Tumor-infiltrating CD8(+) T lymphocytes associated with clinical outcome in anal squamous cell carcinoma.

BACKGROUND: The prognostic significance of tumor-infiltrating CD8(+) T lymphocytes in anal squamous cell carcinoma (SCC) remains unclear. We designed the study to investigate the association between CD8(+) T cells and clinical prognosis among anal SCC patients. METHODS: The density of CD8(+) T cells was assessed by immunohistochemistry. The numbers of CD8(+) T cells were counted and their relationship with clinicopathological factors and survival was explored. RESULTS: A strong positive correlation was noted between intratumoral and peritumoral CD8(+) T cells (r = 0.77, P < 0.001). High intratumoral and peritumoral CD8(+) T cells was associated with well tumor differentiation, early-stage diagnosis, and better prognosis (P < 0.05). Better disease-free survival rates were demonstrated in patients with high CD8(+) T cell density in intratumoral nest (P = 0.01); peritumoral stroma (P = 0.004); and both in combination (P = 0.01). High peritumoral CD8(+) T cell was associated with overall survival (P = 0.025). In HIV-infected patients, high CD8(+) T cell density also had association with disease-free survival (P < 0.05). CONCLUSIONS: High tumor-infiltrating CD8(+) T cell density showed the potential to indicate a favorable effect on prognosis and survival for anal SCC patients.

Disruption of TAK1 in hepatocytes causes hepatic injury, inflammation, fibrosis, and carcinogenesis.

TGF-beta-activated kinase 1 (TAK1) is a MAP3K family member that activates NF-kappaB and JNK via Toll-like receptors and the receptors for IL-1, TNF-alpha, and TGF-beta. Because the TAK1 downstream molecules NF-kappaB and JNK have opposite effects on cell death and carcinogenesis, the role of TAK1 in the liver is unpredictable. To address this issue, we generated hepatocyte-specific Tak1-deficient (Tak1DeltaHEP) mice. The Tak1DeltaHEP mice displayed spontaneous hepatocyte death, compensatory proliferation, inflammatory cell infiltration, and perisinusoidal fibrosis at age 1 month. Older Tak1DeltaHEP mice developed multiple cancer nodules characterized by increased expression of fetal liver genes including alpha-fetoprotein. Cultures of primary hepatocytes deficient in Tak1 exhibited spontaneous cell death that was further increased in response to TNF-alpha. TNF-alpha increased caspase-3 activity but activated neither NF-kappaB nor JNK in Tak1-deficient hepatocytes. Genetic abrogation of TNF receptor type I (TNFRI) in Tak1DeltaHEP mice reduced liver damage, inflammation, and fibrosis compared with unmodified Tak1DeltaHEP mice. In conclusion, hepatocyte-specific deletion of TAK1 in mice resulted in spontaneous hepatocyte death, inflammation, fibrosis, and carcinogenesis that was partially mediated by TNFR signaling, indicating that TAK1 is an essential component for cellular homeostasis in the liver.

John Cunningham virus T-antigen expression in anal carcinoma.

BACKGROUND: Anal carcinoma is thought to be driven by human papillomavirus (HPV) infection through interrupting function of cell regulatory proteins such as p53 and pRb. John Cunningham virus (JCV) expresses a T-antigen that causes malignant transformation through development of aneuploidy and interaction with some of the same regulatory proteins as HPV. JCV T-antigen is present in brain, gastric, and colon malignancies, but has not been evaluated in anal cancers. The authors examined a cohort of anal cancers for JCV T-antigen and correlated this with clinicopathologic data. METHODS: Archived anal carcinomas were analyzed for JCV T-antigen expression. DNA from tumor and normal tissue was sequenced for JCV with viral copies determined by quantitative polymerase chain reaction and Southern blotting. HPV and microsatellite instability (MSI) status was correlated with JCV T-antigen expression. RESULTS: Of 21 cases of anal cancer (mean age 49 years, 38% female), 12 (57%) were in human immunodeficiency virus (HIV)-positive individuals. All 21 cancers expressed JCV T-antigen, including 9 HPV-negative specimens. More JCV copies were present in cancer versus surrounding normal tissue (mean 32.54 copies/µg DNA vs 2.98 copies/µg DNA, P = .0267). There was no correlation between disease stage and viral copies, nor between viral copies and HIV-positive or -negative status (28.7 vs 36.34 copies/µg DNA, respectively, P = .7804). In subset analysis, no association was found between JCV T-antigen expression and HPV or MSI status. CONCLUSIONS: Anal carcinomas uniformly express JCV T-antigen and contain more viral copies compared with surrounding normal tissue. JCV and its T-antigen oncogenic protein, presumably through interruption of cell regulatory proteins, may play a role in anal cancer pathogenesis.

