Ultrastructural and Immunohistochemical Characteristics of Corneal Lenticule Extracted during Correction of Residual Myopia in the Long-Term Period after SMILE

Purpose: To evaluate ultrastructural characteristics of lenticule surface extracted during correction of residual myopia in patients after small-incision lenticule extraction (SMILE). Methods and material: This study had a prospective, consecutive, comparative design. Sixteen patients (16 eyes) underwent additional intervention for residual myopia correction after SMILE. 16 specimens of removed lenticules underwent morphological examination. Markers and reagents were used to determine actin microfilaments, neutral fats and cell nuclei. The tissue was analyzed in layers in 2D slices form, volumetric Z-stacks, or selected areas were formed in orthogonal projections. The surface of the extracted lenticule was analyzed using scanning electron microscopy. Patients’ refractive outcomes were measured postoperatively (1 day;1 and 3 months). Results: Postoperatively uncorrected distance visual acuity (20/20 or better) was in 100% cases 3 months after surgery. Ultrastructural studies have shown the difference in surfaces of the newly formed lenticule. Structural changes of the posterior lenticule surface were characterized by ruptures of collagen fibers on its surface, degenerative changes in keratocytes with signs of colliquation necrosis, cell apoptosis and F-actin in cell cytoplasm. Conclusion: Collagen fibers are immersed in the stroma on the anterior surface of the lenticule. There is no complete structure restoration of collagen fibers explaining the lack of tight adhesion of anterior and posterior surfaces of the intrastromal space even in the long-term postoperative period. There are no degenerative changes of keratocytes on the anterior lenticule surface, that is, their changes in SMILE are reversible in most cases.

Decreased expression of Neurensin-2 correlates with poor prognosis in hepatocellular carcinoma

AIM： To investigate the expression of Neurensin-2 （NRSN2） in hepatocellular carcinoma （HCC） and its prognostic values in predicting survival. METHODS： The expression and prognostic significance of NRSN2 in HCC was examined by performing immunohistochemical analysis using a total of 110 HCC clinical tissue samples, and Western blotting analysis to further confirm the result. RESULTS： Decreased NRSN2 expression was shown in 70.9% cases. Loss of NRSN2 expression in HCC was significantly related to tumor size （P = 0.006）. Larger tumor size was related to negative expression of NRSN2. Patients showing negative NRSN2 expression had a significantly shorter overall survival than those with positive expression （P = 0.008）. Multivariate Cox regression analysis indicated that NRSN2 expression level was an independent factor of survival （P = 0.013）. Western blotting analysis further confirmed decreased expression of NRSN2 in tumor tissues compared with non-tumorous tissues. CONCLUSION： Our study indicated that NRSIV2 could be a tumor suppressor gene for HCC and a candidate biomarker for long-term survival in HCC.

Prognostic factors of primary gastrointestinal stromal tumors： a cohort study based on high-volume centers

Objective： We aimed to evaluate the clinicopathologic characteristics, immunohistochemical expression and prognostic factors of patients with primary gastrointestinal stromal tumors（GISTs）.Methods： Data from 2,570 consecutive GIST patients from four medical centers in China（January2001–December 2015） were reviewed. Survival curves were constructed by the Kaplan-Meier method, and Cox regression models were used to identify independent prognostic factors.Results： Of the included patients, 1,375（53.5%） were male, and the patient age range was 18 to 95（median, 58）years. The tumors were mostly found in the stomach（64.5%）, small intestine（25.1%） and colorectal region（5.1%）.At the time of diagnosis, the median tumor size was 4.0（range： 0.1–55.0） cm, and the median mitotic index per 50 high power fields（HPFs） was 3（range： 0–254）. Of the 2,168 resected patients, 2,009（92.7%） received curative resection. According to the modified National Institutes of Health（NIH） classification, 21.9%, 28.9%, 14.1% and35.1% were very low-, low-, intermediate-and high-risk tumors, respectively. The rate of positivity was 96.4% for c-Kit, 87.1% for CD34, 96.9% for delay of germination 1（DOG-1）, 8.0% for S-100, 31.0% for smooth muscle actin（SMA） and 5.1% for desmin. However, the prognostic value of each was limited. Multivariate analysis showed that age, tumor size, mitotic index, tumor site, occurrence of curative resection and postoperative imatinib were independent prognostic factors. Furthermore, we found that high-risk patients benefited significantly from postoperative imatinib（P〈0.001）, whereas intermediate-risk patients did not（P=0.954）.Conclusions： Age, tumor size, mitotic index, tumor site, occurrence of curative resection and postoperative imatinib were independent prognostic factors in patients with GISTs. Moreover, determining whether intermediate-risk patients can benefit from adjuvant imatinib would be of considerable interest in future studies.

