Prediction of future metastasis and molecular characterization of head and neck squamous-cell carcinoma based on transcriptome and genome analysis by microarrays.

Propensity for subsequent distant metastasis in head and neck squamous-cell carcinoma (HNSCC) was analysed using 186 primary tumours from patients initially treated by surgery that developed (M) or did not develop (NM) metastases as the first recurrent event. Transcriptome (Affymetrix HGU133_Plus2, QRT-PCR) and array-comparative genomic hybridization data were collected. Non-supervised hierarchical clustering based on Affymetrix data distinguished tumours differing in pathological differentiation, and identified associated functional changes. Propensity for metastasis was not associated with these subgroups. Using QRT-PCR data we identified a four-gene model (PSMD10, HSD17B12, FLOT2 and KRT17) that predicts M/NM status with 77% success in a separate 79-sample validation group of HNSCC samples. This prediction is independent of clinical criteria (age, lymph node status, stage, differentiation and localization). The most significantly altered transcripts in M versus NM were significantly associated to metastasis-related functions, including adhesion, mobility and cell survival. Several genomic modifications were significantly associated with M/NM status (most notably gains at 4q11-22 and Xq12-28; losses at 11q14-24 and 17q11 losses) and partly linked to transcription modifications. This work yields a basis for the development of prognostic molecular signatures, markers and therapeutic targets for HNSCC metastasis.

Cyclin L1 (CCNL1) gene alterations in human head and neck squamous cell carcinoma.

We evaluated the expression and amplification of cyclin L1 (CCNL1) gene, a potential oncogene localised in the commonly amplified 3q25-28 region, in human head and neck squamous cell carcinomas (HNSCCs). Overexpression was observed in 55 out of 96 cases (57%) and amplification in nine out of 35 tumours (26%) with no relationships to the clinico-pathological parameters. The Cyclin L1 antibody we developed labels nuclear speckles in tumour cells compatible with a role for CCNL1 in RNA splicing.

Head and neck squamous cell carcinoma transcriptome analysis by comprehensive validated differential display.

Head and neck squamous cell carcinoma (HNSCC) is common worldwide and is associated with a poor rate of survival. Identification of new markers and therapeutic targets, and understanding the complex transformation process, will require a comprehensive description of genome expression, that can only be achieved by combining different methodologies. We report here the HNSCC transcriptome that was determined by exhaustive differential display (DD) analysis coupled with validation by different methods on the same patient samples. The resulting 820 nonredundant sequences were analysed by high throughput bioinformatics analysis. Human proteins were identified for 73% (596) of the DD sequences. A large proportion (>50%) of the remaining unassigned sequences match ESTs (expressed sequence tags) from human tumours. For the functionally annotated proteins, there is significant enrichment for relevant biological processes, including cell motility, protein biosynthesis, stress and immune responses, cell death, cell cycle, cell proliferation and/or maintenance and transport. Three of the novel proteins (TMEM16A, PHLDB2 and ARHGAP21) were analysed further to show that they have the potential to be developed as therapeutic targets.

Cisplatin-induced genes as potential markers for thyroid cancer.

Despite the uncontested role of p53 in cycle arrest/cell death after cisplatin treatment, to date the question whether wild-type p53 confers a resistant or sensitive status on the cell is still a matter of debate. Isogenic and isophenotypic human thyroid papillary carcinoma cell line variants for p53 differently expressed cycle genes after cisplatin treatment. Seven genes (CDC6-related protein, CCNC, GAS1, TFDP2, MAPK10/JNK3, WEE1, RPA1) selected after expression on an Atlas human cell cycle array were analyzed by quantitative real-time PCR. While cisplatin treatment increased their expression in p53 wild-type cells it decreased it in cells with inactivated p53 and had no or less effect on cells with mutated p53. These results show that in a well-defined system, different alterations of p53 can lead to a different regulation of genes and hence to either resistance or sensitivity to cisplatin. Moreover for the first time, MAPK10/JNK3 was identified in human thyroid cells and tissue. Four of the genes (CDC6-related protein, CCNC, GAS1 and TFDP2) were decreased in human papillary carcinoma tissues. Relevance of these genes (especially a decrease in GAS1 in thyroid papillary carcinoma) in various malignant pathologies has already been shown. These genes may be explored as new markers in advanced thyroid cancer such as metastatic and anaplastic forms displaying p53 alterations.

