Serine protease inhibitor Kazal type 1 (SPINK1) as a prognostic marker in stage IV colon cancer patients receiving cetuximab based targeted therapy.

BACKGROUND: Serine peptidase inhibitor Kazal type-1 (SPINK1), a trypsin kinase inhibitor, has well established associations with inflammation, cancer cell proliferation and carcinogenesis. However, the role of SPINK1 has not been investigated in stage IV colorectal cancer (CRC) patients receiving cetuximab based targeted therapy. The aim of this study was to evaluate the use of SPINK1 as a biomarker for predicting how patients with end stage CRC respond to anti-epidermal growth factor receptor (EGFR) therapies. METHODS: Immunohistochemical staining was used for semiquantitative analysis of SPINK1 protein expression in 51 CRC cases. Expression of SPINK1 protein was then analysed to identify correlations with clinicopathological variables. RESULTS: High SPINK1 expression was significantly associated with males (p=0.018). Kaplan-Meier analyses also showed that patients with high SPINK1 expression had significantly longer overall survival compared with controls (p=0.004). Multivariable analyses showed that SPINK1 expression and tumour size were significantly associated with prognosis (HR 0.416 and 0.437; 95% CI 0.217 to 0.797 and 0.236 to 0.810; p=0.008 and p=0.009, respectively) in these stage IV CRC cases. CONCLUSIONS: High SPINK1 expression is associated with a good prognosis in stage IV CRC cases receiving cetuximab based targeted therapy. As SPINK1 expression is also an independent prognostic marker in these patients, it has potential use as a biomarker for clinical decision making and for designing personalised targeted therapies.

Autophagy and Apoptosis Play Opposing Roles in Overall Survival of Esophageal Squamous Cell Carcinoma.

Esophageal cancer is among the most aggressive gastrointestinal tract malignancies, and squamous cell carcinoma is the most common subtype. Although both autophagy and apoptosis involve programmed cell death, autophagy also maintains cell survival by recycling cellular waste. The relationship between autophagy and apoptosis in esophageal squamous cell carcinoma (ESCC) is unclear. Autophagic and apoptotic markers of ESCC were detected by immunohistochemical staining (IHC) in 43 ESCC patients treated during 2007-2011. Chi-square test and Kaplan-Meier method were used to determine how clinicopathological parameters were related to IHC results for LC3B, Beclin-1 and caspase-3 (CASP-3). Correlations among Beclin-1, LC3B, and CASP-3 were analyzed by Spearman rho. The statistical analyses revealed no clinicopathological parameters that significantly correlated with expressions of Beclin-1, LC3B, and CASP-3. However, low CASP-3 expression and high LC3B expression revealed by IHC were predictors of a poor prognosis. Additionally, LC3B expression had a significant negative correlation with CASP-3 expression. Autophagy is antagonistic to apoptosis and predicts poor overall survival in ESCC.

The X-linked inhibitor of apoptosis protein is an independent prognostic marker for rectal adenocarcinoma after preoperative chemoradiotherapy.

Rectal cancer (RC) is a common malignancy of the gastrointestinal tract. Preoperative neoadjuvant concurrent chemoradiotherapy (CCRT) is considered an effective treatment as it down-stages advanced RC. X-linked inhibitor of apoptosis protein (XIAP) and survivin participate in the regulation of apoptosis, a key process in carcinogenesis and tumor progression. However, the prognostic value of concomitant expression of XIAP and survivin in RC patients undergoing neoadjuvant CCRT is not well established. Expression of XIAP and survivin proteins was evaluated by immunohistochemical staining of pre-CCRT and post-CCRT specimens of 58 rectal cancer patients. Clinicopathological parameters were also analyzed. In pre-CCRT biopsy specimens, high survivin expression was significantly associated with perineural invasion (p = 0.025), metastatic status (p = 0.015), and advanced disease stage (I-IV, p = 0.025; early vs. late, p = 0.047). In post-CCRT surgical specimens, high XIAP expression was significantly associated with age (p = 0.037), T stage (p = 0.025), disease stage (p = 0.019), and tumor regression grade (TRG) (p = 0.000). High survivin expression was significantly correlated with T stage (p = 0.044), N stage (p = 0.028), metastasis (p = 0.007), disease stage (p = 0.001), and TRG (p = 0.033). Pearson correlation calculations showed a positive correlation between XIAP and survivin immunoreactivity (p = 0.000). Patients with low XIAP and low survivin expression tended to have significantly longer overall survival (p = 0.006 and 0.001, respectively). In multivariable Cox regression analysis, patients with high XIAP, perineural invasion, and advanced stage had significantly shorter overall survival (p = 0.000, 0.002 and 0.000, respectively). In conclusion, high expression of XIAP and survivin is associated with advanced stage and poor prognosis. Expression of XIAP is positively correlated with that of survivin. These data suggest that XIAP is an independent prognostic marker and might be considered as therapy target for rectal cancer after neoadjuvant therapy.

