Biological markers and DNA flow cytometric analysis in radically resected patients with non-small cell lung cancer. A study of the Perugia Multidisciplinary Team for Thoracic Tumors.

AIMS AND BACKGROUND: The aim of this study was to evaluate the relationship between a panel of biological markers (p53, Bcl-2, HER-2, Ki67, DNA ploidy and S-phase fraction) and clinical-pathological parameters and its impact on outcome in non-small cell lung cancer (NSCLC). METHODS AND STUDY DESIGN: Tumor tissue specimens obtained after surgical resection were collected from consecutive patients with NSCLC. We used an immunocytochemical technique for p53, Bcl-2, HER-2 and Ki67 analysis in fine-needle aspirates obtained from surgical samples that were also evaluated by flow cytometric DNA analysis using a FACScan flow cytometer. RESULTS: From April 2000 to December 2005, 136 patients with radically resected NSCLC were recruited. Median age was 66 years (range, 31-84 years), male/female ratio 117/19, ECOG performance status 0/1 127/4, stage I/II/III 76/25/35, squamous/adenocarcinoma/large-cell/mixed histology 62/49/17/8, smokers yes/no 121/11. Positivity of p53, Bcl-2, HER-2 and Ki67 was detected in 51.4%, 27.9%, 25.0% and 55.8% of the samples, respectively; 82.9% of the cases revealed aneuploid DNA histograms and 56.7% presented an S-phase fraction of more than 12%. Statistically significant associations between high Ki67 and poorly differentiated tumors (P = 0.016) and a smoking history (P = 0.053); p53 positivity and high Ki67 (P = 0.002); HER-2 positivity and adenocarcinoma subtype (P = 0.015) and presence of lymph node involvement (P = 0.006); and Bcl-2 positivity and squamous cell carcinoma subtype (P = 0.058) were observed. At univariate analysis, high Ki67 proved to be the only marker associated with disease-free survival (P = 0.047). After adjusting for stage, none of the examined immunocytochemical markers emerged as an independent factor for disease-free and overall survival; only pathological stage was identified as an independent prognostic factor for disease-free survival (P = 0.0001) and overall survival (P = 0.0001). In the group of 76 patients classified as TNM stage I, high Ki67 was the only marker associated with recurrence of disease (P = 0.05). CONCLUSIONS: Our data do not support a relevant prognostic role of immunocytochemical markers in NSCLC, even if the Ki67 index might have particular relevance to identify patients with more aggressive tumors who are at high risk for disease relapse.

Plasma DNA, microsatellite alterations, and p53 tumor mutations are associated with disease-free survival in radically resected non-small cell lung cancer patients: a study of the perugia multidisciplinary team for thoracic oncology.

INTRODUCTION: This prospective study examined association between circulating plasma DNA, microsatellite alterations (MA), p53 mutations with time to relapse and survival in surgically treated non-small cell lung cancer (NSCLC) patients (pts). METHODS: Plasma samples, adjacent lung tissue, and lung tumor tissue specimens were collected from consecutive patients with stage I-III NSCLC. Blood samples of 66 matched healthy donors with positive smoking history were collected as controls. The plasma DNA amount was determined by real-time PCR. The analysis of MA at loci D3S1300, D3S1289, D3S1266, and D3S2338 on chromosome 3p was performed by radiolabeled PCR. p53 Mutations (exons 5, 6, 7, and 8) were detected by PCR-single-strand conformational polymorphism assay. RESULTS: There were 76 patients, 65 men; median age was 68 years (range, 42-86), 20 had stage I, 40 stage II, and 16 stage III, the majority of pts (48.7%) had squamous-cell histology. Sixty-nine (91%) were smokers and most had good Eastern Cooperative Oncology Group performance status (0/1:72/4). Mean circulating DNA of all pts was 60 ng/ml versus 5 ng/ml in smoker-matched controls (p < 0.0001). In pts without recurrence, mean circulating DNA was 48.5 ng/ml at baseline, 32.8 ng/ml at 3 month, and 20.6 ng/ml at 12 month after surgery. In pts with recurrence, mean circulating DNA at baseline was 97.1 ng/ml. At 3 month after surgery, mean DNA concentration was significantly lower in disease-free pts than in patients with recurrent disease (32.8 versus 292.7 ng/ml; p = 0.0016). MA in at least one locus was found in 39.5% of NSCLC tumors. p53 Genomic mutations were observed in 54.0% of tumor samples. Statistically significant associations were observed between MA and squamous-cell histotype (p = 0.007) and between p53 mutations and lymph node involvement (p = 0.012). MA and p53 mutations were found to be significantly associated with recurrence of disease (p = 0.033 and 0.026, respectively). CONCLUSION: Our results suggest that MA and p53 mutations in tumor DNA have a potential prognostic role for disease recurrence in NSCLC patients, and elevated levels of plasma circulating DNA identify patients with possible systemic disease at diagnosis. This might be proposed as an early detection test of disease recurrence.