Loss of HOP tumour suppressor expression in head and neck squamous cell carcinoma.

We report that homeodomain-only protein (HOP) is expressed in the suprabasal layer of normal upper aerodigestive tract epithelium and expression strongly decreases in hypopharyngeal carcinoma. Interestingly, HOP has very recently been shown to be a tumour suppressor involved in differentiation, suggesting that HOP may have a similar role in head and neck squamous cell carcinoma (HNSSC).

Comparative value of tumour grade, hormonal receptors, Ki-67, HER-2 and topoisomerase II alpha status as predictive markers in breast cancer patients treated with neoadjuvant anthracycline-based chemotherapy.

The aim of this study was to evaluate the predictive value of five different biological factors in breast cancer patients treated with neoadjuvant anthracycline-based chemotherapy: (1) tumour grade scored according to the Elston-Ellis classification, (2) hormonal receptor (HR) status; (3) tumour cell proliferation evaluated by Ki-67 staining, (4) HER-2 and topoisomerase II alpha (TopoIIalpha) expression evaluated by immunohistochemistry (IHC), (5) HER-2 and TopoIIalpha amplification evaluated by real-time polymerase chain reaction (PCR). 119 patients with operable breast cancer were treated with six cycles of FEC (100 5-fluorouracil (5-FU) 500 mg/m2, Epirubicin 100 mg/m2, Cyclophosphamide 500 mg/m2). Tumour response was assessed clinically and by computed tomography (CT) scan, then by pathological assessment. The clinical overall response (OR) was 80%, with 19% of complete responders (CR). The radiological OR was 71%, with 16% of CR. A pathological CR was demonstrated in 13% of the patients according to the Sataloff classification. In the multivariate analysis, the absence of HR expression and Ki-67 > or = 20% were predictive for a clinical CR. A high tumour grade was predictive for a pathological CR. Overexpression or amplification of HER2 or Topollcalpha were not predictive of response.

Differential expression profiling of head and neck squamous cell carcinoma (HNSCC).

Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer in men with an incidence of about 780000 new cases per year worldwide and a poor rate of survival. There is a need for a better understanding of HNSCC, for the development of rational targeted interventions and to define new prognostic or diagnostic markers. To address these needs, we performed a large-scale differential display comparison of hypopharyngeal HNSCCs against histologically normal tissue from the same patients. We have identified 70 genes that exhibit a striking difference in expression between tumours and normal tissues. There is only a limited overlap with other HNSCC gene expression studies that have used other techniques and more heterogeneous tumour samples. Our results provide new insights into the understanding of HNSCC. At the genome level, a series of differentially expressed genes cluster at 12p12-13 and 1q21, two hotspots of genome disruption. The known genes share functional relationships in keratinocyte differentiation, angiogenesis, immunology, detoxification, and cell surface receptors. Of particular interest are the 13 'unknown' genes that exist only in EST, theoretical cDNA and protein databases, or as chromosomal locations. The differentially expressed genes that we have identified are potential new markers and therapeutic targets.

Loss of MDM2 expression in human head and neck squamous cell carcinomas and clinical significance.

