Cancer Nests Research Articles

The histological diversity of adenoid cystic carcinoma demonstrated by double immunohistochemistry for p16 and p63 gene products

Background: Adenoid cystic carcinoma (AdCC) displays morphological diversity within its characteristic tumor cell nests, which are primarily classified into tubular, cribriform, and solid patterns. Such histological diversity should result from the proportional proliferation between neoplastic myoepithelial cells (NMC)/basal cells and ductal epithelial cells. The aim of this study was to clarify the relationship between this morphological diversity and the development of AdCC in the salivary gland using double immunostaining with monoclonal antibodies to p16 and p63 gene products. Methods: We examined 10 samples of formalin-fixed, paraffin-embedded AdCC tissues. Results: In the normal salivary gland, p16 protein (P16) was not expressed in any cells, whereas p63 protein (P63)-positivities were observed in ductal basal cells and in acinar myoepithelial cells. In AdCC foci showing the tubular pattern, P16 expression was localized in the inner cells of the ductal structure, while P63 was localized in the outer NMC of them within the same sections. In tumor foci with the cribriform pattern, P16 was expressed in several cells forming pseudocysts and in some of the inner cells. P63 was expressed in many cells in the cancer nests. In those with the solid pattern, P16 and P63 were intermixed within the same foci. Conclusions: Thus, the double immunohistochemistry for P16 and P63 was useful for observing the morphological diversity of AdCC cells within the same foci on the same tissue section. These data suggest that P16 and P63 may play important roles in the morphological diversity and development of AdCC tissue architectures.

Localization and characterization of lymphatic vessels in oral and cervical squamous cell carcinoma

Lymph node metastasis is considered a factor in determining the prognosis of squamous cell carcinoma (SCC). Both oral and cervical SCC tumor cells prefer lymph vessels as the route of metastasis. D2-40 is a specific marker of lymphatic endothelial cells. This study clarifies the distribution and characteristics of lymphatic vessels in oral and cervical SCCs. Immunohistochemistry was performed in 20 oral and 20 cervical SCCs (10 non-metastatic and 10 metastatic to lymph nodes) using D2-40, CD31, CD34, CD105 and double staining with D2-40 and keratin. Lymphatic vessel density (LVD) was also determined morphologically. Results showed that lymphatic vessels in both types of SCCs were distributed mainly at the superficial region beneath the epithelium. The LVD in each tumor was significantly higher compared to the corresponding normal mucosa. Moreover, the LVD in lymph node metastasis in each tumor was significantly higher compared to their non-metastatic counterparts. Cancer cell invasion was observed in the lymphatic vessels suggesting the existence of lymph node involvement during metastasis. The new lymphatic vessels that proliferated around the cancer nests in both SCCs have endothelial cell characteristics inferred to be associated with early lymphatic development and initial dissemination of cancer cells.

Co-evolution of cancer microenvironment reveals distinctive patterns of gastric cancer invasion: laboratory evidence and clinical significance