Role and cellular source of nicotinamide adenine dinucleotide phosphate oxidase in hepatic fibrosis.

UNLABELLED: Reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate oxidase (NOX) is required for liver fibrosis. This study investigates the role of NOX in ROS production and the differential contribution of NOX from bone marrow (BM)-derived and non-BM-derived liver cells. Hepatic fibrosis was induced by bile duct ligation (BDL) for 21 days or by methionine-choline-deficient (MCD) diet for 10 weeks in wild-type (WT) mice and mice deficient in p47phox (p47phox knockout [KO]), a component of NOX. The p47phox KO chimeric mice were generated by the combination of liposomal clodronate injection, irradiation, and BM transplantation of p47phox KO BM into WT recipients and vice versa. Upon BDL, chimeric mice with p47phox KO BM-derived cells, including Kupffer cells, and WT endogenous liver cells showed a ∼25% reduction of fibrosis, whereas chimeric mice with WT BM-derived cells and p47phox KO endogenous liver cells, including hepatic stellate cells, showed a ∼60% reduction of fibrosis. In addition, p47phox KO compared to WT mice treated with an MCD diet showed no significant changes in steatosis and hepatocellular injury, but a ∼50% reduction in fibrosis. Cultured WT and p47phox KO hepatocytes treated with free fatty acids had a similar increase in lipid accumulation. Free fatty acids promoted a 1.5-fold increase in ROS production both in p47phox KO and in WT hepatocytes. CONCLUSION: NOX in both BM-derived and non-BM-derived cells contributes to liver fibrosis. NOX does not play a role in experimental steatosis and the generation of ROS in hepatocytes, but exerts a key role in fibrosis.

CCR2 promotes hepatic fibrosis in mice.

UNLABELLED: Chemokines and chemokine receptors contribute to the migration of hepatic stellate cells (HSCs) and Kupffer cells, two key cell types in fibrogenesis. Here, we investigate the role of CCR2, the receptor for monocyte chemoattractant protein (MCP)-1, MCP-2, and MCP-3, in hepatic fibrosis. Hepatic CCR2, MCP-1, MCP-2, and MCP-3 messenger RNA expression was increased after bile duct ligation (BDL). Both Kupffer cells and HSCs, but not hepatocytes, expressed CCR2. BDL- and CCl(4)-induced fibrosis was markedly reduced in CCR2(-/-) mice as assessed through collagen deposition, alpha-smooth muscle actin expression, and hepatic hydroxyproline content. We generated CCR2 chimeric mice by the combination of clodronate, irradiation, and bone marrow (BM) transplantation allowing full reconstitution of Kupffer cells, but not HSCs, with BM cells. Chimeric mice containing wild-type BM displayed increased macrophage recruitment, whereas chimeric mice containing CCR2(-/-) BM showed less macrophage recruitment at 5 days after BDL. Although CCR2 expressed in the BM enhanced macrophage recruitment in early phases of injury, CCR2 expression on resident liver cells including HSCs, but not on the BM, was required for fibrogenic responses in chronic fibrosis models. In vitro experiments demonstrated that HSCs deficient in CCR2(-/-) or its downstream mediator p47phox(-/-) did not display extracellular signal-regulated kinase and AKT phosphorylation, chemotaxis, or reactive oxygen species production in response to MCP-1, MCP-2, and MCP-3. CONCLUSION: Our results indicate that CCR2 promotes HSC chemotaxis and the development of hepatic fibrosis.

Gastric adenocarcinoma inducing portal hypertension: a rare presentation.

Advanced gastric cancer usually presents with symptoms due to direct extension into adjacent viscera, distant metastases from lymphatic or hematogenic dissemination and peritoneal seeding. However, portal hypertension as a presentation of metastatic gastric cancer is rare and usually seen in association with other malignancies, e.g. hepatocellular and pancreatic carcinoma. We report a case of signet ring adenocarcinoma of the stomach that presented with esophageal and duodenal varices and bleeding due to portal hypertensive gastropathy. Pagetoid spread of cancer cells likely caused early metastasis and the unusual presentation. We also discussed the pathophysiology of development of portal hypertension in association with malignancies.

Loss of histone variant macroH2A2 expression associates with progression of anal neoplasm.

