DNA copy number loss and allelic imbalance at 2p16 in lung cancer associated with asbestos exposure.

Five to seven percent of lung tumours are estimated to occur because of occupational asbestos exposure. Using cDNA microarrays, we have earlier detected asbestos exposure-related genomic regions in lung cancer. The region at 2p was one of those that differed most between asbestos-exposed and non-exposed patients. Now, we evaluated genomic alterations at 2p22.1-p16.1 as a possible marker for asbestos exposure. Lung tumours from 205 patients with pulmonary asbestos fibre counts from 0 to 570 million fibres per gram of dry lung, were studied by fluorescence in situ hybridisation (FISH) for DNA copy number alterations (CNA). The prevalence of loss at 2p16, shown by three different FISH probes, was significantly increased in lung tumours of asbestos-exposed patients compared with non-exposed (P=0.05). In addition, a low copy number loss at 2p16 associated significantly with high-level asbestos exposure (P=0.02). Furthermore, 27 of the tumours were studied for allelic imbalances (AI) at 2p22.1-p16.1 using 14 microsatellite markers and also AI at 2p16 was related to asbestos exposure (P=0.003). Our results suggest that alterations at 2p16 combined with other markers could be useful in diagnosing asbestos-related lung cancer.

Biological and prognostic role of acid cysteine proteinase inhibitor (ACPI, cystatin A) in non-small-cell lung cancer.

BACKGROUND: Acid cysteine protease inhibitor (ACPI) is an intracellular protein, often linked to neoplastic changes in epithelium and thought to have an inhibitory role in malignant transformation. AIM: To analyse the expression and prognostic role of ACPI in non-small-cell lung cancer (NSCLC). METHOD: Histological samples from 199 patients with resected NSCLC were stained immunohistochemically for the expression of ACPI in normal and preneoplastic bronchial epithelium, and in various types of lung carcinomas. RESULTS: A normal bronchial epithelium showed positive staining for ACPI in the basal cells, whereas the upper two-thirds of the dysplastic epithelium was ACPI positive. High staining for ACPI was found in 74% (91/123) of squamous-cell carcinomas, whereas 16% (8/49) of adenocarcinomas and 30% of (8/27) large-cell carcinomas showed the high expression of ACPI (p<0.001). Among squamous-cell carcinomas, low expression of ACPI was correlated with poor tumour differentiation (p=0.032). In the whole tissue, reduced expression of ACPI was associated with tumour recurrence (p=0.024). In overall survival (OS) and disease-free survival (DFS) analyses, the histological type of the tumour (both p<0.001) and stage of the tumour (p=0.001, p=0.013, respectively) were related to patient outcome. Low expression of ACPI in tumour cells was associated with poor OS and DFS (p<0.041, p=0.004, respectively). In multivariate analysis, ACPI did not retain its prognostic value, whereas the traditional factors were the most important prognostic factors. CONCLUSIONS: ACPI expression is linked with the malignant transformation of the bronchial epithelium and predicts a risk of tumour recurrence as well as poor rate of survival for the patients. However, ACPI does not have any independent prognostic value in NSCLC.

CD44 expression and its relationship with MMP-9, clinicopathological factors and survival in oral squamous cell carcinoma.

We investigated the expression of CD44 and MMP-9 in primary oral squamous cell carcinoma (OSCC) and evaluated their association with each other and clinicopathological factors as well as their prognostic value during long term follow up. Histological samples from 138 OSCC patients were immunohistochemically stained for the expression of CD44 and MMP-9. The staining results were compared with conventional prognostic factors and their impacts to patients' prognosis were also studied with survival analyses. Irregular staining of CD44 in tumour cells was associated with poor tumour differentiation (p=0.003), higher clinical stage (III-IV) (p=0.049), and the presence of T3-4 tumour stage (p=0.03). Strong stromal MMP-9 staining intensity was correlated with poor tumour differentiation (p=0.03). In univariate survival analysis irregular staining of CD44 in tumour cells was related to poor disease free and overall survival (p=0.001 and p<0.001, respectively). In multivariate analysis CD44 staining was a significant independent predictor for overall (p=0.03) and disease free survival (p=0.003). MMP-9 expression showed no statistical significance in survival analyses. Strong stromal staining intensity of MMP-9 correlated with irregular staining of CD44 in tumour cells, but had no prognostic significance in the present cohort. However, irregular staining of CD44 predicted more advanced disease and shortened survival of the patients.