BackgroundCancer invasion results from constant interactions between cancer cells and their microenvironment. Major components of the cancer microenvironment are stromal cells, infiltrating inflammatory cells, collagens, matrix metalloproteinases (MMP) and newly formed blood vessels. This study was to determine the roles of MMP-9, MMP-2, type IV collagen, infiltrating macrophages and tumor microvessels in gastric cancer (GC) invasion and their clinico-pathological significance.MethodsParaffin-embedded tissue sections from 37 GC patients were studied by Streptavidin-Peroxidase (SP) immunohistochemical technique to determine the levels of MMP-2, MMP-9, type IV collagen, macrophages infiltration and microvessel density (MVD). Different invasion patterns were delineated and their correlation with major clinico-pathological information was explored.ResultsMMP2 expression was higher in malignant gland compared to normal gland, especially nearby the basement membrane (BM). High densities of macrophages at the interface of cancer nests and stroma were found where BM integrity was destroyed. MMP2 expression was significantly increased in cases with recurrence and distant metastasis (P = 0.047 and 0.048, respectively). Infiltrating macrophages were correlated with serosa invasion (P = 0.011) and TNM stage (P = 0.001). MVD was higher in type IV collagen negative group compared to type IV collagen positive group (P = 0.026). MVD was related to infiltrating macrophages density (P = 0.040). Patients with negative MMP9 expression had better overall survival (OS) compared to those with positive MMP9 expression (Median OS 44.0 vs 13.5 mo, P = 0.036). Median OS was significantly longer in type IV collagen positive group than negative group (Median OS 25.5 vs 10.0 mo, P = 0.044). The cumulative OS rate was higher in low macrophages density group than in high macrophages density group (median OS 40.5 vs 13.0 mo, P = 0.056). Median OS was significantly longer in low MVD group than high MVD group (median OS 39.0 vs 8.5 mo, P = 0.001). The difference of disease-free survival (DFS) between low MVD group and high MVD group was not statistically significant (P = 0.260). Four typical patterns of cancer invasion were identified based on histological study of the cancer tissue, including Washing pattern, Ameba-like pattern, Spindle pattern and Linear pattern.ConclusionsProteolytic enzymes MMP9, MMP2 and macrophages in stroma contribute to GC progression by facilitating the angiogenesis. Cancer invasion patterns may help predict GC metastasis.

Podoplanin Expression in Cancerous Stroma Induces Lymphangiogenesis and Predicts Lymphatic Spread and Patient Survival

Podoplanin is a mucin-type glycoprotein and a lymphatic endothelial marker. Immunohistochemical staining for podoplanin is currently used as a routine pathologic diagnosis tool in Japan to identify lymphatic invasion of cancer cells. Recent reports suggest that podoplanin and other proangiogenic molecules are expressed in stromal fibroblasts and myofibroblasts. To analyze the distribution of podoplanin expression in tumor stroma and its clinical and biologic significance. We performed immunohistochemistry for podoplanin on tissue microarrays from 1350 cases of 14 common cancer types. Two hundred eighty-seven of 662 cases (43%) showed podoplanin expression in the stromal cells within cancer nests. Stromal podoplanin expression in 14 common cancer types was significantly associated with tumor stage (P < .001), lymph node metastases (P < .001), lymphatic invasion (P = .02), and venous invasion (P < .001). The stromal cells positive for podoplanin were also positive for α-smooth muscle actin but negative for desmin, confirming a myofibroblasts phenotype. In contrast, myofibroblasts in inflammatory fibrotic lung diseases were podoplanin negative. Lymphatic vessel density was greater in the stromas with podoplanin expression than in the stroma lacking podoplanin-expressing stromal cells (P = .01). Survival data were available for non-small cell lung cancer. Stromal podoplanin expression was associated with poorer prognosis in adenocarcinoma (P < .001) and remains statistically significant after adjustment for sex, age, and stage (P = .01). Our data indicate that podoplanin expression in stromal myofibroblasts may function as a proangiogenic biomarker and may serve as a predictive marker of lymphatic/vascular spread of cancer cells and a prognostic marker of patient survival.

Proposed Objective Criteria for “Grade 3” in Early Invasive Colorectal Cancer

To establish objective criteria for "grade 3" (G3) in T1 (TNM staging) colorectal cancer (CRC), a total of 296 T1 CRC cases were reviewed. The incidence of nodal involvement differed most greatly between G3 and non-G3 (21/27 [27%] and 6/162 [3.7%], respectively; P < .0001), when G3 was applied to tumors containing either or both of the following: (1) 10 or more solid cancer nests in the microscopic field of a 4x objective lens and (2) a mucin-producing component fully occupied the microscopic field of a 40x objective lens. Regarding G3, vascular invasion, and tumor budding as indicating the risk of metastasis, nodal involvement rate was 21.0% in the tumors with 1 or more risk factors, whereas it was only 1.7% in the no-risk tumors (P < .0001). In patients treated with local excision only, nodal recurrence occurred in 3 (20%) of 15 risk-positive patients, whereas none of 42 patients without risk factors had nodal recurrence (P = .016). In cases of locally excised T1 CRC, G3 as determined by the proposed criteria, vascular invasion, and budding would comprise a useful combination of parameters for determining the indication for additional laparotomy.