AIMS: The macroH2A histone variants are epigenetic marks for inactivated chromatin. In this study, we examined the expression of macroH2A2 in anal neoplasm from anal intraepithelial neoplasia (AIN) to anal squamous cell carcinoma (SCC). METHODS: AIN and anal SCC samples were analysed for macroH2A2 expression, HIV and human papilloma virus (HPV). The association of macroH2A2 expression with clinical grade, disease recurrence, overall survival and viral involvement was determined. RESULTS: macroH2A2 was expressed in normal squamous tissue and lower grade AIN (I and II). Expression was lost in 38% of high-grade AIN (III) and 71% of anal SCC (p=0.002). Patients with AIN with macroH2A2-negative lesions showed earlier recurrence than those with macroH2A2-positive neoplasm (p=0.017). With anal SCC, macroH2A2 loss was more prevalent in the HPV-negative tumours. CONCLUSIONS: Loss of histone variant macroH2A2 expression is associated with the progression of anal neoplasm and can be used as a prognostic biomarker for high-grade AIN and SCC.

Influence of race on microsatellite instability and CD8+ T cell infiltration in colon cancer.

African American patients with colorectal cancer show higher mortality than their Caucasian counterparts. Biology might play a partial role, and prior studies suggest a higher prevalence for microsatellite instability (MSI) among cancers from African Americans, albeit patients with MSI cancers have improved survival over patients with non-MSI cancers, counter to the outcome observed for African American patients. CD8+ T cell infiltration of colon cancer is postively correlated with MSI tumors, and is also related to improved outcome. Here, we utilized a 503-person, population-based colon cancer cohort comprising 45% African Americans to determine, under blinded conditions from all epidemiological data, the prevalence of MSI and associated CD8+ T cell infiltration within the cancers. Among Caucasian cancers, 14% were MSI, whereas African American cancers demonstrated 7% MSI (P = 0.009). Clinically, MSI cancers between races were similar; among microsatellite stable cancers, African American patients were younger, female, and with proximal cancers. CD8+ T cells were higher in MSI cancers (88.0 vs 30.4/hpf, P<0.0001), but was not different between races. Utilizing this population-based cohort, African American cancers show half the MSI prevalence of Caucasians without change in CD8+ T cell infiltration which may contribute towards their higher mortality from colon cancer.

Detection of multiple human papillomavirus genotypes in anal carcinoma.

UNLABELLED: Infection with human papillomavirus (HPV) is a major risk factor for development of anal squamous cell carcinoma. Despite over 100 genotypes of the virus, HPV 16 and 18 are considered pathogenic as they are seen in the majority of cervical and anal cancers. We have employed a custom microarray to examine DNA for several HPV genotypes. We aimed to determine the accuracy of our microarray in anal cancer DNA for HPV genotypes compared to the DNA sequencing gold standard. METHODS: We utilized a sensitive microarray platform to classify 37 types of mucosal HPVs including 14 known high-risk and 23 low-risk types based on cervical cancer data. We utilized DNA from pathologically confirmed cases of anal squamous cell carcinoma. All samples underwent microarray HPV genotyping and PCR analysis. RESULTS: HPV was detected in 18/20 (90%) anal cancers. HPV genotypes 16 and 18 were present in the majority of specimens, with HPV 16 being the most common. Eighty percent of anal cancers had at least two HPV types. Ten percent of cases (2/20) tested negative using our microarray; DNA sequencing confirmed the lack of presence of HPV DNA in these samples. CONCLUSIONS: Microarray technology is an accurate way to screen for various genotypes of HPV in anal cancer, with 100% correlation with genomic DNA detection of HPV. The majority of anal cancers in our study associated with pathogenic HPV 16 and/or 18. Other HPV genotypes are present simultaneously with HPV 16 and 18, and might contribute to its pathogenesis.

Relationship of EMAST and microsatellite instability among patients with rectal cancer.

BACKGROUND: Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature identified in 60% of sporadic colon cancers and may be linked with heterogeneous expression of the DNA mismatch repair (MMR) protein hMSH3. Unlike microsatellite instability-high (MSI-H) in which hypermethylation of hMLH1 occurs followed by multiple susceptible gene mutations, EMAST may be associated with inflammation and subsequent relaxation of MMR function with the biological consequences not known. We evaluated the prevalence of EMAST and MSI in a population-based cohort of rectal cancers, as EMAST has not been previously determined in rectal cancers. METHODS: We analyzed 147 sporadic cases of rectal cancer using five tetranucleotide microsatellite markers and National-Cancer-Institute-recommended MSI (mononucleotide and dinucleotide) markers. EMAST and MSI determinations were made on analysis of DNA sequences of the polymerase chain reaction products and determined positive if at least two loci were found to have frame-shifted repeats upon comparison between normal and cancer samples from the same patient. We correlated EMAST data with race, gender, and tumor stage and examined the samples for lymphocyte infiltration. RESULTS: Among this cohort of patients with rectal cancer (mean age 62.2 ± 10.3 years, 36% female, 24% African American), 3/147 (2%) showed MSI (three males, two African American) and 49/147 (33%) demonstrated EMAST. Rectal tumors from African Americans were more likely to show EMAST than Caucasians (18/37, 49% vs. 27/104, 26%, p = 0.014) and were associated with advanced stage (18/29, 62% EMAST vs. 18/53, 37%, non-EMAST p = 0.02). There was no association between EMAST and gender. EMAST was more prevalent in rectal tumors that showed peri-tumoral infiltration compared to those without (30/49, 60% EMAST vs. 24/98, 25% non-EMAST, p = 0.0001). CONCLUSIONS: EMAST in rectal cancer is common and MSI is rare. EMAST is associated with African-American race and may be more commonly seen with metastatic disease. The etiology and consequences of EMAST are under investigation, but its association with immune cell infiltration suggests that inflammation may play a role for its development.

