologic and clinical prognostic factors; only overexpression of HER2 receptor (score 2+/3+) on the primary tumor was associated with a higher incidence of CK-19 mRNA-positive cell detection (P = .033). Multivariate analysis revealed that detection of peripheral blood CK-19 mRNA-positive cells was associated with early clinical relapse (P < .00001) and disease-related death (P = .008). CONCLUSION: Detection of peripheral-blood CK-19 mRNA-positive cells is an independent predictive and prognostic factor for reduced disease-free interval and overall survival, respectively, in node-negative breast cancer patients.

Abstract: BACKGROUND: The purpose of this study was to investigate the toxicity and efficacy of the sequential administration of gemcitabine (GMB) in combination with cisplatin (CDDP) followed by docetaxel (Taxotere) as first-line treatment of advanced urothelial carcinoma. PATIENTS AND METHODS: Patients [aged </=70 years and performance status (PS) (Eastern Cooperative Oncology Group) 0-2] with previously untreated locally advanced/recurrent or metastatic urothelial carcinoma were eligible. Study treatment consisted of GMB (1000 mg/m(2), days 1 and 8) and CDDP (70 mg/m(2), day 1) (GP regimen), every 21 days for a total of four cycles followed by docetaxel (D; 100 mg/m(2), day 1) every 21 days for four cycles. RESULTS: Thirty-eight patients with a median age of 67 years were enrolled; 67% of them had PS 0 and 87% stage IV disease. Patients received a median of four GP and four D cycles per patient. Grade 3-4 neutropenia occurred in 27% and 63% patients with GP and D, respectively. Grade 3-4 thrombocytopenia occurred in 11% of patients, only with the GP regimen. Other toxic effects were mild. There was no toxic death. The objective response rate was 55.2% [95% CI: 39.45%-71.07%]. Five patients had complete response (13.15%) and 16 patients had partial response (42.1%), while nine patients had disease stabilization (23.7%) (intention-to-treat analysis). After a median follow-up period of 13 months (range 1.5-40.5 months), the median time to progression was 6.8 months (range 1-40.5 months), the median overall survival 13 months (range 1.5-40.5 months), and the 1-year survival rate 55.3%. CONCLUSION: The sequential administration of GP followed by D is active and well tolerated as first-line treatment of advanced urothelial carcinoma and merits to be further evaluated.

Abstract: Cisplatin or carboplatin plus paclitaxel is considered the standard first-line treatment in ovarian cancer. Attempts to maximize tumor cytoreduction with first-line chemotherapy by incorporating new promising agents led to sequential drug administration with two or three doublets. In the present study, we aimed to evaluate the activity and the tolerance of two sequential doublets (paclitaxel/carboplatin and liposomal doxorubicin/carboplatin) administered as first-line treatment in patients with FIGO III/IV ovarian cancer. Treatment consisted of four cycles of carboplatin (6 AUC) plus paclitaxel (175 mg/m2; PC regimen) followed by four cycles with carboplatin (6 AUC) plus liposomal doxorubicin (40 mg/m(2); LD/C regimen) every 3 weeks. Forty-one patients in FIGO III or IV were enrolled. In an intention-to-treat analysis, 20 (49%) complete (CR) and 12 (29%) partial (PR) responses were achieved (overall response rate, ORR: 78%; 95% confidence interval, CI: 64.1-91.9%); with the PC regimen (164 cycles); 7 (17%) patients have stable (SD) and 2 (5%) progressive (PD) disease. The LD/C regimen (124 cycles) was administered in 36 (88%) patients because of 2 early deaths and 3 patient withdrawals. Three additional patients, 2 with PR and 1 with SD after PC chemotherapy) achieved a CR. Upon completion of the LD/C chemotherapy there were 18 (44%) patients with CR and 9 (22%) with PR (ORR=66%; 95% CI: 64-92%). The median duration of response was 27 months and the median time to progression 20 months. The probability of 2-year survival was 67%. Grade 3 and 4 neutropenia was observed in 34 and 14.6% of the patients, respectively, during the PC regimen, while during the treatment with LD/C the percentages for grade 3 and 4 neutropenia were 44.4 and 19.4%, respectively. Febrile neutropenia occurred only in patients treated with the PC regimen (4.9%). The incorporation of liposomal doxorubicin in this sequential doublet schedule of first-line treatment of ovarian carcinoma created a feasible and active regimen. Prospective randomized studies are required to assess its efficacy on patient survival.

Abstract: PURPOSE: To evaluate the efficacy and tolerance of irinotecan (CPT-11) in combination with bolus and continuous infusion of 5-fluorouracil (5-FU) and leucovorin (LV) (FOLFIRI regimen) as first-line treatment of elderly patients with metastatic colorectal cancer (MCC). METHODS: Thirty consecutive, previously untreated patients with metastatic colorectal cancer, aged (median 76 years; range 70-84) were enrolled. The performance status (WHO) was 0 in 8, 1 in 16 and 2 in 6 patients; 19 (63%) patients had prior surgery and 8 (27%) adjuvant chemotherapy. CPT-11 (180 mg/m(2) as a 90 min i.v. infusion) was administered on day 1, LV (200 mg/m(2) as a 2-hour i.v. infusion), 5-FU (400 mg/m(2)/d i.v. bolus followed by 600 mg/m(2)/d as a 22-hour i.v. continuous infusion) were given on days 1 and 2 every 2 weeks. RESULTS: Complete response was achieved in one (3.3%) patient and partial response in 10 (33.3%) (overall response rate: 36.6%; 95% C.I.: 26.6-48.4%); 11 (36.6%) patients had stable disease and, 8 (26.6%) disease progression. The median duration of response was 7.5 months and the median time to disease progression 7.0 months. After a median follow-up period of 17 months, the median overall survival was 14.5 months. Main toxicities were: grade 3-4 neutropenia (n = 6; 20%), grade 3 thrombocytopenia (n = 1; 3.3%), grade 2 anemia (n = 9; 30%), grade 3-4 diarrhea (n = 5; 17%) and grade 3 asthenia (n = 3; 10%). There was one treatment-related death due to neutropenic sepsis. CONCLUSIONS: The FOLFIRI combination is an active regimen with manageable toxicity as front-line treatment in patients above 70 years of age.

Abstract: OBJECTIVES: Capecitabine (CAP) and oxaliplatin (OX) have shown interesting activity in a wide range of solid tumors. A phase I study was conducted in order to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of their combination in patients with refractory solid tumors. PATIENTS AND METHODS: Thirty-three pretreated patients with histologically confirmed inoperable neoplasms were enrolled. The patients' median age was 64 years, 21 were males, and 27 had a WHO performance status of 0-1. OX was administered on days 1 and 8, as a 3-hour intravenous infusion, at escalated doses ranging from 50 to 70 mg/m(2). CAP was administered orally for 14 consecutive days, at escalated doses ranging from 1,200 to 2,100 mg/m(2)/day. Treatment was repeated every 3 weeks. RESULTS: At the dose of 2,100 mg/m(2) (Xeloda) and 70 mg/m(2) (OX), all 3 enrolled patients presented DLT (grade 3 diarrhea, grade 3 asthenia and grade 3 neurotoxicity, respectively), and, thus, the recommended MTD for future phase II studies are 2,000 mg/m(2) for CAP and 70 mg/m(2 )for OX. A total of 145 treatment cycles were administered. Toxicity was very mild. Grade 2/3 neutropenia was observed in 4 (3%) treatment cycles. The main nonhematologic toxicities were grade 2/3 nausea/vomiting (7 cycles; 5%), grade 2/3 neurotoxicity (10 cycles; 7%), grade 2/3 asthenia (8 cycles; 5.5%) and grade 2/3 diarrhea (6 cycles; 4%). There was no treatment-related death. One (4%) complete remission, 2 (8%) partial remissions, and 9 (36%) cases of stable disease were observed among 25 evaluable patients. CONCLUSIONS: The results demonstrate that CAP and OX can be safely combined at clinically relevant doses and that this regimen merits further evaluation.