The transforming potential of the MDM2 oncogene has been attributed to the overproduction of the protein. In order to investigate regulation of MDM2 expression in head and neck squamous cell carcinomas, we analysed MDM2 gene amplification, and mRNA and protein expression in tumour specimens from 62 patients, in cell lines, and in normal epithelium adjacent to tumours or obtained from healthy patients. Additionally, TP53-induced MDM2-P2 transcription was evaluated and compared with TP53 status. MDM2 gene amplification and mRNA over-expression is infrequent, 7 and 9%, respectively. The predominant transcript codes for full-length MDM2 protein (90kD) and the level of alternatively spliced forms is not significant. We show that only 47% of tumours exhibit MDM2 immunostaining in more than one third of the neoplastic cells, and thus more than half of the tumours display no or low levels of MDM2 protein. In contrast, MDM2 protein is always detectable in basal and parabasal cells of morphologically normal epithelium outside the invasively growing tumour, as well as in a normal uvula sample. Similarly, the total amount of MDM2 transcripts analysed by reverse transcriptase-polymerase chain reaction is reduced in tumour samples compared to normal tissues, essentially due to a decrease in P2 transcript levels. The relationship between mutated p53 status and low levels of MDM2 found in cell lines is also observed to a certain extent in primary tumour samples. Overall, there is a high frequency of TP53 mutation and under-expression of MDM2 in the head and neck tumours. Moreover, a significant association of decreased MDM2 expression is observed with advanced tumour stage and 3 years survival.

G3BP is overexpressed in human tumors and promotes S phase entry.

The expression of the human Ras-GTPase activating protein (GAP)-binding protein (G3BP) was studied in human tumors and cell lines of different origins. Northern blot analysis and immunoblotting experiments showed enhanced expression of G3BP in all tumor samples as compared to healthy tissue. The enhanced expression does not seem to be related to the tumor site or to the stage of development of the cancer. In light of the proposed functions of G3BP, its increased expression in tumors suggest that it plays a role in dedifferentiation and proliferation processes. We also show that G3BP promotes S phase entry in cultured fibroblasts deprived of serum and that this function is dependent on the presence of the RNA binding domain of the protein.

Bcl-2/Bax protein ratio predicts 5-fluorouracil sensitivity independently of p53 status.

p53 tumour-suppressor gene is involved in cell growth control, arrest and apoptosis. Nevertheless cell cycle arrest and apoptosis induction can be observed in p53-defective cells after exposure to DNA-damaging agents such as 5-fluorouracil (5-FU) suggesting the importance of alternative pathways via p53-independent mechanisms. In order to establish relationship between p53 status, cell cycle arrest, Bcl-2/Bax regulation and 5-FU sensitivity, we examined p53 mRNA and protein expression and p53 protein functionality in wild-type (wt) and mutant (mt) p53 cell lines. p53 mRNA and p53 protein expression were determined before and after exposure to equitoxic 5-FU concentration in six human carcinoma cell lines differing in p53 status and displaying marked differences in 5-FU sensitivity, with IC(50)values ranging from 0.2-22.6 mM. 5-FU induced a rise in p53 mRNA expression in mt p53 cell lines and in human papilloma virus positive wt p53 cell line, whereas significant decrease in p53 mRNA expression was found in wt p53 cell line. Whatever p53 status, 5-FU altered p53 transcriptional and translational regulation leading to up-regulation of p53 protein. In relation with p53 functionality, but independently of p53 mutational status, after exposure to 5-FU equitoxic concentration, all cell lines were able to arrest in G1. No relationship was evidenced between G1 accumulation ability and 5-FU sensitivity. Moreover, after 5-FU exposure, Bax and Bcl-2 proteins regulation was under p53 protein control and a statistically significant relationship (r = 0.880, P = 0.0097) was observed between Bcl-2/Bax ratio and 5-FU sensitivity. In conclusion, whatever p53 status, Bcl-2 or Bax induction and Bcl-2/Bax protein ratio were correlated to 5-FU sensitivity.

Detection of prostate-specific antigen- or prostate-specific membrane antigen-positive circulating cells in prostatic cancer patients: clinical implications.