Vascular endothelial growth factor, p53, Rb, Bcl-2 expression and response to chemotherapy in advanced non-small cell lung cancer.

Vascular endothelial growth factor (VEGF) increases microvascular permeability and stimulates endothelial cell growth. p53 Overexpression has been associated with resistance to cisplatin-based chemotherapy in patients (pts) with NSCLC. The aim of this study was to evaluate the predictive role of VEGF for chemotherapy response, its relationship with p53, Rb, Bcl-2 and hemoglobin levels and its impact on overall survival in pts with advanced NSCLC. Bronchial or fine-needle biopsy specimens from 85 pts with NSCLC obtained before chemotherapy were analyzed using an immunohistochemical method for VEGF, p53, Rb and Bcl-2. There were 73 males and 12 females with a median age of 62.6 years. The majority of pts (48%) had squamous cell histology. Ten pts had stage IIIA, 25 stage IIIB and 50 stage IV. Thirty six (43%) pts had positive immunostaining for VEGF, 37 (44%) had positive p53, 53 (62%) had negative Rb and 4 (5%) had positive Bcl-2. VEGF was negatively correlated with Rb (r(s) = 0.26; P = 0.015), positively with Bcl-2 (r(s) = 0.22; P = 0.42), whereas no statistically significant correlation with p53, age, stage and histological type was found. In a logistic regression model, adjusting for treatment, VEGF expression was not associated with chemotherapy response (odds ratio (OR) = 1.01; P = 0.085 ), unlike p53 positivity and Rb negativity ( OR = 4.0, P = 0.005; OR = 2.6, P = 0.016, respectively). A statistically significant higher VEGF expression was detected in the subgroups defined, using as cut-off value Hb median level (13.3g/dl) (chi-square = 5.00; ; one d.f.; P = 0.025). At a median follow-up time of 8.4 years, 2-year survival was 21%. After adjustment for stage and chemotherapy treatment, VEGF expression was not associated with a better overall survival (OR = 1.06; P = 0.80), unlike Bcl-2 positivity showed a statistically significant effect (OR = 0.28; p = 0.02). Our results suggest that VEGF is weakly correlated with regulators of apoptosis and has not been shown to be an independent predictive factor for resistance to cisplatin-based chemotherapy and prognostic for survival.

The clinical relevance of Bcl-2, Rb and p53 expression in advanced non-small cell lung cancer.

PURPOSE: The aim of this study was to evaluate the impact of Bcl-2, retinoblastoma (Rb) and p53 proteins on overall survival of 102 patients with locally advanced and metastatic NSCLC who underwent cisplatin-based chemotherapy. MATERIALS AND METHODS: Paraffin-embedded bronchial biopsy and fine-needle biopsy specimens were evaluated by an immunostaining method. RESULTS: Median age of analyzed patients was 61 years. Male/female ratio was 88/14. There were 10 (10%) patients with stage IIIA, 37 (36%) with stage IIIB and 55 (54%) with stage IV NSCLC. Only 15 (15%) tumor specimens had no detectable alterations for analyzed factors. Forty-six samples (45%) had positive immunostaining for p53, 61 (60%) had negative immunostaining for Rb and 8 (8%) had positive immunostaining for Bcl-2. Median and 5-year survival of analyzed population was 12 months and 6%, respectively. In univariate analysis Bcl-2 overexpression, stage (III versus IV) and normal lactate dehydrogenase (LDH) serum levels were associated with better overall survival (P<0.02, 0.001 and 0.03). In multivariate analysis, only stage was identified as an independent predictive factor. CONCLUSION: High frequency of Rb and Bcl-2 loss was detected in patients with advanced NSCLC. P53, Rb and Bcl-2 have not been shown to be independent predictors of survival even if Bcl-2 might have a particular relevance in patients with advanced NSCLC and should be better explored in this setting.

Gemcitabine and cisplatin as induction chemotherapy for patients with unresectable Stage IIIA-bulky N2 and Stage IIIB nonsmall cell lung carcinoma: an Italian Lung Cancer Project Observational Study.

