HtrA1, a potential predictor of response to cisplatin-based combination chemotherapy in gastric cancer.

AIMS: HtrA1 is a member of the HtrA (high-temperature requirement factor A) family of serine proteases. HtrA1 plays a protective role in various malignancies due to its tumour suppressive properties. The aim of this study was to determine HtrA1 expression as a predictor of chemoresponse in patients with advanced gastric cancer. METHODS AND RESULTS: HtrA1 expression was determined by immunohistochemistry on specimens of primary gastric cancer from 80 patients treated consecutively with cisplatin-based combination chemotherapy. Response to chemotherapy was assessed according to Response Evaluation Criteria in Solid Tumours (RECIST) criteria. Our population consisted of males/females [51/29; median age 64 years (range 32-82)]. A complete or partial response was observed in 71.4% [95% confidence interval (CI) 54.7-88.2], 66.7% (95% CI 47.8-85.5) and 28.6% (95 CI 11.8-45.3) of tumours showing high, medium and low HtrA1 expression, respectively. A statistically significant association between HtrA1 expression and the clinical response was observed (P = 0.002). The median overall survival for patients with high/medium expression was 17 months compared to 9.5 months for patients with low HtrA1 expression (P = 0.037). CONCLUSIONS: Identification of HtrA1 in gastric cancer prior to chemotherapy indicates that levels of HtrA1 could be used to predict response to platinum-based combination therapies. Further assessment of HtrA1 expression is highly warranted in large, prospective studies.

Prognostic role of human equilibrative transporter 1 (hENT1) in patients with resected gastric cancer.

Nucleoside transporter proteins are specialized proteins that mediate the transport of nucleosides and nucleoside analog drugs across the plasma membrane. The human equilibrative nucleoside transporter 1 (hENT1) is a member of these proteins and mediates cellular entry of gemcitabine, cytarabine, and fludarabine. The hENT1 expression has been demonstrated to be related with prognosis and activity of gemcitabine-based therapy in breast, ampullary, lung, and pancreatic cancer. We investigated the immunohistochemical expression of hENT in tumor samples from 111 patients with resected gastric adenocarcinoma, correlating these data with clinical parameters and disease outcomes. None of the patients received chemotherapy or radiation therapy before or after surgery as a part of an adjuvant or neoadjuvant program. On univariate survival analysis, the hENT1 expression was associated with overall survival (OS) and disease free survival (DFS). Specifically, those patients with overexpression of hENT1 showed a shorter OS (P = 0.021) and a shorter DFS (P = 0.033). Considering only the node positive patients, higher hENT levels were associated with significantly shorter median DFS (21.7 months; 95% CI 11.1-32.4) compared with patients with low expression of hENT1. The hENT1 expression was defined, in the lymph-node positive patients, as an independent prognostic factor (P = 0.019). Furthermore, considering only patients with diffuse or mixed tumors and lymph-node positive, the expression of hENT1 was strongly related with DFS and OS. Immunohistochemistry for the hENT1 protein carries prognostic information in patients with resected gastric cancer and holds promise as a predictive factor in chemotherapy decisions.

Intratumoural FOXP3-positive regulatory T cells are associated with adverse prognosis in radically resected gastric cancer.

We investigated the clinical significance of tumour-infiltrating FOXP3-positive regulatory T cells (Tregs) in radically resected (R0) gastric cancer. From a single-institution database, tumors of 110 patients who underwent R0 resection for stage II-III disease were studied for FOXP3-positive Tregs by immunohistochemistry. The observed median number of FOXP3-positive Tregs was used as the cut-point in analyses (<6 versus >or=6 count). Tregs were significantly higher in gastric carcinomas than in normal tissue (P = 0.0001). Tregs count >or=6 was significantly associated with vascular/lymphatic/perineural invasion (VELIPI) in the tumour (P = 0.03). Multivariate analysis showed association between adverse relapse-free survival and grading 3, stage III, VELIPI and Tregs count >or=6 (P = 0.02). Adverse overall survival was associated with grading 3, stage III, VELIPI and Tregs count >or=6 (P = 0.006). FOXP3-positive Tregs may be a novel marker for identifying high-risk gastric cancer patients. Present findings deserve additional investigation as Tregs may also represent an innovative therapeutic target.

Immunohistochemical study of dendritic cells in foetal skin and lymph-nodes supporting the hypothesis for the neural crest origin of Langerhans cells.

An immunohistochemical study on foetal skin and lymph-nodes was undertaken in 35 foetuses with the aim to obtain some evidence supporting the hypothesis for the neural crest origin of the Langerhans cells (LCs).Specimens from the axillary and inguinal areas including the skin, subcutaneous tissues and lymph-nodes have been examined. For immunohistochemistry a panel of antibodies were used such as CD1, Langerin, Melan A, HMB45, S100 protein, CD21, subsets for B and T lymphocytes, CD68, Myeloperoxidase, CD31 and CD34. Immunohistochemistry using Melan-A or HMB45 resulted on the detection of melanocytes in the basal layers of epidermis during the first months of gestation. At the same time CD1 allowed the identification of LCs as scattered starry cells in the basal and supra-basal of epidermis layers. Similarly, in the subcutaneous lymph-nodes, LCs could be demonstrated already from the 4th month of foetal life, likely in relation to their known migratory and circulating ability (veiled cells). Immunohistochemical results demonstrated that LCs are intimately correlated to melanocytes during the foetal life, they and both become apparent in the same gestational time and similarly increase in number during the last months of foetal life. The neural crest origin for melanocytes has been universally accepted; the same site origin for Langerhans cells and their biological meaning are discussed. Probably LCs, which can be observed in embryonic tissues of ovary teratomas, in the epidermis of foetal skin and lymph-nodes, and from literature reported in the epidermis of all living beings, represent ancestral cells related to the T lymphocyte compartment acting as a primary defence mechanism against invading micro-organisms of the natural outer habitat.

The relationship of Langerhans cells to melanocytes and Schwann cells in mature cystic teratomas of the ovary.

Mature cystic teratomas have been widely studied relative to their tissue components derived from all 3 embryonic layers, and immunohistochemical methods have demonstrated a variety of neurohormonal polypeptides. To our knowledge, Langerhans cells (LCs), which are a peculiar component of epidermis, have not been reported in ovarian teratomas. The origin of these cells is still a matter of debate, ranging from bone marrow stem cells to neural elements. Thirty mature teratomas of the ovary were studied by immunohistochemistry using CD1 (specific against dendritic LCs), S100 protein (against LCs and melanocytes), and melan-A and HMB45 (against melanocytes). