[Molecular targets in colon cancer]. / Molekulare Targets beim Kolonkarzinom.

Colorectal cancer is the second leading cause of cancer death in Switzerland. The nihilism that dominated the treatment of these patients for decades has been replaced by a measure of enthusiasm, given recent therapeutic advances. New anticancer drugs such as irinotecan and oxaliplatin have changed the standard chemotherapy treatment of metastatic colorectal cancer. However, the real hype has come from molecular targeted therapy. Identification of cellular processes characteristic of colon cancer has permitted therapeutic targeting with favorable therapeutic index. Inhibition of the epidermal growth factor receptor in the clinic has provided proof of principle that interruption of signal transduction cascades in patients has therapeutic potential. Angiogenesis, especially the vascular endothelial growth factor pathway, has been proven to be another highly successful molecular target. In this article, we will review molecular targets, which are under active clinical investigation in colon cancer.

A randomized phase II trial of capecitabine and two different schedules of irinotecan in first-line treatment of metastatic colorectal cancer: efficacy, quality-of-life and toxicity.

BACKGROUND: To determine the efficacy, impact on quality-of-life (QoL) and tolerability of two different irinotecan administration schedules in combination with capecitabine as first-line treatment of metastatic colorectal cancer. PATIENTS AND METHODS: We carried out a randomized phase II trial to select one of the following treatment regimens for further investigation: weekly irinotecan at a dose of 70 mg/m(2) days 1, 8, 15, 22, 29 (arm A) or 3-weekly irinotecan at a dose of 300/240 mg/m(2) day 1 and days 22 (arm B) in combination with capecitabine 1000 mg/m(2) twice daily days 1-14 and days 22-35 every 6 weeks. RESULTS: Seventy-five patients with good performance status entered the trial. The two arms were well balanced for relevant patient and disease characteristics. The most frequent toxic effects were grade 3/4 diarrhea (arm A: 34%, B: 19%), grade 3/4 neutropenia (A: 5%, B: 19%) and grade 2/3 alopecia (A: 26%, B: 65%). Other grade 3/4 toxic effects were rare (<5%). Response rates were 34% [95% confidence interval (CI) 20% to 51%] in arm A and 35% (95% CI: 20% to 53%) in arm B. Median time to progression was 6.9 (4.6-10.1) and 9.2 (7.9-11.5) months and median overall survival was 17.4 (12.6-23.0+) and 24.7 (16.3-26.4+) months. Patients with an objective tumor response reported better physical well-being (P < 0.01), mood (P < 0.05), functional performance (P < 0.05) and less effort to cope (P < 0.05) compared with the non-responders and stable disease patients. CONCLUSIONS: The primary end point of this study was the objective response rate and based on the statistical design of the trial, the 3-weekly irinotecan schedule was selected over weekly irinotecan administration. The 3-weekly irinotecan schedule also seemed advantageous in terms of grade 3/4 diarrhea, time to progression, overall survival and patient convenience, but the study was not designed to detect differences in these parameters. In addition, tumor response was shown to have a beneficial effect on QoL indicators.

5-Fluorouracil as protracted continuous intravenous infusion can be added to full-dose docetaxel (Taxotere)-cisplatin in advanced gastric carcinoma: a phase I-II trial.

BACKGROUND: A phase I-II multicenter trial was conducted to define the maximum tolerated dose (MTD) according to tolerance and toxicity (primary objective), as well as to describe the clinical activity, in terms of response and survival (secondary objectives), of a combination of 5-fluorouracil (5-FU) in protracted continuous intravenous infusion (p.i.v.) with docetaxel and cisplatin for patients with advanced gastric cancer. PATIENTS AND METHODS: Patients with measurable unresectable and/or metastatic gastric carcinoma, World Health Organization performance status < or =1, normal hematological and renal functions, adequate hepatic function and not pretreated for advanced disease by chemotherapy, received up to eight cycles of a combination of docetaxel on day 1, cisplatin on day 1 and 5-FU p.i.v. on days 1-14 (TCF) every 3 weeks, which was escalated up to the MTD, defined by the occurrence of dose-limiting toxicity in two patients in one dose level. RESULTS: Fifty-two patients were accrued and treated (43 in the phase I part of the trial and nine additional at the recommended dose level). A median of five cycles/patient was given. The recommended dose of TCF was docetaxel 85 mg/m(2) on day 1, cisplatin 75 mg/m(2) on day 1 and 5-FU p.i.v. 300 mg/m(2)/day on days 1-14. Grade > or =3 toxicities were neutropenia 79%, alopecia 46%, fatigue 23%, mucositis 10%, diarrhea 19%, nausea/vomiting 13%, neurological 4% and palmar-plantar 2%. Ten non-fatal febrile neutropenia episodes were recorded in eight patients. There were no treatment-related deaths. Among 41 patients with measurable disease (79%), we observed one complete and 20 partial responses for an overall intent-to-treat response rate of 51% (95% confidence interval 35-67%). Five patients (20%) had stable disease for > or =12 weeks (four cycles). The median overall survival was 9.3 months. CONCLUSIONS: 5-FU p.i.v. at 300 mg/m(2)/day for 2 weeks out of three could be safely added to the docetaxel-cisplatin (TC) combination, but the dose of docetaxel had to be reduced to 75 mg/m(2) in a subsequent phase II trial. This drug regimen seems to be very active in advanced gastric cancer. Comparison with both TC and ECF in a randomized SAKK trial is ongoing.

Adjuvant therapy after excision and radiation of isolated postmastectomy locoregional breast cancer recurrence: definitive results of a phase III randomized trial (SAKK 23/82) comparing tamoxifen with observation.

BACKGROUND: Adjuvant systemic treatment for patients with isolated locoregional recurrence (ILRR) of breast cancer is based on a single reported randomized trial. The trial, conducted by the Swiss Group for Clinical Cancer Research, compared tamoxifen (TAM) with observation after complete excision of the ILRR and proper radiotherapy. We performed a definitive analysis of treatment outcome at >11 years of follow-up, after the majority of the patients had a subsequent event of interest. Patient and methods One hundred and sixty-seven patients with 'good-risk' characteristics of disease were randomized. 'Good-risk' was defined as estrogen receptor expression in the ILRR, or having a disease-free interval of >12 months and a recurrence consisting of three or less tumor nodules, each

Cyclin D1 overexpression in bronchial epithelia of patients with lung cancer is associated with smoking and predicts survival.

PURPOSE: Cyclin D1 is overexpressed in almost 60% of resectable non-small-cell lung cancer (NSCLC). In the absence of cyclin D1 gene amplification, overexpression is characterized by allelic imbalanced transcript levels. METHODS: The aims were to study cyclin D1 expression by immunohistochemistry and allelic balance of transcripts in tumor-free bronchial epithelia from patients with resectable NSCLC by using monoclonal antibodies (48 patients and 288 sites), microdissection/reverse transcriptase polymerase chain reaction/restriction fragment length polymorphism analyses (24 patients and 144 sites). Derived data were related to patient characteristics-in particular, smoking habits. RESULTS: In 167 (58%) of 288 sites, cyclin D1 was overexpressed, with cytoplasmic and nuclear sublocalization in 53% and 7% of all sites, respectively. Nuclear overexpression was more frequent in premalignant versus normal or hyperplastic epithelia (55% v 3%; P <.0001). Allele-specific expression imbalances were found in 69 (48%) of 144 sites; in particular, those in which cyclin D1 was overexpressed (P =.004). In 14 (58%) of 24 patients, balanced or imbalanced transcript ratios and degree of expression were consistent at all sites for the same patient, whereas in another 10 patients, transcript balances and cyclin D1 expression patterns varied across the sites. Nuclear cyclin D1 expression in at least one site (14 of 48 patients) was linked to heavy smoking (> 40 pack-years; P =.02) and shorter overall survival (P =.01). CONCLUSION: Allele-specific, probably damage-driven, deregulation of the cyclin D1 gene may precede and perhaps facilitate the spread of preneoplastic clones across the bronchial epithelial surface in a significant number of patients. Cyclin D1 expression at multiple bronchial sites may identify a subgroup of heavy-smoking patients with poor outcome.

Activity of raltitrexed and gemcitabine in advanced pancreatic cancer.

BACKGROUND: Gemcitabine has evolved as standard therapy in advanced pancreatic cancer since the demonstration of a significant clinical benefit. Phase II trials have shown that gemcitabine can be successfully combined with thymidylate synthase (TS) inhibitors such as continuous-infusion 5-fluorouracil (5-FU). However, continuous-infusion 5-FU is inconvenient because of the need for a central venous access. The aim of this study was to assess the efficacy and safety of gemcitabine in combination with raltitrexed (Tomudex), a novel and selective TS inhibitor that has the advantage of a 3-weekly treatment interval and manageable toxicity. PATIENTS AND METHODS: Chemotherapy-naïve patients with measurable advanced pancreatic cancer were treated with raltitrexed 3 mg/m(2) as a 15-min infusion on day 1 and gemcitabine 1000 mg/m(2) on days 1 and 8, every 21 days. RESULTS: Twenty-five eligible patients (17 male, eight female) with metastatic (21 patients) or locally advanced (four patients) disease entered the study. The median number of courses per patient was four (range 1-14). One patient was not evaluable for response. There were three partial remissions [12%; 95% confidence interval (CI) 2.6% to 31.2%] and nine stable disease situations (36%; 95% CI 18.0% to 57.5%), while the tumours of 12 patients (48%; 95% CI 27.8% to 68.7%) showed progressive disease after three treatment cycles. WHO grade 3/4 toxicity was rare and symptomatic in only one patient, who experienced grade 4 diarrhoea and grade 3 nausea and vomiting. Symptomatic benefit was seen in 12 patients. Median survival was 185 days (95% CI 129-241) with six patients still alive. CONCLUSIONS: The efficacy of raltitrexed plus gemcitabine is limited, but compares well with other chemotherapy treatment options in advanced pancreatic cancer. However, this combination is convenient and symptomatic toxicity is rare. Thus, raltitrexed and gemcitabine should be investigated further in combination with drugs interfering with specific molecular targets.

Mitomycin C induces apoptosis and caspase-8 and -9 processing through a caspase-3 and Fas-independent pathway.

Caspase-3 activity has been described to be essential for drug-induced apoptosis. Recent results suggest that in addition to its downstream executor function, caspase-3 is also involved in the processing of upstream caspase-8 and -9. To test the absolute requirement for caspase-3, we examined mitomycin C (MMC)-induced apoptosis in the caspase-3 deficient human breast cancer cell line MCF-7. MMC was used as anticancer drug since this agent was preferentially active compared to chemotherapeutic compounds with differing mechanisms of action such as cisplatin, docetaxel, or lovastatin. MMC treatment led to pronounced caspase-8, -9, and -7 processing and early morphological features of apoptosis within 48 h. This could be inhibited by the broad-spectrum caspase inhibitor z-VAD.fmk and to a lesser extent by z-IETD.fmk and z-LEHD.fmk, which have a certain preference for inhibiting caspase-8 and -9, respectively. MMC induced apoptosis in MCF-7 cells was not mediated by the death receptor pathway as demonstrated by experiments using the inhibiting anti-Fas antibody ZB4 and transfections with CrmA, a viral serpin inhibitor of caspase-8, and the dominant negative Fas-associated death domain (FADD-DN). Stable expression with Bcl-2 significantly prevented the processing of caspase-9 but also of caspase-8 and blocked the induction of apoptosis. Thus, we provide evidence that caspase-3 activity is dispensable for MMC-induced apoptosis and for caspase-8 and -9 processing in MCF-7 cells.

Patient preference and pharmacokinetics of oral modulated UFT versus intravenous fluorouracil and leucovorin: a randomised crossover trial in advanced colorectal cancer.

The aim of this study was to determine the patient's preference for oral UFT/leucovorin (LV) or intravenous (i.v.) 5-fluorouracil (5-FU)/LV chemotherapy in metastatic colorectal cancer and to compare 5-FU exposure with these two treatment options. A total of 37 previously untreated patients with advanced colorectal cancer were randomised to start treatment with either oral UFT 300 mg/m2/day plus oral LV 90 mg/day for 28 days every 5 weeks or i.v. 5-FU 425 mg/m2/day plus LV 20 mg/m2/day for 5 days every 4 weeks. For the second treatment cycle, patients were crossed-over to the alternative treatment regimen. Prior to the first and after the second therapy cycle, patients were required to complete a therapy preference questionnaire (TPQ). The pharmacokinetics of 5-FU were determined by taking blood samples on days 8, 15 or 22 and 28 for UFT and on days 1 and 5 for i.v. 5-FU. 36 patients were eligible. 84% of the patients preferred oral UFT over i.v. 5-FU. After having experienced both treatment modalities, patients indicated taking the medication at home, less stomatitis and diarrhoea, and pill over injection as the most important reasons for their preference. The area under the plasma concentration curve (AUC) for 5-FU after UFT administration was 113 microM x min on day 8, 114 on day 15 and 98 on day 28; the peak levels (Cmax) were 1.2, 1.3 and 1.0 microM, respectively. The AUC for the 5-FU/LV courses was 3083 microM x min for day 1 and 3809 for day 5 (P=0.002). The Cmax was 170.1 and 196.2 microM (P=0.06) and the clearance 2.6 and 1.9 l/min, respectively (P=0.002). Patients with metastatic colorectal cancer clearly preferred oral over i.v. chemotherapy treatment. This choice was most importantly influenced by convenience and toxicity considerations. Although i.v. bolus 5-FU leads to higher peak 5-FU concentrations and AUC values compared with oral UFT, this pharmacokinetic advantage of i.v. 5-FU seems to translate mainly into higher toxicity as seen in large randomised studies comparing oral UFT/LV with i.v. 5-FU/LV. Oral UFT/LV compares favourably with i.v. 5-FU/LV in terms of toxicity and patient's preference and leads to prolonged 5-FU exposure, which is comparable to continuous i.v. 5-FU treatment.

Alterations of cell cycle regulators are less frequent in advanced non-small cell lung cancer than in resectable tumours.

Prognosis of lung cancer is related to stage of disease at time of diagnosis. In this study we examine alterations of pathways governing the cell cycle, in particular pRb-cyclinD1-p16 alpha and p53-p14ARF, in a series of NSCLC (n=92) at different stages at diagnosis. Using immunohistochemistry, we assessed the expression of the retinoblastoma protein (pRb), cyclin D1, p16 alpha, p53 and p14ARF. Tumours in stage I-IIIA (resectable) were more likely to have alterations in the pRb-cyclinD1-p16 alpha pathway than tumours in advanced stage (IIIB-IV) (90% versus 63%, P=0.002). pRb and p14ARF were more frequently downregulated in resectable tumours (P< or =0.03), and cyclin D1, p16 alpha, and p53 were altered at a similar frequency in resectable and advanced tumours. In 12 patients, metastatic sites (5 lymph node, 3 bone, 2 brain and 2 gastrointestinal metastases) were available for comparison with the primary tumour: 19 altered protein expressions were found to be concordant, six additional alterations (in 4 patients) were found in the metastases only, especially in lymph node metastases (3 patients). Compared with normal protein expression, both pathway alterations were associated with a longer survival (P=0.02). In a multivariate analysis (Cox regression) this difference was not maintained after adjustment for age, stage and tumour differentiation. Cyclin D1 was the sole protein with independent prognostic value in resectable tumours: the relative risk of local relapse was 4.7 in tumours without cyclin D1 overexpression (P=0.02, Cox regression analysis). No protein studied had a predictive significance for response after chemotherapy in non-resectable tumours. These results demonstrate a strong correlation between stage and pathway alterations, cell cycle regulators being less likely altered in advanced NSCLC. Tumours with defects in these control pathways tend therefore to remain localised and to metastasize at a later phase in tumour development. This finding might be an explanation for distinct biological behaviour (e.g. chemotherapy response) of resectable versus advanced disease.

The bmi-1 oncoprotein is differentially expressed in non-small cell lung cancer and correlates with INK4A-ARF locus expression.

Genes of the polycomb group function by silencing homeotic selector genes that regulate embryogenesis. In mice, downregulation of one of the polycomb genes, bmi-1, leads to neurological alterations and severe proliferative defects in lymphoid cells, whilst bmi-1 overexpression, together with upregulation of myc-1, induces lymphoma. An oncogenic function has been further supported in primary fibroblast studies where bmi-1 overexpression induces immortalization due to repression of p16/p19ARF, and where together with H-ras, it readily transforms MEFs. It was the aim of this study to assess the expression of bmi-1 in resectable non-small cell lung cancer (NSCLC) in association with p16 and p14ARF (=human p19ARF). Tumours (48 resectable NSCLC (32 squamous, 9 adeno-, 2 large cell, 4 undifferentiated carcinomas and 1 carcinoid); stage I, 29, II, 7, III, 12; T1, 18, T2, 30; differentiation: G1 12, G2 19, G3 17) were studied by immunohistochemistry for protein expression and by comparative multiplex PCR for gene amplification analysis. In tumour-free, normal lung tissue from patients, weak - moderate bmi-1 staining was seen in some epithelial cells, lymphocytes, glandular cells and in fibroblasts, whereas blood, endothelial, chondrocytes, muscle cells and adipocytes did not exhibit any bmi-1 expression. In tumours, malignant cells were negative/weakly, moderately and strongly positive in 20, 22 and 6 cases, respectively. As assessed by multiplex PCR, bmi-1 gene amplification was not the reason for high-level bmi-1 expression. Tumours with moderate or strong bmi-1 expression were more likely to have low levels of p16 and p14ARF (P = 0.02). Similarly, tumours negative for both, p16 and p14ARF, exhibit moderate-strong bmi-1 staining. 58% of resectable NSCLC exhibit moderate-high levels of bmi-1 protein. The inverse correlation of bmi-1 and the INK4 locus proteins expression (p16/p14ARF) supports a possible role for bmi-1 misregulation in lung carcinogenesis.

Activity of gemcitabine and continuous infusion fluorouracil in advanced pancreatic cancer.

BACKGROUND/OBJECTIVES: Gemcitabine has been shown to improve survival and quality of life parameters compared to fluorouracil alone in advanced pancreatic cancer [J Clin Oncol 1997;15:2403-2413]. However, fluorouracil was given as a weekly bolus in that study and other administration schedules might be more effective. The objective of this trial was to determine the activity and toxicity of gemcitabine in combination with continuous infusion (CI) fluorouracil in advanced pancreatic cancer. PATIENTS AND METHODS: Chemotherapy-naïve patients with measurable advanced adenocarcinoma of the pancreas were treated with gemcitabine 1,000 mg/m(2) intravenously weekly x 3 followed by 1 week of rest every 4 weeks and 200 mg/m(2)/day CI fluorouracil until disease progression or limiting toxicity. RESULTS: Twenty-five patients were evaluable for response and toxicity. Objective partial responses were documented in 5 patients (20%; 95% confidence interval 6.8-40.7%) and disease stabilization or minor responses in 13 patients (52%; 31.3-72.2%). Toxicity was mild with grade 2/3 leucopenia in 26%, stomatitis in 15%, nausea in 6%, diarrhea in 3%, and hand-foot syndrome in 2% of the treatment cycles. In 3 patients a catheter thrombus occurred and in 1 patient the treatment had to be stopped due to asthenia. The performance status improved in 39% of the patients and 65% benefitted in terms of a decrease in pain intensity or consumption of analgesics. CONCLUSION: This phase II trial confirms a significant antitumor activity and a beneficial clinical effect of gemcitabine plus CI fluorouracil in advanced pancreatic cancer. The combination is well tolerated and it will have to be shown whether oral fluoropyrimidines can increase the practicability of this treatment without impairing efficacy.

Docetaxel (Taxotere)-cisplatin (TC): an effective drug combination in gastric carcinoma. Swiss Group for Clinical Cancer Research (SAKK), and the European Institute of Oncology (EIO).

PURPOSE: A multi-centric trial was performed to explore the clinical activity, in terms of response and toxicity (primary objectives), duration of response and survival (secondary objectives), of docetaxel with cisplatin in advanced gastric cancer (AGC). PATIENTS AND METHODS: Patients with measurable unresectable and/or metastatic gastric carcinoma, performance status < or = 1, normal hematological, hepatic and renal functions and not pretreated for advanced disease by chemotherapy received up to eight cycles of TC (docetaxel 85 mg/m2 dl, cisplatin 75 mg/m2 dl) q3w. Dose escalation to 100 mg/m2 was performed in five patients and was discontinued for excessive toxicity. RESULTS: Forty-eight patients were accrued. A median of 5 cycles/patient was given. We observed 2 complete and 25 partial responses for an overall intent to treat response rate of 56% (95% CI: 41%-71%). Twelve patients had stable disease for > or = 9 weeks (3 cycles). The median time to progression and overall survival were 6.6 and 9 months, respectively. Grade > or = 3 toxicities were neutropenia 81%, anemia 32%, thrombocytopenia 4%, alopecia 36%, fatigue 9%, mucositis 9%, diarrhea 6%, nausea/vomiting 4%, neurologic 2%, and one anaphylaxis precluding treatment administration. We recorded nine episodes of non-fatal febrile neutropenia in eight patients, two of them with docetaxel at 100 mg/m2. There were no direct treatment-related deaths. CONCLUSIONS: TC is active in AGC with a high response rate in a multicentric trial. Despite its hematotoxicity, this regimen is well tolerated and can be recycled as originally planned in 78% of the cases. These results may serve as basis for further developments of docetaxel containing regimens in this disease.