OBJECTIVES: To evaluate the clinical benefit from using circulating prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) mRNA detection in prostate cancer staging and in follow-up. METHODS: Nested reverse transcriptase-polymerase chain reaction (RT-PCR) assays were performed on RNA extracted from blood drawn from 56 patients with prostate cancer before any treatment. Additionally, assays were done on posttreatment samples from 50 patients who were followed up by serum PSA level, to determine whether any relationship exists between RT-PCR results and tumor recurrence. The prostate cell specificity of assays was evaluated by analysis of 21 blood samples from women or cystoprostatectomized men. RESULTS: With PSM RT-PCR assay, good sensitivity and prostate cell specificity could not be attained together, since high PSM mRNA illegitimate expression has been shown in some healthy donor bloods. For this reason, only PSA RT-PCR assay was used as a clinical marker. PSA mRNA was detected in peripheral blood of 4 out of 31 patients with clinically localized prostate cancer. It showed no relationship to the pathologic stage, but significant relationship to metastatic status, lymph node involvement and Gleason score. During follow-up, circulating PSA mRNA was detected in 8 out of 17 (47%) patients in treatment failure and in only 1 out of 33 (3%) successfully treated patients, with significant relationship between RT-PCR results and concomitant serum PSA levels. CONCLUSION: Our study reveals no significant advantage to PSA RT-PCR assay (1) in improving the staging of clinically localized prostate cancer or (2) in follow-up treatment failure, as compared to the usual recurrence marker (serum PSA). Additional investigations are needed to determine the ultimate significance and the management of patients with positive PSA RT-PCR assays.

[Long-term prognostic role of steroid receptors in cancer of the breast]. / Rôle pronostique à long terme des récepteurs stéroïdiens dans le cancer du sein.

We screened for the prognostic value of estrogen receptor (ER) and progesterone receptor (PR) through a multicentric study of 2,257 operable breast cancer patients who did not received adjuvant therapy. Three hundred and seven local-regional recurrences, 105 metachronous contralateral breast cancer, 589 metastases and 537 deaths from cancer had been diagnosed with a median follow-up of 8.5 years. A total of 69% of the tumors were ER positive and 54% PR positive. For statistical analysis, 1,665 patients were studied because of complete clinical and biological data. In univariate analysis, ER and PR status were of prognostic value for the metastases-free interval (MFI) and the overall survival (OS). In multivariate analysis (Cox proportional hazard model), only the ER status showed a significant difference between positive and negative groups regarding the MFI and OS. By using Cox regression model with time-dependent covariates, we show that the predictive value of ER status of the primary tumor decreases by approximately 20% per year, losing its significance after 8 years of follow-up. These results show that ER and PR status have a relatively limited predictive value and their major interest remain in the domain of therapeutic decision.

Amplification of 11q13 DNA markers in head and neck squamous cell carcinomas: correlation with clinical outcome.

This study was performed on 282 patients with primary head and neck squamous cell carcinomas to evaluate the prognostic importance of 11q13 amplification. Amplification of the 11q13 DNA markers, HST-1/FGF-4 and BCL-1, evaluated by Southern and slot blot hybridisation, was detected in 52% of tumours. 11q13 amplification was associated with tumour site since this alteration occurred in 76% of tumours arising in the hypopharynx, versus 40% in the other sites (P = 0.0007). 11q13 amplification was also significantly related to the presence of involved neck lymph nodes (P = 0.013). The relationship between 11q13 amplification and risk of progression was studied in two subgroups of head and neck cancer patients with regard to treatment modalities. The presence of 11q13 amplification in the tumour was not significantly associated with a shorter event-free survival (P = 0.82) and crude survival (P = 0.61) of the 201 patients treated by surgery and postoperative radiotherapy. Similarly, absence of a relationship was observed for the group of 79 patients treated by surgery alone. These results confirm that 11q13 amplification is a prominent event in head and neck squamous cell carcinoma, indicating that it may be a common genetic event in the development of these neoplasms, but is not a reliable prognostic marker.

Prognostic value of steroid receptors after long-term follow-up of 2257 operable breast cancers.