Enhanced apoptosis correlates with poor survival in patients with laryngeal cancer but not with cell proliferation, bcl-2 or p53 expression.

The purpose of the current study was to analyse apoptosis and bcl-2 expression in laryngeal squamous cell carcinoma (SCC) with special reference to their prognostic significance, correlation with the clinical and pathological characteristics as well as cell proliferation and p53 accumulation. 172 patients with primary laryngeal SCC were followed-up for a median of 67 months. The volume corrected apoptotic (A/V) index was analysed using an in situ end labelling method (TUNEL) in 85 randomly selected patients. The expression of bcl-2 and p53 was analysed with monoclonal antibodies. The proliferative activity was measured both with Ki-67 (MIB-1) antibody and the volume corrected mitotic (M/V) index. The A/V index was not associated with p53 (P = 0.6) or bcl-2 (P = 0.6) expression or with proliferative parameters (P = 0.9 for M/V and P = 0.3 for MIB-1). The 10-year overall survival in patients with a high A/V index was poorer when compared with patients with a low index (47% versus 81%, P = 0.005), while accumulation of bcl-2 had no prognostic significance (P = 0.5). In Cox multivariate analysis of the whole cohort, stage (P < 0.0005) and histological grade (P = 0.04) were predictors of overall survival. In the subset of patients with an A/V index available, predictors of survival were stage (P = 0.05), A/V index (P = 0.02) and histological grade (P = 0.04). A high A/V index was an independent predictor of poor survival in laryngeal SCC. This effect was not associated with tumour cell proliferation. Accumulations of p53 and bcl-2 were not associated with apoptosis. Expression of bcl-2 lacks any prognostic significance in laryngeal SCC. We propose that assessment of the A/V index may help in selecting patients with poor prognosis.

Reduced expression of CD44v3 variant isoform is associated with unfavorable outcome in non-small cell lung carcinoma.

The expression of CD44 standard form (CD44s) and variant isoforms v3 and v6 was analyzed in 233 resected non-small cell lung carcinoma (NSCLC) specimens by immunohistochemistry (IHC), and the mRNA status of CD44v3 and CD44v6 in a cohort of samples was determined by in situ hybridization (ISH) and further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of CD44s, CD44v3, and CD44v6 was correlated with clinicopathologic variables and survival. The expression of CD44v3 and v6 was reduced in 97% and 90% of the adenocarcinomas and in 86% and 74% of the large cell/anaplastic carcinomas, respectively, as compared with squamous cell carcinomas, where they were reduced in 53% and 51% of the cases (P = .0001 and P = .004 for v3 and v6). The corresponding values for CD44s were 92%, 70%, and 51%, respectively (P = .011). The reduced CD44s and CD44v6 expression was associated with lymph node metastases (P = .03 and P = .005, respectively) and the reduced expression of CD44s also with advanced stage (P = .04). Recurrences during the follow-up were more often found within the tumors showing reduced expression of CD44v3 (P = .04). Combining ISH and IHC results showed that CD44v3 and v6 mRNA were not always processed into protein, suggesting a regulation disturbance posttranscriptionally since malignant transformation of cells has occurred. In survival analyses, the reduced expression of CD44s and CD44v3 was associated with a shortened disease-free survival (P = .04 and P = .01, respectively). In multivariate analysis, CD44v3 retained its independent prognostic value (P = .03). These results emphasize the value of CD44, and especially the v3 variant isoform in the behavior of NSCLC.

Prognostic value of epidermal growth factor expression in breast cancer.