Histopathologic features of rim enhancement MRI for breast infiltrating ductal carcinoma

Objective To investigate the histopathologic features of rim enhancement MRI of breast infiltrating ductal carcinoma (IDC).Methods Routine and dynamic contrast enhanced MRI was used to examine 65 patients with breast lumps.Thirty of the 65 patients who were pathologically confirmed to have breast IDC were included in the present study.The manifestations of dynamic contrast enhanced MRI and the histopathologic parameters of the masses (the size of cancer nest,stroma type,microvessel density and degree of fibrosis) were observed.And the relationship of MR findings with the above-mentioned histopathologic features was analyzed.Results Peripheral rim enhancement was obvious in IDC and a type-Ⅲ(wash-out) time/signal intensity course was the dominant type (17/30,56.7%).The average enhancement rate during the first post-contrast minute(SI1%)was higher than 75% in IDC.The 30 IDCs fell into small (13,43%),medium(12,40%),and large(5,17%) groups according to the size of cancer nest;and into delicate(5,17%),narrow(16,53%),and broad(9,30%) groups according to the cancer stroma type.Early rim enhancement was associated with a small cancer nest (P0.05),a high ratio of peripheral-to-central microvessel density,and a low ratio of peripheral-to-central fibrosis (P0.01).Delayed rim enhancement was significantly associated with narrow stroma (P0.05).Conclusion Rim enhancement and washout sign on contrast-enhanced MR imaging of the breast IDC are associated not only with angiogenesis,but also with various histological features of the cancinoma,including the size of cancer nests,width of stroma,and degree of fibrosis.

Effects of E-selectin and their ligands on the adhesive metastasis of hepatocellular carcinoma

To explore the effects of E-selectin, ICAM-1 and their ligands on the adhesive metastasis of hepatocellular carcinoma (HCC), and to select possible anti-adhesion drugs for hepatocellular carcinoma metastasis. 78 HCC patients were analyzed with the correlation of clinical features to the expression levels of E-selectin, sLeX, sLeA and CD44v6 in the tumor tissue. The adhesion between HepG2 and endothelial cell lines was examined by solid phase adhesion assay in vitro. Two kinds of drugs were accessed for their anti-adhesion ability. The positive rate of E-selectin in vascular endothelia cells adjacent to cancer nest is 70.51%, and which of sLeX, sLeA, CD44v6 within tumor cells is 64.10%, 69.23%, 62.90% respectively. The patients' life span is closely related with the positive expression of sLeX, sLeA, CD44v6 (P = 0.008, 0.001, 0.022). The positive expression of E-selectin, sLeX and sLeA is significantly correlated to portal vein tumor thrombus (PVTT), preoperative extrahepatic metastasis, and satellite foci, but not to the size of tumor and AFP. The level of CD44v6 expression is significantly correlated to patient's survival time. The expression levels of E-selectin and ICAM-1 are remarkably higher after ED25 and ECV304 cell lines be activated. Meanwhile the adhesive ability of HepG2 to endothelial cell is mediated. Dexamethasone, tanshinone IIA are able to block this adhesion at low concentration. The expression levels of E-selectin, sLeX, sLeA and CD44v6 are closely correlated with clinical features. E-selectin, ICAM-1 and their ligands are important molecules of hepatocellular carcinoma and endothelial cells to tumor adhesive metastasis. Dexamethasone, tanshinone II A can be hopefully used as anti-adhesion drugs.

Use of T-cell infiltrate to predict long-term survival following local therapy in tongue cancer patients.