Influence of target gene mutations on survival, stage and histology in sporadic microsatellite unstable colon cancers.

High-frequency microsatellite unstable (MSI-H) colon tumors develop as a consequence of mutations at repetitive sequences in target genes. TGFBR2 and ACVR2, encoding TGFbeta superfamily receptors, and the proapoptotic gene BAX are frequent targets for frameshift mutation. We analyzed the effect of these mutations on survival and histology in 2 separate cohorts. Forty-eight MSI-H Dukes B2 colon tumors from a cohort of 172 patients had mutations in TGFBR2, BAX and ACVR2 correlated with patient survival. Further, 54 population-based MSI-H colon cancers of all stages from a cohort of 503 patients had mutations correlated with tumor stage, grade and size. Of 44 amplifiable MSI-H Dukes B2 tumors, 70% harbored TGFBR2, 63% BAX and only 4.5% ACVR2 mutations. While mutation alone did not influence survival, concomitant mutation of TGFBR2 and BAX was associated with an improved prognosis in Dukes B2 patients (p=0.05). ACVR2 mutations were more frequent in the second, population-based cohort (stage II: 32.5%, p<0.05). While no target gene mutation correlated with stage in this cohort, poor histological grade and large tumor volume were associated with mutant ACVR2, but not TGFBR2 or BAX mutations, and likely accounts for the lower prevalence of ACVR2 mutations in the first, well-differentiated Dukes B2 cohort. Because target gene mutations did not correlate with stage, they likely occur early in the pathogenesis of MSI-H cancers. Mutations in TGFBR2 and BAX may improve survival in MSI-H Dukes B2 patients, and mutations of ACVR2 may augment histological changes consistent with poor tumor grade that is characteristic of MSI-H colon cancers, and increase tumor size.

Use of 5-fluorouracil and survival in patients with microsatellite-unstable colorectal cancer.

BACKGROUND & AIMS: 5-Fluorouracil improves mortality in stage III colorectal cancer patients. In vitro studies suggest that microsatellite instability influences cell survival after 5-fluorouracil treatment. We investigated the survival influence of 5-fluorouracil in patients with microsatellite instability-high tumors. METHODS: We collected data and tumors on 204 consecutive stage II and III colorectal cancer patients from registries at the University of California and Veterans Administration hospitals in San Diego, California, from 1982 to 1999. Archival DNA was extracted, and microsatellite instability was assessed by National Cancer Institute-recommended markers. Cox proportional hazard modeling was used to determine survival associations for microsatellite instability and 5-fluorouracil treatment status. RESULTS: We identified 36 microsatellite instability-high (17.6%) and 168 non-microsatellite instability-high tumors (82.4%). Microsatellite instability-high tumors were significantly associated with proximal colon location, presence of mucin, and surrounding lymphoid reaction. Univariate and multivariate analyses showed no survival difference between microsatellite instability-high and non-microsatellite instability-high groups (hazard ratio, 1.04; P = 0.88). Dichotomized by use of 5-fluorouracil, there was increased risk of death in patients who received no adjuvant chemotherapy (hazard ratio, 2.02; P = 0.02). However, the benefit of 5-fluorouracil was different between microsatellite instability-high and non-microsatellite instability-high groups. Patients with non-microsatellite instability-high tumors who received 5-fluorouracil had better survival compared with patients who were not treated (P < 0.05). Conversely, patients with microsatellite instability-high tumors who were treated with 5-fluorouracil had no survival difference compared with patients without treatment (P = 0.52). CONCLUSIONS: There is improved survival in patients with non-microsatellite instability-high tumors after 5-fluorouracil-based chemotherapy that does not extend to patients with microsatellite instability-high tumors. The microsatellite instability status of a patient's colorectal cancer may indicate differences in 5-fluorouracil-based chemosensitivity; this is consistent with in vitro studies.