A series of 198 female breast cancer biopsies were analysed immunohistochemically for the expression of epidermal growth factor (EGF), with special emphasis on its prognostic significance. A total of 67/198 tumours (33.8%) were EGF-positive, 24 (12%) of which showed strong expression of EGF. EGF was usually expressed in the cytoplasm of the cancer cells but, in 22 cases, the normal ducts adjacent to the cancer showed positive staining as well. Strong EGF expression was related to distant metastases at diagnosis (P = 0.04). Oestrogen(ER)- and progesterone-receptor(PR)-negative tumours showed EGF positivity with equal frequency (P = 0.05 in both). Axillary lymph node status, histological type, tumour size, histological grade, S-phase fraction, mitotic index or cancer recurrence did not show any statistical correlation with EGF expression. Tumour size (P = 0.007), axillary lymph node involvement (P = 0.003) and ER content (P = 0.03) were independent prognostic factors in multivariate survival analysis, whereas EGF positivity, as an independent factor, had no effect on survival. In univariate analysis, however, EGF positivity predicted a more favourable outcome in axillary-lymph-node-positive tumours (P = 0.04). The results suggest that immunohistochemical assessment of EGF expression has hardly any clinical significance in addition to the well-established prognostic factors in breast cancer.

Reduced expression of alpha-catenin, beta-catenin, and gamma-catenin is associated with high cell proliferative activity and poor differentiation in non-small cell lung cancer.

AIMS: To investigate the expression of catenins (alpha, beta, and gamma) in non-small cell lung carcinoma (NSCLC) and its relation to clinicopathological factors and prognosis. METHODS: The expression of catenins was analysed immunohistochemically in 261 patients with resected NSCLC, diagnosed between 1978 and 1996 in eastern Finland: The cell proliferation index of the tumours was analysed by means of an image analyser. The staining results were compared with clinicopathological characteristics and survival. RESULTS: Normal catenin staining was found significantly more often in adenocarcinomas than in squamous cell carcinomas or anaplastic/large cell carcinomas. Reduced staining of alpha-catenin, beta-catenin, and gamma-catenin was related to poor differentiation of the tumour. The tumours with reduced staining of beta-catenin or gamma-catenin often had higher cell proliferation activity. Nuclear staining of beta-catenin and gamma-catenin was found in 16 (7%) and 29 (13%) cases, respectively. This nuclear staining correlated directly with increased cell proliferation and inversely with membranous staining. In survival analyses the predictors of overall and disease free survival were stage and tumour type. The expression of catenins did not affect survival. CONCLUSIONS: The expression of alpha-catenin, beta-catenin, and gamma-catenin is related to histological type and differentiation in NSCLC, although catenins have no independent prognostic value. However, this study supports the important role of the nuclear accumulation of beta-catenin and gamma-catenin in highly proliferative cells.

Nuclear beta catenin expression is related to unfavourable outcome in oropharyngeal and hypopharyngeal squamous cell carcinoma.

AIMS: To investigate the expression of alpha, beta, and gamma catenins in oropharyngeal and hypopharyngeal squamous cell carcinoma and their relations to each other, as well as to clinical data, tumour differentiation, and prognosis. METHODS: Primary tumours for analysis were obtained from 138 patients diagnosed with squamous cell carcinoma of the oropharynx or hypopharynx between 1975 and 1998 in eastern Finland. Immunohistochemistry was used to evaluate the expression of alpha, beta, and gamma catenins. The expression patterns of all catenins were related to clinical data and survival. RESULTS: The expression patterns of all three catenins were significantly interrelated. Reduced gamma catenin expression was significantly associated with poor histological differentiation. No association was found between alpha or beta catenin expression and clinicopathological characteristics. In univariate analysis, patients whose tumours had nuclear beta catenin expression had shorter overall survival than patients with no nuclear expression. In Cox multivariate analysis, nuclear beta catenin expression, tumour status (T class), and Karnofsky performance index were independent prognostic factors of overall survival. CONCLUSIONS: Reduced expression of gamma catenin is associated with dedifferentiation in primary squamous cell carcinoma of the oropharynx and hypopharynx. The fact that nuclear beta catenin expression independently predicts short overall survival suggests that it might be a valuable prognostic marker in pharyngeal squamous cell carcinoma.