5547 Background: Oral tongue squamous cell cancer (OTSCC) is one of the most aggressive subtypes in head and cancers. Currently, the most reliable prognostic indicator for OTSCC patients is the status of cervical lymph nodes. Identification of differences in tumor microenvironment might be of prognostic value in this type of cancer. Of those, the clinicopathologic significance of the tumor-infiltrating lymphocytes (TIL) in various human cancers has been an issue of great interest. However, importance of TIL in OTSCC has rarely been reported. The purpose of this study was to investigate whether the degree of TIL in the surgical specimens of OTSCC can be used to predict the clinical outcome following resection. Methods: We performed an immunohistochemical study to identify and count the number of CD4+ T cells, Foxp3+ regulatory T cells, Ki-67+ cells, and CD105+ cells in cancer nests and stroma in surgical specimens obtained from 53 OTSCC patients. Microvessel density (MVD) was measured with a light microscope in a single area of invasive tumor (200x field or 0.74 mm2) representative of the highest microvessel density. Correlation between the degree of TIL and clinicopathological variables, and survival rate were analyzed. Results: Patients with high TIL in cancer nests had significantly longer survival time than those with low TILs (5-year survival rate; 70% vs 33.9%, p=0.039). High density of TIL was observed more frequently in the patients who were current smokers or alcohol drinkers. Subsequent multivariate analysis revealed that degree of TIL was an independent prognostic factor in the patients with smoking associated cancers only (p=0.023 in Cox proportional hazard model), but not in non-smokers. In addition, higher MVD was observed in patients with high TIL. However, ratio of foxp3+ regulatory T cell did not correlate with survival in our patient series. Conclusions: Our data show that total T cell infiltrate in the invasive tumor is significantly correlated with overall survival time in patients with OTSCC after resection. Significant impact on survival of TIL was found only in patients with smoking-related cancer, suggesting immune response may play an important role in prognosis of smoking-related OTSCC. No significant financial relationships to disclose.

Osteoclast-type giant cell tumor of minor salivary gland with mucin-rich salivary duct carcinoma: a case report of unusual histology with immunohistochemical analysis

Salivary giant cell tumor (GCT) is exceedingly rare. This article presents an additional rare case of salivary GCT with salivary duct carcinoma (SDC). The patient was a 40-year-old Japanese male. The peripheral region of the tumor showed SDC and partly revealed a mucin accumulation with cancer nests, which was a mucin-rich variant of SDC. In the central region of the tumor, mononuclear ovoid tumor cells contained osteoclastic-type giant cells. SDC showed immunopositivity for gross cystic disease fluid protein-15 (GCDFP-15), androgen receptor (AR), and Her-2, whereas the giant cell lesion was negative for GCDFP-15, AR, and Her-2. Mononuclear cells in salivary GCT showed immunopositivity for epithelial membrane antigen and p53. The salivary GCT was thought to be neoplastic and derived from epithelial cells. The present case is the first de novo case of intraoral salivary GCT with a mucin-rich variant of SDC.

Expression of integrin-linked kinase in oral leukoplakia and early invasive carcinoma

To investigate the transmutation and significance of integrin-linked kinase (ILK) in oral leukoplakia and early invasive squamous cell carcinoma. Immunohistochemistry accompanied with periodic acid Schiff reaction (PAS) histochemistry was applied to detect the expression of ILK in 19 normal oral mucosa, 43 simple hyperplasia, 84 epithelial dysplasia, 54 early invasive carcinoma. Among the oral leukoplakia and early invasive squamous cell carcinoma, dramatic positive expression of ILK was found in 163/181 (90%), but was negative in normal mucosa. ILK staining in stroma exhibited a significantly direct correlation with the exacerbated epithelial dysplasia (chi(2) = 41.585, P < 0.001). Along with the oral carcinogenesis, there was an obvious tendency of change-overed ILK staining from superfacial to the basal orientation, the stratum basale diverted into positive, and compared with scattering in cytoplasm, the location of ILK was prone to cytomembrane in epithelium. In severe dysplasia and carcinoma in situ, there was a positive correlation between the basal layer and stroma expression of ILK (P = 0.029). It was not only a weaker tincture of ILK in cancer nest than lesional epithelia, but also an augmentation of stroma staining in early invasive squamous cell carcinoma [76% (41/54)] by contrast with severe dysplasia [45% (18/40)]. The pivotal role of ILK in the regulation of oral carcinogenesis is considerable, however, the precise mechanisms through which ILK affects cellular processes remain to be fully characterized.