Serine Protease Inhibitor Kazal Type 1 (SPINK1) Promotes Proliferation of Colorectal Cancer Through the Epidermal Growth Factor as a Prognostic Marker.

Serine protease inhibitor Kazal type-1 (SPINK1), a trypsin kinase inhibitor, is involved in inflammation, cell proliferation and carcinogenesis. The role and association between SPINK1, EGFR and Ki-67 in colorectal adenoma (CRA) and colorectal cancer (CRC) are still unknown. In this study, we used immunohistochemical stain to evaluate expression of SPINK1, EGFR and Ki-67 proteins in 30 CRA and 53 CRC patients semiquantitatively, and then analyzed their correlation with clinicopathologic parameters. Our results revealed that SPINK1 expression was noted in the upper and basal parts of the crypts in CRA and was more intensely related with cellular atypia. EGFR expression was found in 13 out of 30 adenomas, including 9 out of 15 adenomas with dysplasia or synchronous CRC (60 %), and 4 out of 15 adenomas without dysplasia (26.7 %). In CRC, high SPINK1 expression was significantly associated with males (p = 0.041) and advanced disease stage (p = 0.015). EGFR positivity was significantly correlated with higher T stage (p = 0.004) and disease stage (stage I-IV, p = 0.017; early vs. late, p = 0.015). Pearson's correlation showed positive correlation between the SPINK1 intensity and EGFR immunoreactivity (p = 0.011), and Ki-67 and SPINK1 intensity or percentage (p = 0.017 and p = 0.039 respectively). In Kaplan-Meier analyses, patients with high SPINK1 intensity tended to have shorter overall survival (p = 0.03). Concomitant expression of high SPINK1 intensity and EGFR was also identified as being associated with poor prognosis (p = 0.015). In conclusion, high SPINK1 expression is associated with advanced stage and poor prognosis. There is positive correlation between high SPINK1 expression, EGFR immunoreactivity, and high Ki-67 labeling index. The SPINK1 protein seems to play a role in tumor proliferation and malignant transformation through the EGFR pathway. SPINK1 may serve as a prognostic biomarker in therapeutic targeting in the future.

Improved technique for manually constructing tissue microarrays for large-core arrays.

Tissue microarrays were originally developed to enable alignment of multiple tissue cores in a single paraffin block and to enable high-throughput laboratory analysis. However, a major drawback is the loss of tissue cores during slide preparation, especially when sectioning the tissue block. Tissue cylinders directly aligned in the metal box without preheating tend to detach from the surrounding paraffin, which results in incomplete or folded tissue sections. The proposed solution is preheating all tissue cylinders on a hot plate to facilitate fusion between the paraffin within the core and the paraffin surrounding the core. In this study, 6 tissue microarray blocks were constructed from 528 tissue cores extracted from various formalin-fixed, paraffin-embedded human tissue samples. The tissue cores in the arrays revealed good homogenization with the surrounding paraffin wax, and the tissue sections were obtained intact. Both hematoxylin-eosin and immunohistochemical staining confirmed satisfactory results. This simple and economical method is easily performed in the laboratory without expensive instrumentation.

Utility of the capsule-based technique for cell block preparation - in body fluids and Liqui-PREP™ specimens.

