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Gene expression profile of N+ and N0 HNSCC

ABSTRACT: Head and neck squamous cell carcinomas (HNSCCs) is the seventh most common solid malignancy in the United States, accounting for more than 47,000 new cancer cases. Surgery of patients clinically diagnosed with lymph node metastasis (N+) also involves neck dissection, which causes disfigurement and pain. However, after histological examination, more than 30% of clinically N+ patients turn out to be metastasis-free (N0). Clinically negative lymph node patients have occult node metastasis in up to 50% of the cases. Within two years of follow-up these patients may or may not develop metastatic disease. On the other hand, around 50% of N0 patients do not have occult node metastasis. Therefore, many N0 patients undergo neck dissection unnecessarily. Due to limitations in detecting lymph node metastasis before surgery, both N+ and N0 patients may receive inappropriate treatment. This indicates that a better understanding of the biology of HNSCC is urgently needed. Despite tumor complexity, many studies have tried unsuccessfully to test single genes to be used as prognostic markers in HNSCC. A group of tumor samples can be characterized in terms of the behavior of modules. These modules include clusters of coexpressed genes, such as genes that belong to the same pathway or functional category. Cancer is a multifaceted phenomenon that involves activation and/or disruption of various cellular processes. Thus, the identification of pathways or group of genes such as those involved in the development of lymph node metastasis and recurrent disease may help understanding the complex biology of cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE18020 | GEO | 2010/05/28

SECONDARY ACCESSION(S): PRJNA129041

REPOSITORIES: GEO

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Project description:Head and neck squamous cell carcinomas (HNSCCs) is the seventh most common solid malignancy in the United States, accounting for more than 47,000 new cancer cases. Surgery of patients clinically diagnosed with lymph node metastasis (N+) also involves neck dissection, which causes disfigurement and pain. However, after histological examination, more than 30% of clinically N+ patients turn out to be metastasis-free (N0). Clinically negative lymph node patients have occult node metastasis in up to 50% of the cases. Within two years of follow-up these patients may or may not develop metastatic disease. On the other hand, around 50% of N0 patients do not have occult node metastasis. Therefore, many N0 patients undergo neck dissection unnecessarily. Due to limitations in detecting lymph node metastasis before surgery, both N+ and N0 patients may receive inappropriate treatment. This indicates that a better understanding of the biology of HNSCC is urgently needed. Despite tumor complexity, many studies have tried unsuccessfully to test single genes to be used as prognostic markers in HNSCC. A group of tumor samples can be characterized in terms of the behavior of modules. These modules include clusters of coexpressed genes, such as genes that belong to the same pathway or functional category. Cancer is a multifaceted phenomenon that involves activation and/or disruption of various cellular processes. Thus, the identification of pathways or group of genes such as those involved in the development of lymph node metastasis and recurrent disease may help understanding the complex biology of cancer. Samples were obtained during surgery and neck dissection of 81 patients with primary untreated HNSCC at the Head and Neck Surgery Department from the Hospital AC Camargo (São Paulo, Brazil) between 1998 and 2003. Diagnosis of HNSCC was determined by biopsy. All patients signed a pre-informed consent and the study was approved by our institutional review board. After surgery, all patients were treated with adjuvant radiotherapy. Tumor samples were snap-frozen in liquid nitrogen. Before RNA extraction, diagnosis was confirmed by hematoxylin-eosin staining. Frozen samples were hand dissected for removal of normal cells, necrosis, and infiltrating inflammatory cells. Total RNA was extracted using TRIzol. RNA quality was accessed by spectrophotometry and gel electrophoresis. To be considered as high-quality, the RNA had to have a 260/280 ratio higher than 1.7 and a 18S/28S rRNA ratio ~2. Amplifictaion of the mRNA was done using a T7-based protocol and cDNA was indirectly labeled with Alexa Dye 555 or 647. Samples and a common RNA reference were hybridized overnight at 42oC in dye-swap to a 4,800-element in-house printed microarray, enriched with cancer-related ESTs derived from the Human Cancer Genome Project. After washing, slides were scanned on a confocal laser scanner and data were extracted.

Project description:Purpose: Presence of pelvic lymph node metastases is the main prognostic factor in early stage cervical cancer patients, primarily treated with surgery. Aim of this study was to identify cellular tumor pathways associated with pelvic lymph node metastasis in early stage cervical cancer. Experimental Design: Gene expression profiles (Affymetrix U133 plus 2.0) of 20 patients with negative (N0) and 19 with positive lymph nodes (N+), were compared with gene sets that represent all 285 presently available pathway signatures. Validation immunostaining of tumors of 274 consecutive early stage cervical cancer patients was performed for representatives of the identified pathways. Results: Analysis of 285 pathways resulted in identification of five pathways (TGF-β, NFAT, ALK, BAD, and PAR1) that were dysregulated in the N0, and two pathways (β-catenin and Glycosphingolipid Biosynthesis Neo Lactoseries) in the N+ group. Class comparison analysis revealed that five of 149 genes that were most significantly differentially expressed between N0 and N+ tumors (P<0.001) were involved in β-catenin signaling (TCF4, CTNNAL1, CTNND1/p120, DKK3 and WNT5a). Immunohistochemical validation of two well-known cellular tumor pathways (TGF-β and β-catenin) confirmed that the TGF-β pathway (positivity of Smad4) was related to N0 (OR:0.20, 95%CI:0.06-0.66) and the β-catenin pathway (p120 positivity) to N+ (OR:1.79, 95%CI:1.05-3.05). Conclusions: Our study provides new, validated insights in the molecular mechanism of lymph node metastasis in cervical cancer. Pathway analysis of the microarray expression profile suggested that the TGF-β and p120-associated non-canonical β-catenin pathways are important in pelvic lymph node metastasis in early stage cervical cancer. For the microarray experiment, we selected fresh frozen primary cervical cancer tissue, containing at least 80% tumor cells, of patients with histologically confirmed N0 (n=20) and of patients with N+ (n=19). The N0 and N+ groups were matched for age, FIGO stage and histology (all squamous cell carcinoma).

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Gene expression profile of N+ and N0 HNSCC

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