A histopathological study of 102 autopsy cases of primary lung cancer with respect to pleural carcinomatosis

Visceral pleural lesions in primary lung cancer were studied histopathologically in 102 autopsy cases. Pleural carcinomatosis was defined as the presence of cancer cells in the visceral pleura and/or pleural space. Ipsilateral and contralateral pleural carcinomatosis in the primary lung cancer were compared. They were found to be similar in that both were closely related with cancer cell infiltration of blood vessels in the lung parenchyma, lymphangiosis carcinomatosa in the lung parenchyma and multiple extrathoracic organ metastases. However, differences were observed. In the ipsilateral visceral pleura, cancer cells were thought to infiltrate the interstitium in the vascular layer of the visceral pleura from lymphatic vessels in the vascular layer of the visceral pleura with the following frequency; 3.6:8.52:6.52:1. In the contralateral visceral pleura, however, cancer cells were thought to infiltrate the interstitium in the vascular layer of the visceral pleura from lymphatic vessels in the vascular layer of the visceral pleura, the pleural space, the subpleural lung parenchyma and blood vessels in the vascular layer of the visceral pleura with the following frequency; 4:1.9:12.25:1. Disruption of the elastic layer of the visceral pleura was also studied. Twenty-eight of 66 pleural carcinomatosis cases showed no disruption of the elastic layer, and greater than 1-mm disruption was found in only 7 cases. The author thought that the disruption in these few cases was related to the manner of cancer-cell infiltration of the visceral pleura. When cancer cells spread to the visceral pleura from the lung parenchyma, they lost intercellular adhesiveness in four adenocarcinoma cases. Thus we suspected that cancer cells lost intercellular adhesiveness and became single cells or small cancer nests when infiltrating the visceral pleural from the lung parenchyma, and single cells or small cancer nests entered through the elastic layer of the visceral pleura. Therefore only small disruptions are found in the visceral pleura of pleural carcinomatosis cases.

Expression of calreticulin is associated with infiltration of T-cells in stage IIIB colon cancer

To investigate the correlation between expression of calreticulin and infiltration of lymphocytes in stage IIIB colon cancer. Sixty-eight pathologically-confirmed specimens were obtained from stage IIIB (T3N1M0) colon cancer patients who underwent radical resection between January 1999 and May 2002 at the Cancer Center of Sun Yat-Sen University, Guangzhou, China. Immunohistochemical analysis was performed to show infiltration of lymphocytes and expression of calreticulin in colon cancer. Association between calreticulin expression, infiltration of lymphocytes, and 5-year survival rate of patients was assessed. The expression level of calreticulin was lower in cancer nest than in its adjacent normal epithelium since 61.8% (42/68) of the samples were stained with calreticulin in colon cancer. The expression of calreticulin in colon cancer was associated with the infiltration of CD45RO+ cells rather than with that of CD3+ cells. In addition, the stronger expression of calreticulin and the higher infiltration of CD3+ and CD45RO+ cells in colon cancer were associated with the higher 5-year survival rate of patients. Expression of calreticulin is associated with infiltration of T-cells, which implies that a low expression level of molecular marker may represent a new mechanism underlying immune escape in colon cancer.