Expression of transcription factor AP-2 in colorectal adenomas and adenocarcinomas; comparison of immunohistochemistry and in situ hybridisation.

AIMS: To investigate whether the three different AP-2 isoforms are expressed differently in colorectal adenomas and carcinomas. METHODS: The study comprised 43 randomly selected patients diagnosed and treated at Kuopio University Hospital in 1996 for colorectal adenocarcinoma (n = 30) and colorectal adenoma (n = 13). The expression of AP-2alpha, AP-2beta, and AP-2gamma was analysed by immunohistochemistry (IHC) and the mRNA status of AP-2alpha was determined by in situ hybridisation (ISH) and confirmed by reverse transcription polymerase chain reaction (RT-PCR). AP-2 expression patterns were correlated with clinicopathological variables. RESULTS: In adenomas and carcinomas, AP-2beta cytoplasmic positivity was higher than that of AP-2alpha or AP-2gamma. AP-2alpha expression was reduced in advanced Dukes's stage carcinomas. In high grade carcinomas, both AP-2alpha and AP-2gamma expression was reduced. ISH demonstrated increased AP-2alpha values in high grade carcinomas. Seven of 30 carcinoma specimens displayed a moderate or strong mRNA signal, despite being negative for AP-2alpha protein. RT-PCR from AP-2alpha mRNA and protein positive tumours confirmed that the positive signal in ISH originated from the exon 2 of TFAP2A. CONCLUSIONS: AP-2alpha was reduced in advanced Dukes's stage adenocarcinomas. Together with reduced AP-2gamma expression in high grade carcinomas, this might contribute to tumour progression. The discrepancy between mRNA and protein expression suggests that post-transcriptional regulatory mechanisms might modify the availability of functional AP-2alpha protein in colorectal carcinoma.

Expression of epidermal growth factor receptor (EGFR) in breast cancer as related to clinical, prognostic and cytometric factors.

A series of 213 female breast carcinomas were analysed immunohistochemically for the expression of epidermal growth factor receptor (EGFR), with special emphasis on its possible prognostic significance. A total of 114/213 tumors (53.5%) were EGFR positive. EGFR was almost exclusively expressed in the cytoplasm of the cancer cells, but in a few cases, the cell membranes showed EGFR positive staining as well. EGFR expression was related to the histological grade of the tumours in that a linear decrease of the staining was found in parallel with the decreasing tumour differentation (P = 0.024). On the other hand, axillary lymph node status (P = 0.95), histological type (P = 0.60), tumor size (P = 0.87), DNA-index (P = 0.56), S-phase (P = 0.80), mitotic index (P = 0.72), or estrogen (ER) and progesterone receptor (PR) content (P = 0.45) did not show any statistical correlation with the EGFR expression. EGFR positivity as an independent factor, had little (if any) effect on the patients prognosis. Tumor size (P = 0.004), axillary lymph node involvement (P = 0.024) and PR positivity (P = 0.008) were the single most significant prognostic factors in multivariate survival analysis. The results indicate that, in clinical breast cancer, immunohistochemical assessment of EGFR provides no prognostic information additional to the well established prognostic factors.

Prognostic value of hyaluronan expression in non-small-cell lung cancer: Increased stromal expression indicates unfavorable outcome in patients with adenocarcinoma.