Abstract: BACKGROUND: The purpose of this study was to evaluate the prognostic significance of the molecular detection of cytokeratin 19 (CK-19) mRNA-positive cells in the peripheral blood of women with operable breast cancer after the completion of adjuvant chemotherapy. PATIENTS AND METHODS: Blood from 161 patients with stage I and II breast cancer, obtained after the completion of adjuvant chemotherapy, was tested by nested RT-PCR for CK-19 mRNA detection. Using univariate and multivariate analyses possible interactions with other prognostic factors and association of CK-19 mRNA detection with risk of relapse, disease-free interval (DFI) and overall survival were investigated. RESULTS: After completion of adjuvant chemotherapy, 27.3% of patients had peripheral blood CK-19 mRNA-positive cells; there was no association of this finding with any other prognostic factors or the type of chemotherapy regimen used. For patients with less than four involved axillary lymph nodes the risk of relapse was 3.81 [95% confidence interval (CI) 1.06-13.71] times higher, and the DFI was significantly reduced (P = 0.028) if CK-19 mRNA-positive cells were detectable in the blood after the completion of adjuvant chemotherapy. In contrast, for patients with four or more involved lymph nodes, the presence of CK-19 mRNA-positive cells after adjuvant chemotherapy did not significantly affect the risk of relapse or DFI. Furthermore, the risk of relapse was higher (hazards ratio 3.70; 95% CI 1.09-13.89) and the DFI was reduced (P = 0.022) for patients with detectable CK-19 mRNA-positive cells following adjuvant cyclophosphamide, methotrexate and 5-fluorouracil (CMF) as compared with epirubicin, cyclophosphamide and 5-fluorouracil (FEC) or sequential taxotere-epirubicin and cyclophosphamide (T/EC) chemotherapy. CONCLUSIONS: The detection of CK-19 mRNA-positive cells in the peripheral blood after adjuvant chemotherapy may be of clinical relevance for patients with early breast cancer and less than four involved axillary lymph nodes.

Abstract: PURPOSE: To evaluate the prognostic significance of molecular detection of cytokeratin 19 (CK-19) mRNA-positive cells by nested reverse transcriptase polymerase chain reaction (RT-PCR) in the peripheral blood of women with stages I and II breast cancer before adjuvant chemotherapy. PATIENTS AND METHODS: The sensitivity and specificity of CK-19 mRNA detection by nested RT-PCR were investigated using MCF-7 and ARH-77 cells and blood from healthy women and patients with hematologic malignancies, metastatic colorectal cancer, and early and metastatic breast cancer. Peripheral blood from 148 patients with operable breast cancer, obtained before initiation of any adjuvant therapy, was tested for the presence of CK-19 mRNA-positive cells. RESULTS: The nested RT-PCR assay for CK-19 mRNA detected one MCF-7 tumor cell in 10(6) normal peripheral blood mononuclear cells in four of five experiments; no signal was detected with the CK-19-negative ARH-77 cells. CK-19 mRNA was detected in the peripheral blood of 3.7% of healthy blood donors, 14.3% of patients with hematologic malignancies, and 3.2% of patients with metastatic colorectal cancer. Detection rates for CK-19 mRNA-positive cells in the bone marrow/blood of patients with early or metastatic breast cancer were 63%/30% and 74%/52%, respectively. For stages I and II breast cancer, detection of CK-19-positive cells in the peripheral blood before adjuvant therapy was associated with reduced disease-free interval (P =.0007) and overall survival (P =.01). In multivariate analysis, detection of peripheral-blood CK-19-positive cells was an independent prognostic factor for disease relapse and death. CONCLUSION: Molecular detection of CK-19 mRNA-positive cells by RT-PCR in the peripheral blood of patients with stages I and II breast cancer before initiation of adjuvant therapy has independent prognostic value as a marker of poor clinical outcome.