The prognostic value of oestrogen receptor (ER) and progesterone receptor (PR) was estimated through a multicentric study of 2257 operable breast cancer patients followed up for a median of 8.5 years. None of the patients had received adjuvant therapy. The series included 33.3% stage I patients, 57.1% stage II, 5.7% stage IIIa and 2.4% stage IIIb. At the end point of the study 589 metastases and 537 deaths from cancer were recorded. Receptor measurements were performed by radiolgand assay according to a uniform protocol. A total of 68.8% of the tumous were ER positive and 54.0% PR positive ( > or = 10 fmol mg-1 cytosol protein). In univariate analysis, ER and PR status (positive/negative) were of prognostic value (P < 0.001) for the disease-free interval (DFI), the metastases-free interval (MFI) and the overall survival (OS). The OS of the patients after a first metastasis was also significantly different between ER-positive and -negative tumours (P < 0.001). In multivariate analysis (Cox proportional hazard model, 1665 patients), only the ER status showed a significant difference (P < 0.01) between positive and negative groups regarding the DFI, MFI and OS. By using Cox non-proportional, time-dependent models, we show that the predictive value of ER status of the primary tumour decreases by approximately 20% per year, losing its significance after 8 years of follow-up. Overall, when compared with TNM and histological grading, ER and PR status have a low prognostic value, their major interest remaining solely in the domain of therapeutic decision.

Rflp analysis of the L-myc oncogene in head and neck-cancer - relationship study with susceptibility and disease progression.

The L-myc DNA restriction fragment length polymorphism (RFLF), revealed by EcoRI digestion, has been evaluated in a case-control study including 161 head and neck cancer (HNSCC) patients and 160 normal healthy individuals with similar smoking and alcohol habits. No significant difference in the distribution of L-myc genotypes (LL, LS or SS) was found between the two populations implying thus no predisposition to head and neck tumour by either allele. There was no significant association between L-myc genotypes and the usual clinicopathological features such as T staging, differentiation status and lymph node involvement. Moreover, follow-up data from 154 patients was obtained and correlated with the L-myc pattern. No significant difference was observed in metastasis occurrence, multiple cancer incidence and survival data in the patients classified according to the L-myc genotypes; only a trend to preferentially develop metastasis in lung for patients with S allele was noted. In conclusion, our data shows that the L-myc typing does not contribute to HNSCC risk or prognosis assessment. A review of L-myc RFLP published studies shows contradictory results even on the same type of tumour and emphasizes the lacunae in understanding the biological role of L-myc for valid interpretation of L-myc allelic associations with cancer susceptibility or prognosis.

Alteration of the C-met oncogene locus in human head and neck-carcinoma.

The c-met proto-oncogene encodes the receptor for the hepatocyte growth factor/scatter factor (HGF/SF) which induces eel proliferation and motility. We have analysed the genetic alteration involving the c-met locus on chromosome 7q31 using the pMet H polymorphic probe, in tumor and normal DNA from 87 patients with head and neck squamous cell carcinomas (HNSCC). We report the observation of loss of heterozygosity (LOH) at this locus in 23% of informative cases, contrasting with the previously reported 40% LOH detected in breast cancer. Further, gain of genetic material was also observed in 13% of the HNSCCs. The alterations of c-met gene were not significantly associated with standard pronostic features including tumor size and lymph node status. Involvement of the c-met locus in allelic imbalance, either loss or gain of genetic material, is relatively consistent with complex karyotype patterns detected in head and neck squamous cell carcinomas through previous cytogenetic studies.

Frequent amplification of 11q13 DNA markers is associated with lymph node involvement in human head and neck squamous cell carcinomas.

Amplification of 11q13 DNA markers, particularly hst-1/FGF4 oncogene and the bcl-1 locus, was evaluated in 178 head and neck squamous cell carcinomas (SCCs) by Southern blot and slot blot hybridisation. Coamplification of hst-1/FGF4 and bcl-1 genes was found in 57% of primary tumours and in 60% of the 89 metastatic lymph nodes tested. The pattern of amplification was significantly similar in matched sets of primary SCCs and metastatic lymph nodes. Levels of amplification, quantified by densitometric analysis of slot blots, ranged from 2 to 18-fold normal gene dosage. Also, c-myc oncogene (8q24) was found amplified less frequently, since 7% of 169 SCCs tested contained amplification of this gene, the level of which ranged from 2 to 8-fold. Hst-1/bcl-1 gene amplification was observed more frequently in the tumours arising from the hypopharynx. Coamplification of hst-1 and bcl-1 genes was significantly positively associated with tumours with nodal involvement (P = 0.001). Incidence of hst-1/bcl-1 gene amplification is higher in the tumours with a clinical stage III or IV. Hst-1/bcl-1 gene amplification was not related to tumour differentiation or local invasiveness. This prospective study shows that amplification of 11q13 DNA markers is a prominent event occurring in head and neck SCC and may contribute to the pathogenesis and evolution of a subset of patients bearing this type of cancer.