OBJECTIVE: The aim of this study was to find a better technique for the cell block preparation. STUDY DESIGN: We developed a novel capsule-based technique and applied it to different cell block preparation materials including plasma thromboplastin (PT) clot and agarose gel-embedding medium, in order to concentrate the cells in a limited field, then compared the result with the widely used, modified alcohol fixative preparation technique based on specimen adequacy and diagnostic accuracy. Twenty-eight nongynecologic body fluids and 41 gynecologic Liqui-PREP™ (LGM International Inc., Fort Lauderdale, Fla., USA) cytology specimens were collected for cell block preparation. We performed routine hematoxylin and eosin staining on the cell block sections, which were scored according to four specimen adequacy parameters: background, cellularity, nuclear preservation and cytoplasmic preservation. The differences between the diagnostic results of cell block slide and conventional cytology smear were also presented. RESULTS: The capsule-based PT clot technique in nongynecologic body fluids provided more cellularity in the cell block sections and reduced atypical diagnosis when compared to conventional smears. CONCLUSION: We suggest the capsule-based PT clot technique is a better technique for nongynecologic body fluids in the preparation of a satisfactory cell block.

A simple and economical method for the manual construction of frozen tissue arrays.

Tissue microarray has been developed to enable multiple cores of tissue in one or more new paraffin blocks. Currently, almost all tissue microarrays are made by coring cylindrical tissues from formalin-fixed and paraffin-embedded tissues. The disadvantages of formalin-fixed and paraffin-embedded tissues include the poor preservation of antigenicity of certain proteins and mRNA degradation induced by the fixation and embedding process. However, frozen tissue array construction presents technical difficulties, and tissue array devices are expensive, particularly for small- and medium-sized laboratories. We describe a simple manual method for producing well-aligned tissue arrays by a capsule freeze method that allows us to successfully perform hematoxylin-eosin and immunohistochemical stain. All 120 tissue samples were collected and constructed into blocks by this capsule freeze method. The capsules were not affected during the sectioning process, and the capsule material always disappeared during the aqueous steps of the stain processing. The frozen tissue arrays were smoothly sectioned without the use of a tape transfer system and immunohistochemical study was performed with satisfactory results. This alternative method can be applied in any laboratory, and is both simple and economical.

Use of caveolin-1, thyroid transcription factor-1, and cytokeratins 7 and 20 in discriminating between primary and secondary pulmonary adenocarcinoma from breast or colonic origin.

The objectives of this study were firstly to compare the immunostaining patterns of antibodies against caveolin-1, thyroid transcription factor-1 (TTF-1), cytokeratin 7 (CK7) and cytokeratin 20 (CK20) in primary and secondary pulmonary adenocarcinomas of breast or colonic origin, and secondly, to investigate their use alone and in combination, in distinguishing between primary and secondary lung adenocarcinomas from breast or colonic origin. Of the 49 lung adenocarcinoma specimens that were enrolled in this study, 30 were primary pulmonary adenocarcinomas, and 19 (9, breast origin; 10, colonic origin) were metastatic pulmonary carcinomas. Immunohistochemical staining was used to detect the expression of caveolin-1, TTF-1, CK7, and CK20. Primary pulmonary adenocarcinoma most often had the CK7-positive/CK20-negative immunohistochemical phenotype and was either TTF-1 positive or caveolin-1 negative. Secondary pulmonary adenocarcinoma of breast origin most often had the CK7-positive/CK20-negative immunohistochemical phenotype and was either TTF-1 negative or caveolin-1 positive, while secondary pulmonary adenocarcinoma of colonic origin most often had the CK20-positive/CK7-negative immunohistochemical phenotype and was either TTF-1 negative or caveolin-1 positive. The results suggest that caveolin-1, TTF-1, or CK7/CK20 alone did not distinguish reliably between primary and secondary pulmonary adenocarcinomas originating from breast or colon. The use of a panel of antibodies that includes TTF-1, caveolin-1, and CK7/CK20 may have higher sensitivity in discriminating between primary adenocarcinomas and metastatic lung adenocarcinomas from breast or colonic origin.