Hepatoma-derived growth factor expression as a prognostic marker in cervical cancer

AIM： To examine the association of hepatoma-derived growth factor （HDGF） expression with the prognosis of patients with cervical cancer of the uterus （CC）. METHODS： HDGF is a unique nuclear growth factor, and it may play an important role in the development and progression of carcinoma. HDGF expression in 88 CC patients aged 23 to 76 years （median, 54 years） was analyzed by immunohistochemistry. A rabbit polyclonal antibody against the C-terminal amino acids （aa 231-240） of the human HDGF sequence was used as primary antibody at a dilution of 1：5000. This specific anti-HDGF antibody was purified using C-terminal peptide-conjugated Sepharose columns. Staining of endothelial cells in the noncancerous areas of each specimen was used as an internal positive control. Samples with more than 80% of tumor cells showing positive immunoreactivity in both the nucleus and cytoplasm were regarded as HDGF index level 2,more than 80% positive immunoreactivity in either the nucleus or cytoplasm as level 1, and less than 80% in both the nucleus and cytoplasm as level 0. The chi-square test and Fisher’s exact probability test were used to examine the relationship between HDGF expression and clinicopathologic parameters, and statistical significance was examined by the log-rank test. Multivariate analysis of factors related to survival was performed using Cox’s proportional hazards regression model. Statistical signifcance was set at P 〈 0.05. RESULTS： The five-year overall survival rate was 82.9%. Fourteen patients died due to tumors, nine of whom had tumor recurrence at 2-21 mo （median, 10 mo） after surgery. Tumor recurrence in five patients was determined at the time of the patients’ deaths. Nineteen cases were regarded as HDGF index level 0, 11 as level 1, and 58 as level 2. Patients with level 2 expression showed higher rates of histological classification of keratinized squamous cell carcinomaand adenosquamous carcinoma （44.8% of level 2 patients and 13.3% in levels 0 and 1）, deep invasion （pT2-4 in 65.5% of level 2 patients, and 30.0% in levels 0 and 1）, the presence of lymphatic invasion （50.0% in level 2, and 20.0% in levels 0 and 1）, and the presence of lymph node metastasis （37.9% in level 2, and 6.7% in levels 0 and 1）. Patients with an HDGF index of level 2 CC showed poorer 5-year overall survival rates than those with level 0 or 1 CC （74.0% and 100%, respectively, P = 0.0036）. Univariate analysis revealed that histological classification （P = 0.04）, depth of tumor invasion （P = 0.0001）, vascular invasion （P = 0.004）, and lymph node metastasis （ P = 0.0001） were significant factors affecting overall survival in addition to HDGF expression. Multivariate analysis revealed HDGF expression level and lymph node metastasis as independent prognostic factors for overall survival （P = 0.0148 and P = 0.0197, respectively）. The prognostic signifcance of HDGF was further analyzed in pT1 andsignifcance of HDGF was further analyzed in pT1 and pT2-4 patient groups, respectively. Among patients with pT1 CC, one the 39 analyzed patients died during the study, and no difference was observed among patients with HDGF index level 0, 1, or 2 CC. However, prognostic signifcance of the HDGF index was observed in the pT2-4 patient group, in which the mortality rates of patients with HDGF index level 2 CC and those with level 0 or 1 CC signifcantly differed （P = 0.0463）. CONCLUSION： The HDGF expression level is of prognostic signifcance in CC.

Stem/progenitor cells in fetuses and newborns:overview of immunohistochemical markers

Microanatomy of the vast majority of human organs at birth is characterized by marked differences as compared toadult organs, regarding their architecture and the cell types detectable at histology. In preterm neonates, thesedifferences are even more evident, due to the lower level of organ maturation and to ongoing cell differentiation.One of the most remarkable finding in preterm tissues is the presence of huge amounts of stem/progenitor cells inmultiple organs, including kidney, brain, heart, adrenals, and lungs. In other organs, such as liver, the completelydifferent burden of cell types in preterm infants is mainly related to the different function of the liver duringgestation, mainly focused on hematopoiesis, a function that is taken by bone marrow after birth. Our preliminarystudies showed that the antigens expressed by stem/progenitors differ significantly from one organ to the next.Moreover, within each developing human tissue, reactivity for different stem cell markers also changes duringgestation, according with the multiple differentiation steps encountered by each progenitor during development. Abetter knowledge of stem/progenitor cells of preterms will allow neonatologists to boost preterm organmaturation, favoring the differentiation of the multiple cells types that characterize each organ in at term neonates.

The Expression and Location of Midkine in Gastric Carcinomas of Chinese Patients

Midkine （MK）, a heparin-binding growth factor, can regulate cell growth, survival and differentiation. MK is expressed at high levels in a variety of human carcinomas. Recently, the urine and serum MK concentration was analyzed in gastric cancer patient. However, the association of the cytokine mRNA expression with the categorical clinicopathological variables of the tumors and the location of its protein expression in the tumor tissues are still elusive. MK mRNA expression from the surgically resected specimens of healthy gastric tissues （9 cases）, gastric cancer tissues and the matched non-cancerons tissues adjacent to the cancer （37 cases） were assessed by reverse transcriptase-polymerase chain reaction （RT-PCR） and real-time PCR. Immunohistochemical analysis was performed to locate MK in gastric cancer. The expression of MK mRNA in gastric cancer was much higher in tumor tissues than that in the non-cancerons tissues and control tissue samples. And its expression was significantly associated with the pTNM stage and distant metastasis, but not with the differentiation grade, tumor size and nodal involvement. MK protein was ubiquitous in the tumor, especially in the adenoid part of tumors. In addition, it was found in the cytoplasm of tumor cells and highly concentrated in nucleus and nucleolns. The expression level and location of MK in gastric tumor tissues of Chinese Patients may be related to the tumor genesis and progression. Further study is necessary on the mechanism of MK in gastric tumorigenesis and tumor growth.