Influence of mastectomy techniques on estrogen and progesterone receptor analysis in carcinoma of the breast.

Gradual tumor tissue devascularization during mastectomy is thought to decrease estrogen (ER) and progesterone (PgR) receptor activity. To determine whether or not hormone receptor values could be influenced by different mastectomy techniques, 62 patients with carcinoma of the breast had a Tru-cut needle (Baxter Healthcare Corporation) biopsy (premastectomy sample) and underwent modified radical mastectomy (postmastectomy sample) either before (group 1, 40 patients) or after (group 2, 22 patients) axillary lymph node dissection. When the two surgical procedures were compared in 33 patients in whom it could be assessed, no significant tendency (p = 0.51 for ER and p = 0.36 for PgR) for the postmastectomy sample to have hormone receptors levels less than samples taken at biopsy was detected. Overall, in the two groups (44 assessable patients), comparison with respect of each patient, between premastectomy and postmastectomy samples showed that the variations in either ER or PgR receptor values, determined by immunoenzymatic assays, were not statistically significant (p = 0.32 for ER and p = 0.21 for PgR). The current results indicated the relative stability of steroid receptors during the two modified radical mastectomy procedures and suggested that a systematic reference determination of hormone receptors on biopsy before modified radical mastectomy is unnecessary.

Increased stromelysin 3 gene expression is associated with increased local invasiveness in head and neck squamous cell carcinomas.

Matrix metalloproteinases are believed to play an important role in tumor invasion and metastasis. To examine the expression of the stromelysin 3 (ST3) gene, a new member of the matrix metalloproteinase gene family, 111 head and neck squamous cell carcinomas and 21 metastatic lymph nodes were analyzed by Northern blot. ST3 gene expression was observed in 106 carcinomas and 19 metastatic nodes, but in only 2 of 60 samples of corresponding normal tissue tested in parallel. ST3 RNA, by in situ hybridization, and ST3 protein, by immunohistochemical analysis, were specifically detected in fibroblastic cells immediately surrounding invasive cancer cells. This fibroblastic expression of the ST3 gene is characteristic among the matrix metalloproteinase genes known to be overexpressed in head and neck carcinomas, since stromelysin 2 transcripts were specifically detected in neoplastic cells, and type I collagenase transcripts in both neoplastic cells and stromal fibroblasts. Furthermore, there was a highly significant positive correlation (P < 0.0001) between ST3 RNA levels and local invasiveness by the cancer cells, suggesting that enhanced expression of the ST3 gene may contribute to the neoplastic phenotype in head and neck carcinomas.

Expression of collagenase-related metalloproteinase genes in human lung or head and neck tumours.

We address the question as to whether increased metalloproteinase production might be related to the high regional recurrence rate of some carcinomas, and particularly head and neck squamous-cell carcinomas (SCC). Northern blot of total RNA prepared from 26 lung carcinomas, 107 head and neck carcinoma samples and corresponding normal tissue samples demonstrates the frequent and sometimes concomitant over-expression of the 2 stromelysin genes, the type-I collagenase gene and the pump-I gene in the head and neck tumour tissue samples. In these SCC, over-expression of the 2 stromelysin genes and the type-I collagenase gene (but not the pump-I gene) is associated with a high degree of tumour differentiation. Moreover, a tumour with high levels of the stromelysin mRNAs is more likely to show high local invasiveness, suggesting that the stromelysins may be implicated in the clinical course of head and neck tumours. Evaluation of the corresponding mRNA levels may prove a useful indicator for predicting the clinical aggressiveness of these tumours.