Apparent Diffusion Coefficient in Invasive Ductal Breast Carcinoma: Correlation with Detailed Histologic Features and the Enhancement Ratio on Dynamic Contrast-Enhanced MR Images

Purpose. To investigate the correlation of Apperent Diffusion Coefficient (ADC) values in invasive ductal breast carcinomas with detailed histologic features and enhancement ratios on dynamic contrast-enhanced MRI. Methods and Materials. Dynamic MR images and diffusion-weighted images (DWIs) of invasive ductal breast carcinomas were reviewed in 25 (26 lesions) women. In each patient, DWI, T2WI, T1WI, and dynamic images were obtained. The ADC values of the 26 carcinomas were calculated with b-factors of 0 and 1000 s/mm2 using echoplanar DWI. Correlations of the ADC values were examined on dynamic MRI with enhancement ratios (early to delayed phase: E/D ratio) and detailed histologic findings for each lesion, including cellular density, the size of cancer nests, and architectural features of the stroma (broad, narrow, and delicate) between cancer nests. Results. The mean ADC was 0.915 ± 0.151 × 10−3 mm2/sec. Cellular density was significantly correlated with ADC values (P = .0184) and E/D ratios (P = .0315). The ADC values were also significantly correlated to features of the stroma (broad to narrow, P = .0366). Conclusion. The findings suggest that DWIs reflect the growth patterns of carcinomas, including cellular density and architectural features of the stroma, and E/D ratios may also be closely correlated to cellular density.

Prognostic significance of natural killer cell infiltration in hepatocellular carcinoma

Several studies have Shown the correlation of high numbers of tumor-infiltrating natural killer (NK) cells with a good prognosis for cancer patients. This study was to investigate the impact of NK cell infiltration on the survival and prognosis of patients with hepatocellular carcinoma (HCC) after resection. The proportion of infiltrating NK cells of HCC patients was measured using flow cytometry, and the expression of CD56+ (NK) cells was investigated using immunohistochemistry. Prognostic values of intratumoral and peritumoral NK cell densities were evaluated by Kaplan-Meier method and Cox regression. The level of NK cells was significantly lower in tumor-infiltrating lymphocytes (TIL) of HCC patients than in nontumor-infiltrating lymphocytes (NIL) [(11.8 +/- 8.1)% vs. (18.0+/-7.9)%, P=0.002]. The density of NK cells was also significantly lower in cancer nests than in peritumoral lesions (2.3 +/- 2.6 vs. 8.5 +/- 4.5 cells per field, P<0.001). Patients with low intratumoral NK cells had shorter disease-free survival (P=0.027) and overall survival (P=0.005) than patients with high intratumoral NK cells. In contrast, NK cells in the peritumoral area showed no prognostic significance for either disease-free survival or overall survival. Multivariate Cox proportional hazards analysis showed that intratumoral NK cell density was an independent prognostic factor of prolonged overall survival (hazard ratio = 2.658, P=0.019). Low NK cells infiltration could predict poor prognosis in patients with HCC.

Tyrosine-phosphorylation of the 12th armadillo-repeat of β-catenin is associated with cadherin dysfunction in human cancer

Tyrosine phosphorylation of beta-catenin, an intracytoplasmic cadherin-binding protein, causes disruption of the cadherin-mediated cell adhesion system in cancer cells. We identified that the c-erbB-2 protein activated by TGFalpha was capable of binding to the 12th armadillo repeat (arm12) of beta-catenin with increased phosphorylation-state level. We produced a monoclonal antibody, 4G7, which recognizes a phosphorylated-tyrosine residue (Tyr-654) located at arm12 of beta-catenin. Tyrosine phosphorylation of the arm12 was detected after stimulation with TGFalpha in TMK-1. Immunoprecipitation analyses using 4G7, anti-beta-catenin, alpha-catenin, the c-erbB-2 gene product and phosphotyrosine antibodies indicated that tyrosine phosphorylation of the arm12 was closely correlated with dissociation between E-cadherin/beta-catenin and alpha-catenin or the c-erbB-2 gene product, but not with dissociation between beta-catenin and E-cadherin. Immunocytochemical staining of TMK-1 cells using the 4G7 antibody revealed that tyrosine phosphorylation of the arm12 was localized at cell-cell contact sites of cancer cells transiently and only after TGFalpha treatment. Immunohistochemical localization of the 4G7 antibody was observed in cancer cells at the invasive front where they detached from cancer nests. These findings indicated that the tyrosine phosphorylation of arm12 is a key marker of cadherin dysfunction and the 4G7 antibody may be useful in screening for a beta-catenin phosphorylation ligand or tyrosine kinases.