BACKGROUND: The objective of this trial was to evaluate the activity and safety of one of the newer platinum-based doublets as a neoadjuvant regimen in patients with unresectable Stage IIIA-bulky N2 and Stage IIIB nonsmall cell lung carcinoma (NSCLC). METHODS: From June 1996 to April 2000, 129 consecutive patients with locally advanced NSCLC were treated with gemcitabine, 1000 mg/m(2) on Days 1 and 8 and cisplatin, 70 mg/m(2) on Day 2 (GC) of a 21-day treatment cycle, for 4 cycles, as part of a combined-modality approach. RESULTS: After induction chemotherapy, 80 patients (62%; 95% confidence interval, 53.6-70.4%) achieved a partial response, 43 patients (33%) had stable disease, and 6 patients (5%) had disease progression during chemotherapy. Forty patients (31%), were considered resectable and underwent thoracotomy. Complete resectability was obtained in 38 patients (29%), with 2% of patients achieving a pathologic complete response. After surgery, 9 patients with Mountain Classification Stage IIIA NSCLC and 9 patients with Stage IIIB NSCLC received definitive adjuvant radiotherapy. Forty-six of 52 patients with Stage IIIB disease and 24 of 37 patients with Stage IIIA disease who were not considered suitable for surgery received definitive radiotherapy. The median time to disease progression was 11.4 months, the median survival was 19.4 months (range, 1.2-55.2 + months), and the 1-year survival rate was 74%. The lungs (33%) and the brain (21%) were the main sites of recurrence. Major toxicity was comprised of Grade 3-4 thrombocytopenia, which occurred in 34 patients (27%). CONCLUSIONS: GC administered according to a 3-week schedule was a highly active and safe regimen in patients with primary, unresectable, locally advanced NSCLC.

Relevance of p53, bcl-2 and Rb expression on resistance to cisplatin-based chemotherapy in advanced non-small cell lung cancer.

PURPOSE: Tumors with p53 overexpression have been associated with enhanced resistance to cisplatin-based chemotherapy in a few and small studies involving non-small cell lung cancer. The relationships and interactions between p53, Rb and bcl-2 immunostaining, clinical parameters and response to cisplatin-based chemotherapy were evaluated in the present study. EXPERIMENTAL DESIGN: Histological specimens obtained by bronchial or fine-needle biopsy from patients who underwent cisplatin-based chemotherapy between 1992 and 1999 were evaluated by immunostaining. RESULTS: There were 102 patients, 88 men. Median age was 63 years; 47 had stage III and 55 stage IV disease. Forty-six tumor samples (45%) had positive immunostaining for p53, 61 (59%) had negative immunostaining for Rb and 8 (8%) had positive immunostaining for bcl-2. The response rate of the group with p53 positive immunostaining was 26% versus 57% of the p53 negative group (P=0.004). In multivariate analyses p53 positive immunostaining was identified as an independent predictive factor for resistance to cisplatin-based chemotherapy (P=0.006). CONCLUSIONS: Our study confirmed an association of p53 immunostaining and response rate of patients treated with cisplatin-based chemotherapy.

Long-term results of induction chemotherapy followed by concurrent chemotherapy and thoracic irradiation in limited small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is a chemoresponsive tumor but overall survival remains poor even in limited disease (LD). With the aim of eradicating chemoresistant tumor cells and reducing toxicity, we investigated in this phase II trial the feasibility and outcome of a sequential approach of induction chemotherapy (CT) followed, in responding patients with LD-SCLC, by intensified platinum-based CT and concurrent thoracic irradiation (TI). MATERIALS AND METHODS: We treated 55 consecutive LD-SCLC patients with three 21-day cycles of cyclophosphamide, epiadriamycin and vincristine (CEV) as induction CT. In 44 (80%) patients there was an objective response and they received treatment intensification consisting of TI and concomitant CT with carboplatin and etoposide plus recombinant granulocite colony stimulating factor. Twenty-five (57%) patients were submitted to twice-daily thoracic irradiation (TDTI; 1.5 Gy per fraction, to a total dose of 45 Gy) and 19 (43%) to once-daily thoracic irradiation (ODTI; 2 Gy per fraction, to a total dose of 50 Gy). RESULTS: Median follow up was 75 months (range, 42-102). Of 44 patients submitted to intensification with TI plus CT, 32 (73%) had a complete and 12 (27%) a partial response. Median overall survival of all 55 patients was 17 months with actuarial survival probabilities of 2 and 5 years, 32 and 25%, respectively. Analysis of patient sub-groups showed a 5-month median survival in non-responders, 19 in TDTI and 17 in ODTI patients, respectively. Two and 5 year survival probabilities were 0% in non-responders, 40 and 35% in TDTI and 39 and 21% in ODTI patients, respectively. At present, 13 of 44 responders are still alive, of which nine (20%) have been progression-free from 45 to 93 months (median 60). Treatment failure was registered in 31 (70%) of 44 patients who received both induction and intensification treatment. One-half of patients had intrathoracic recurrence, eight of which only local and the remaining seven local and distant. Fourteen (32%) patients had brain metastases. Grade 3-4 neutropenia occurred in 24 (55%) patients with no differences between treatment groups. Grade 3 esophagitis was registered in four (9%) patients: in 3/25 (12%) and 1/19 (5%) of those who received TDTI and ODTI, respectively (P=not significant). Acute radiation pneumonitis occurred in three (12%) patients submitted to TDTI. No clinically debilitating pulmonary fibrosis, permanent esophageal stricture or toxic death was observed. CONCLUSIONS: In LD-SCLC patients late concurrent CT plus TI is feasible and effective. Our long-term results are similar to the best reported in the literature. Despite the high incidence of complete response obtained, however, one-half of the patients had intrathoracic relapse and one-third brain metastases.