p21 is associated with cyclin D1, p16INK4a and pRb expression in resectable non-small cell lung cancer.

p21 (p21WAF1/CIP1) is involved in cell cycle regulation, as an inhibitor of cyclin dependent kinases (CDK2, CDK4 and CDK6). However, subsequent in vitro studies have suggested that p21 may influence this process by an additional mechanism, in particular through the regulation of cyclin D1 subcellular localisation. This study of primary resectable non-small cell lung cancer (NSCLC) was designed to examine p21 functions in association with the expression of cyclin D1 (including its subcellular localisation), p16INK4a and pRb. p21 expression was examined in 50 NSCLC (stage I-IIIA) and in several normal lung samples all of which had previously been studied for cyclin D1 (DNA, RT-PCR, immunostaining), p16INK4a (DNA, RT-PCR, immunostaining), and pRb (immunostaining). As assessed by immunoblotting and immunostaining, p21 was expressed at low levels in normal lung tissue with immunoreactivity seen in a small number of bronchial epithelial cells only. In NSCLC, p21 expression (> or =10% of positive cells) was observed in 42% (21/50) of cases. High p21 expression was associated with well differentiated tumours (p = 0.01) and cyclin D1 nuclear staining (p = 0.02). Furthermore, we found an inverse correlation with p16INK4a (p = 0.004) and a direct correlation with pRb expression (p = 0.02). Risk of relapse was associated with p16INK4a and p21 status with no relapse in patients with normal p16INK4a and p21. Our results confirm that a large number of NSCLC have a low level of p21 expression. The associations of p21 and nuclear cyclin D1, pRb, p16INK4a support the relevance of pathways linked to lung carcinogenesis that involve p21 but may act in addition to direct CDK inhibition.

A randomized crossover trial assessing patient preference for two different types of portable infusion-pump devices.

BACKGROUND: A variety of anticancer agents are better tolerated and more effective if given as continuous compared to bolus administration. Portable pump devices are needed to allow outpatient continuous infusion. Different types of portable pumps are available and we tested patient preference in a randomized crossover design. PATIENTS AND METHODS: Patients on continuous infusion fluorouracil were randomly assigned to start treatment with an elastomeric infusor (Baxter) or a mechanical, electronically controlled pump (CADD-1, Pharmacia) and crossed over to the alternative model after three weeks. After exposure to both pump types patients were asked to indicate their preferred device. RESULTS: After 10 patients the study was closed because all study participants preferred the elastomeric pump (P < 0.01). Reasons were pump weight (100%), smaller pump size (89%), interference with daily activities (89%), user friendliness (56%), impact on sleep (44%), and lack of technical problems (22%). Although the mechanical pump required more handling time for the first two refillings, the learning curve suggested about equal time requirement thereafter. CONCLUSION: In the interest of patient comfort, the disposable elastomeric infusor is an acceptable alternative to the more accurate electronically controlled pumps especially for drugs with a short half-life and a favorable toxicity profile.

Spontaneous apoptosis and the expression of p53 and Bcl-2 family proteins in locally advanced head and neck cancer.