Endoscopic resection for T1a-MM and T1b-SM1 squamous cell carcinoma of the esophagus.

In Japan, the first paper on endoscopic resection (ER) for squamous cell carcinoma (SCC) of the esophagus confined to the mucosa was reported as endoscopic mucosal resection (EMR) in 1988. Since publication of that article, ER has been recommended as the standard treatment for squamous and mucosal cancer of the esophagus. T1a-EP and T1a-LPM esophageal cancer seldom involves lymph node metastasis. However, in cases of T1a-MM and T1b-SM1 esophageal cancer with lymph node metastasis (10% to 30%), the indication of ER is limited. The risk factors for lymph node metastasis in T1a-MM and T1b-SM1 esophageal cancer were cleared by clinical and pathological studies. Endoscopic findings such as type 0-I or type 0-III, size of 50mm or more, and pathological findings such as lymphatic permeation, venous permeation, poorly differentiated SCC and INFb or INFc were suggestive of high risk for lymph node metastasis. In addition, histopathological findings of small cancer nests, defined as "budding" or "droplet infiltration," suggest frequent lymph node metastasis. In cases of T1a-MM and T1b-SM1 esophageal cancer with high risk of lymph node metastasis, adjuvant therapy including chemoradiotherapy and radical esophagectomy are recommended after ER. A recent advance in ER for esophageal cancer is the establishment of endoscopic submucosal dissection (ESD). It has allowed us to perform an en-block resection of a large mucosal lesion of the esophagus and detailed histopathological examination. However, ESD requires more difficult manipulation than EMR. The indication of EMR or ESD is sought.

The role of mitochondrial DNA alterations in esophageal squamous cell carcinomas

The study objective was to evaluate the roles of mitochondrial DNA alterations in esophageal squamous cell carcinoma, with emphasis on the changes in the copy number and D310 variants of mitochondrial DNA. Paired samples microdissected from esophageal muscles, noncancerous esophageal mucosa, cancerous esophageal squamous cell carcinoma nests, and metastatic lymph nodes of 72 patients with esophageal squamous cell carcinoma were subjected to DNA extraction. The copy number and D310 variants of mitochondrial DNA were determined by quantitative real-time polymerase chain reaction and direct sequencing, respectively. Fifty-six patients (77.8%) with somatic D310 mutations had lower survival probability (P = .027). From noncancerous esophageal mucosa to cancerous esophageal squamous cell carcinoma nests and metastatic lymph nodes, the D310 variants were decreased from 2.2 to 1.7 and 1.5, respectively, with a trend to homoplasmy (P = .0009). Concurrently, the mitochondrial DNA copy number was increased from 0.159 to 0.192 and 0.206, respectively, (P = .024), especially in cigarette smokers (P = .014) and heavy wine drinkers (P = .005). Notably, a decrease in D310 variants (1.5, P < .001) and an increase in the incidence of the homoplasmic D310 pattern (P = .005) were observed in the matched esophageal muscle tissues. Among the 56 esophageal squamous cell carcinoma cancer nests with somatic D310 mutations, 51 (91.1%) had D310 variants in association with their corresponding noncancerous esophageal mucosa, including 36 (64.3%) fully related and 15 (26.8%) partially related pairs. We demonstrated that somatic D310 mutations and increase in the copy number of mitochondrial DNA are of clinical importance in esophageal squamous cell carcinoma. We also propose a model of DNA instability and clonal expansion during the carcinogenesis and progression of esophageal squamous cell carcinoma from the viewpoint of mitochondrial DNA transmission.

Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1

Macrophages (Mφ) are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. We have recently found that tumors can alter the normal developmental process of Mφ to trigger transient activation of monocytes in peritumoral stroma. We showed that a fraction of monocytes/Mφ in peritumoral stroma, but not in cancer nests, expresses surface PD-L1 (also termed B7-H1) molecules in tumors from patients with hepatocellular carcinoma (HCC). Monocytes activated by tumors strongly express PD-L1 proteins with kinetics similar to their activation status, and significant correlations were found between the levels of PD-L1+ and HLA-DRhigh on tumor-infiltrating monocytes. Autocrine tumor necrosis factor α and interleukin 10 released from activated monocytes stimulated monocyte expression of PD-L1. The PD-L1+ monocytes effectively suppressed tumor-specific T cell immunity and contributed to the growth of human tumors in vivo; the effect could be reversed by blocking PD-L1 on those monocytes. Moreover, we found that PD-L1 expression on tumor-infiltrating monocytes increased with disease progression, and the intensity of the protein was associated with high mortality and reduced survival in the HCC patients. Thus, expression of PD-L1 on activated monocytes/Mφ may represent a novel mechanism that links the proinflammatory response to immune tolerance in the tumor milieu.

Expression of dendritic cell phenotypic antigens in cervical lymph nodes of patients with hypopharyngeal and laryngeal carcinoma

The purpose of this study was to assess the presence of dendritic cell phenotypic antigens in the cervical lymph nodes of patients with hypopharyngeal and laryngeal carcinoma, and to assess the significance of such antigens in the tumour immune reaction. Immunohistochemical staining of cervical lymph nodes was performed using antibodies against cell surface markers such as S-100 protein and cluster of differentiation 1a and 83 glycoproteins. Two hundred and seventy-four cervical lymph nodes obtained at surgery from 37 patients with hypopharyngeal carcinoma and 31 patients with laryngeal carcinoma were thus evaluated. The number of dendritic cells positive for each phenotypic antigen was significantly greater in non-metastatic lymph nodes than in metastatic lymph nodes. In the metastatic lymph nodes, cluster of differentiation 1a glycoprotein positive dendritic cells were predominantly detected in the cancer 'nest', whereas mature dendritic cells staining for cluster of differentiation 83 glycoprotein were prominent in the peritumour area. In the metastatic lymph nodes, in contrast to the cluster of differentiation 1a glycoprotein positive dendritic cells, the degree of infiltration of cluster of differentiation 83 glycoprotein positive dendritic cells was significantly higher in the peritumour area than in the cancer nest. There was a significant difference in survival status, comparing patients with different degrees of dendritic cell infiltration for each type of phenotypic antigen. Dendritic cells may play different roles in tumour immunity against hypopharyngeal and laryngeal carcinoma. The phenotypic antigens of dendritic cells may thus constitute important indices with which to predict the prognosis of patients with hypopharyngeal and laryngeal carcinoma.

Proteomic profiling in pancreatic cancer with and without lymph node metastasis

The aim of the study was to observe different protein profiles in pancreatic cancer with and without lymph node metastasis (LNM), and search for novel LNM-associated proteins, which would help to understand the metastatic mechanisms and provide targets for therapeutic interventions. Cancer nests were manually miscrodissected from 8 LNM and 7 non-LNM pancreatic cancer tissues, and the protein extracts were then separated by difference gel electrophoresis (DIGE) and identified by MALDI-TOF-TOF. Four differently regulated proteins, ezrin, radixin, moesin, and c14orf166, were selected for further validation by Western blot and immunohistochemistry. In DIGE analysis, we identified 18 up-regulated proteins and 15 down-regulated proteins in LNM pancreatic cancer nests compared with non-LNM ones. Western blot and immunohistochemical analyses confirmed that radixin, moesin and c14orf166, but not ezrin, had significantly higher expression levels in LNM pancreatic cancers than in non-LNM controls. In conclusion, the specific protein profiles found in this study might provide new insights into the mechanism of lymph node metastasis. For the first time, c14orf166 was identified asa novel metastasis-associated protein, and the roles of radixin, moesin and c14orf166 in cancer metastasis deserve further investigations.