The prognostic value of hyaluronan (HA) was analyzed in a large number of patients (n = 261) with non-small-cell lung cancer (NSCLC) by staining archived tumor samples with a biotinylated HA-specific probe. The level of HA in the tumor cells and surrounding stroma was scored and compared with parallel CD44 stainings, clinicopathological factors and survival data. Adenocarcinomas were characterized by a low percentage of HA-positive cells with low staining intensity compared with squamous-cell and large-cell/anaplastic carcinomas. The HA signal in the peri-tumoral stroma was often higher than that in the uninvolved stroma in all subgroups of NSCLC. CD44 and HA associated with the cancer cells showed a strong positive correlation with each other. In the whole tumor material, dominated by squamous-cell carcinomas (n = 168), recurrences were more often found in cases showing a low percentage of cancer cell-associated HA. However, within the adenocarcinoma subgroup (n = 68), a high percentage of cell-associated HA was correlated with poor tumor differentiation. Also specific for the adenocarcinoma subgroup was the increased number of recurrences in cases with a strong stromal HA signal. In survival analysis of the whole material (n = 189), a low percentage of HA-positive cancer cells was associated with a shortened disease-free survival (DFS) together with stage and tumor type. However, in the subgroup of patients with adenocarcinoma (n = 49), a strong stromal signal for HA predicted poor DFS. The level of HA in the stroma of adenocarcinomas retained its prognostic value in Cox's multivariate analysis. These results indicate that the frequency and intensity of HA has a significant prognostic value in NSCLC, particularly when the histological subtypes are analyzed as separate entities.

Expression of hyaluronan in normal and dysplastic bronchial epithelium and in squamous cell carcinoma of the lung.

A series of 85 lung/bronchial tissue samples from 76 patients consisting of normal, metaplastic and dysplastic epithelium and different types of lung carcinomas were analyzed for the distribution of hyaluronan (HA), using a biotinylated hyaluronan binding complex as an HA-specific probe. The normal pseudo-stratified columnar bronchial epithelium was either negative for HA or displayed a weak staining around the basal cells. The epithelia of serous and mucous bronchial glands were HA negative whereas the submucosal connective tissue was strongly positive. In metaplastic, dysplastic and carcinoma in situ lesions the whole epithelium from basal to uppermost cells expressed HA on plasma membranes. Epithelial HA was also found in squamous cell carcinomas, but not in adenocarcinomas, carcinoid tumors or small cell carcinomas of the lung. Whereas epithelial HA was present in all lesions of the squamous cell type, the staining intensity displayed great local variability in 50% of the cases with severe dysplasia, carcinoma in situ and squamous cell carcinomas. In squamous cell carcinomas, such an irregular staining pattern was significantly associated with poor differentiation. Our results indicate that the expression of HA in different bronchial lesions and lung tumors is restricted to those showing squamous cell differentiation, being absent from other types of lung carcinomas. The increase of HA depleted areas in poorly differentiated squamous cell carcinomas emphasizes the important role of HA in tumor differentiation. HA on carcinoma cell surface may influence tumor growth and metastatic behavior.

Reduced CD44 standard expression is associated with tumour recurrence and unfavourable outcome in differentiated thyroid carcinoma.

CD44 was detected with an antibody recognizing all forms of CD44 (CD44 standard) and others specific for its v3 and v6 variant isoforms; their prognostic value was evaluated in 213 patients with differentiated thyroid carcinoma (DTC). The staining patterns of CD44 standard (s) and CD44v6 in tumour tissue were quite similar, 176 cases (83%) being highly positive for CD44s and 153 cases (72%) for CD44v6. Only 18 (9%) tumours showed high expression of CD44v3. Papillary carcinomas were significantly more often high expressors of CD44s and CD44v6 than follicular carcinomas (p<0.001 for both). Age older than 60 years, distant metastases, and advanced pTNM stage were related to loss of expression of CD44s (p<0.001, p=0.021, and p=0.003, respectively). Tumour recurrence and cancer-related mortality were related to the reduced level of CD44s (p=0.049 and p=0.042). CD44v3 did not associate with any of the clinicopathological factors. In univariate analysis, CD44s was the only significant prognostic factor for disease-free survival (p=0.0488). In multivariate analysis, CD44s and thyroglobulin level were significant prognostic factors for disease-free survival (p=0.040 and p<0.001, respectively). The reduced level of CD44s in DTC patients seems to be an independent prognostic factor for unfavourable disease outcome.