Laryngeal And Hypopharyngeal Squamous Cell Carcinoma Of The Biological Markers Of Screening And Serum Diagnostic Prediction Model

LSCC is one of the commonest primary head and neck malignancy, representing approximately 1% of all malignancies. LSCC is often preceded by premalignant laryngeal lesions (PMLL) such as keratosis, moderate dysplasia, severe dysplasia of vocal cord and laryngeal papilloma. Multiple studies have shown that increasing T and N stage correlate with lower survival. LSCC patients diagnosed in T1N0M0 stage have a five-year survival at a rate of 90-95%, and T2N0M0 cancer at a rate of 80-90%, in the advanced stage T3-T4N0M0 the five-year survival is decreased to 70%, but with the presence of nodal metastasis T1-T4N1-3M0 the five-year survival is lower than 50%[l]. Patients with early stage carcinoma of the larynx can be managed highly successfully in terms of survival, organ preservation, and treatment related morbidity with larynx-sparing surgery. However, survival rates and quality of life are invariably poor in those patients with stage III, IV disease who were usually treated with near-total, total or extended laryngectomy, synchronous neck dissection and postoperative radiotherapy. So detection of LSCC at early disease stages is paramount to successful clinical therapy. In patients with LSCC, and particularly of the supraglottis, management of the regional lymphatics is a crucial component of the overall treatment plan. So, metastasis-associated serum biomarkers for LSCC are also urgently needed.Unlike many other cancers of the head and neck area, carcinoma of the hypopharynx are rarely found early when they are small and localized to the site of the primary lesion. In addition, tumors located in hypopharynx especially in postcricoid area are often hidden from physical examinations such as mirror, flexible fiberoptic laryngoscopy. Multiple studies have shown that increasing T and N stage correlate with lower survival. The presence of nodal metastasis indicates the poorer prognosis. As reported by Edmund, patients with HSCC diagnosed in T1-2N0M0 stage have a five-year survival rate of 70-80%, in the advanced stage T1-T4N1-3M0 the five-year survival rate is decreased to 20% [1]. So, early diagnosis is very important to improve the prognosis of hypopharyngeal carcinoma.A major obstacle to screen for a diagnostic biomarker is the tremendous molecular heterogeneity that exists for nearly all human cancers, suggesting that simultaneous screening of multiple biomarkers will be required to improve the early detection/diagnosis of cancer. Since proteins are, for the most part, the mediators of acell's function, the study of the changes in proteins that result from a pathological lesion, such as complex multistep malignant process, would appear to be a rich source of potential cancer. A new proteomic approach for the detection of cancer which is called surface enhanced laser desorption/ ionization time-of-flight mass spectrometry and ProteinChip technology, have been developed. One of the key features of SELDI-TOF MS is its ability to provide a rapid protein expression profile from a variety of biological and clinical samples. Up to now, there are little published data on the use of this technique coupled with decision tree algorithm in studies of HSCC, LSCC, PMLL and lymph node metastasis. This work was aim to discover the novel biomarkers to separate LSCC ,HSCC vs the healthy controls and to establish predictive model to diagnose LSCC and HSCC. The effectiveness of the the decision tree model was then tested using double-blind sample dataset through Biomarker Pattern Software. The study is also designed to identify specific protein profiles that accompany neoplastic transformation in the PMLL. Finally, serum biomarkers to lymph node metastasis were also investigated.Part One: Establishment of the platform of protein chip SELDI-TOF- MSThe objective of this part of research was to establish a reliable protein chip SELDI-TOF-MS platform and to optimize the technological conditions in the experiments. Serum and cancer cell lysate samples from 8 cases of LSCC patients were collected respectively, special protein or peptide spectra was determined by SELDI-TOF-MS measurement after treating the sample onto four types of Protein Chips(WCX2 H4 SAX2, IMAC3) for each case. According to the protein profiling results, suitable parameters and protein chips in serum and cell lysate were screened respectively, and the reliability and stability of the technology were assessed.RESULTS:1. Most of the proteins detected by protein chip range from 1100 to 30000 at the Mass/charge (m/z) value.2. Analysis of serum protein fingerprints showed WCX2 Chip combined the most proteins, followed by IMAC3 Chip, and less in H4 and SAX2 Chips.3. Analysis of cell lysate protein fingerprints showed WCX2 Chip combined themost proteins, followed by H4 Chip, and less in IMAC3 and SAX2 Chips.4. The coefficient variation (CV) of protein intensity and