BACKGROUND: Apoptotic cell death plays a key role in the pathogenesis, aggressiveness, and therapy responsiveness of cancer. The suicidal machinery of apoptosis is genetically controlled. Proteins of the Bcl-2 family as well as p53 are important regulators of apoptosis. OBJECTIVE: To assess the rate of spontaneous apoptosis and the expression of p53 and Bcl-2 family proteins in locally advanced squamous cell carcinomas of the head and neck. DESIGN: Twenty-six patients with locally advanced squamous cell carcinoma of the head and neck were included in the study. The expression of p53, Bcl-2, Mcl-1, Bax, and Bak was assessed by immunohistochemical analysis. The terminal deoxytransferase-mediated deoxyuridine nick end-labeling assay was used to quantify apoptosis by flow cytometry. RESULTS: Tumor cells containing immunostaining for the antiapoptotic proteins Bcl-2 and Mcl-1 were present in 4 (15%) and 24 (92%) of the cases evaluated, respectively, whereas immunopositivity for the proapoptotic proteins Bax and Bak was found in 9 (35%) and 24 (92%) of the samples. Immunoreactivity to p53 was detected in 20 (77%) of the samples. There was a positive correlation between the expression of Bcl-2 and Bax and between Mcl-1 and Bak. A low fraction of apoptotic cells (<2.5%) in the pretreatment tumor samples was significantly correlated with increased 2-year survival in these patients. CONCLUSIONS: Our results establish the frequent expression of the Bcl-2 family proteins Bcl-2, Mcl-1, Bax, and Bak in locally advanced head and neck cancer. In addition, this study suggests that the apoptotic fraction in pretreatment tumor samples might be of prognostic importance for the outcome in these patients.

Expression of apoptosis regulatory proteins of the Bcl-2 family and p53 in primary resected non-small-cell lung cancer.

Proteins of the Bcl-2 family as well as p53 are important regulators of apoptosis. Alterations in the expression of these proteins can contribute to the formation of cancer, as well as influence tumour response to chemo- and radiotherapy. We used antibodies specific for the human Bcl-2, Mcl-1, Bax, Bak and p53 proteins to examine the expression of these apoptosis-regulating genes in 49 archival specimens of patients with radically resected non-small-cell lung cancer (NSCLC). Tumour cells containing immunostaining for the antiapoptotic proteins Bcl-2 and Mcl-1 were present in 31% and 58% of the cases evaluated, respectively, whereas immunopositivity for the proapoptotic proteins Bax and Bak was found in 47% and 58% of the samples. p53 immunopositivity was detected in 61% of the samples. The expression of Bcl-2 and p53 and the expression of Mcl-1 and Bax showed a positive association (P = 0.02 and P = 0.06 respectively), whereas the expression of Bax was inversely related to p53 (P = 0.008). The expression of Bcl-2 had a negative influence on relapse-free survival in this population of primary resected NSCLC patients (P = 0.02). The expression of p53 and Bcl-2 was significantly associated with metastasis-free survival (P < 0.01). Only patients with p53-positive tumours developed metastases during the follow-up period. Our results establish the frequent expression of the Bcl-2 family proteins Bcl-2, Mcl-1, Bax and Bak in NSCLC. It can be expected that Bcl-2 family members have no straightforward impact on clinical outcome in this disease because their interactions in the regulation of apoptosis are complex.

Expression of p16INK4a/p16alpha and p19ARF/p16beta is frequently altered in non-small cell lung cancer and correlates with p53 overexpression.

The CDKN2 locus expresses two different mRNA transcripts, designated alpha and beta. The protein product of the alpha transcript is the cell cycle inhibitor and tumour suppressor p16INK4a. The beta transcript is translated in an alternate reading frame (ARF) and in humans encodes a 15 kDa protein (p19ARF). Immunohistochemical and Western analysis of p16INK4a has shown that the protein is downregulated in a significant number of tumours, but less is known on the expression of the p19ARF. We have examined the expression of p16INK4a and p19ARF in resectable non-small cell lung cancer (NSCLC) by immunostaining (n=49) and multiplex RT-PCR (n=28). In order to investigate the mechanism responsible for p16INK4a downregulation, exon 1alpha methylation was analysed in a PCR-based assay. Of 49 tumours examined by immunostaining, 24 and 20 tumours expressed p16INK4a and p19ARF at nil to low levels, respectively. p19ARF was localized primarily to the nuclei of tumour cells, but was also seen to varying degrees in nuclei of lymphocytes, chondrocytes, fibroblasts, and epithelial cells. No tumour with normal p16INK4a had decreased p19ARF expression. Among 16 tumours with nil to low p16INK4a expression, 11 tumours exhibited full methylation of at least one site within exon 1alpha and these tumours showed normal p19ARF expression. In contrast, no methylation of exon 1alpha was observed in five tumours which also lacked p19ARF. In normal lung, p16INK4a and p19ARF were not expressed at detectable levels, the multiplex RT-PCR results were balanced, and sites within exon 1alpha were strongly methylated. In tumours, imbalanced multiplex RT-PCR data (p16INK4a