m/z value was 0.184731 and 0.000242, respectively, within the same protein chip.5. The coefficient variation (CV) of protein intensity and m/z value was 0.214244 and 0.000055, respectively, between the protein chips.Part Two: SELDI-TOF MS Profiling of Serum for detection of pre-malignant laryngeal lesions, laryngeal squamous cell carcinoma and the Progression to lymph node metastasisProteomic profiling of serum is an emerging technique to identify new biomarkers indicative of disease severity and progression. Our study was to assess the use of surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify multiple serum protein biomarkers for early detection of laryngeal squamous cell carcinoma(LSCC), to establish predictive model, and to accurately distinguish LSCC patients without lymph node metastasis with the patients with lymph node metastasis. A cohort of 309 serum samples with LSCC (n=142), PMLL (n=57). and normal control (n=110) were consented into this study. These serum samples were randomly divided into training set (including 89 LSCC patients at stage I-II and 65 normal controls, 57 PMLL, 30 LSCC patients with lymph node metastasis) and blind testing set (including 53 LSCC patients at stageIII-IV and 45 normal controls). Serum protein profiles on weak cationic exchange (WCX2) were performed by SELDI-TOF MS and then analyzed by Biomarker Wizard software. The Decision Tree classification algorithm and blind validation were determined by Biomarker Pattern software(BPS). Results:1. A panel of 18 biomarkers Ranging from 2000-30000( m/z) was selected based on their collective contribution to the optimal separation between stage I- II LSCC patients and healthy controls.2. Among them 1 candidate protein peak with the m/z value of 4176 were selected to establish predictive model by BPS with sensitivity of 86.52 % and specificity of 84.62%. The ability to detect LSCC patients was evaluated using blinding test data in stages III and IV cancer patients. An sensitivity of 84.91 %, specificity of 82.22% were validated in blind testing set.3. The malignant shift was discovered to begin even in the pre-malignant stage with the comparison of PMLL to LSCC and healthy control.4. Meanwhile 14 potential biomarkers could differentiate LSCC patients with or without lymph node metastasis (P<0.05)Part Three: SELDI-TOF MS Profiling of Serum for detection of of hypopharyngeal squamous cell carcinomaWe used SELDI-TOF MS to find new potential biomarkers and to establish predictive model for early detection of hypopharyngeal squamous cell carcinoma (HSCC). 100 serum samples including 48 from HSCC patients, 52 from normal controls which were divided into training set and blind testing set were treated on WCX2 and IMAC3 protein chip, serum protein or peptide pattern were detected by SELDI-TOF-MS. The data of spectra were analyzed by Biomarker Wizard software to screen serum proteome biomarkers of HSCC. While decision tree classification algorithm and blind validation were determined by Biomarker Pattern software (BPS).Results:1. Ranging from 2000-50000(m/z), 13 potential biomarkers from the WCX2 surface could differentiate HSCC patients from normal controls (P<0.05).2. Ranging from 2000-50000(m/z), 21 potential biomarkers from the IMAC surface could differentiate HSCC patients from normal controls (P<0.05).3. Among them 4 candidate protein peaks with the m/z value of 7796,4216, 5927, 5361 were selected to establish predictive model by BPS with sensitivity of 94 % and specificity of 89%. An sensitivity of 92 %, specificity of 82% were validated in blind testing set.CONCLUSIONS1. SELDI-TOF MS was an ideal technological platform for proteomic research because of high reproducibility and stability. WCX2 protein chip could be suitable for the research of serum proteomics, while WCX2 H4 suitable for the research of cellular proteomics. Quality control and standardization of analysis conditions could be key issue for the reliability of outcome.2. The high sensitivity and specificity achieved by the serum protein biomarkers show great potential for the early detection of LSCC. SELDI-TOF MS serumprofiling also is able to distinguish LSCC patients with or without lymph node metastasis The differential expression of proteins among normal, PMLL and cancer cells might provide the prediction for the changes from normal to PMLL to malignant and to lymph node metastasis.3. The combination of SELDI with bioinformatics tools could help find serum proteome biomarkers and establish predictive model for early detection of HSCC.NOVELTY1. Establishment of tissue and serum protein fingerprints in HSCC and LSCC.2. For the first time, through the application of this technology, we identified protein expression changes in HSCC patients, PMLL patients and lymph node metastasis.3. Establish predictive model of HSCC and LSCC for the first time.4. Screening of serum protein biomarkers related to formation and aggravation of HSCC and LSCC, which may shed further light on the field.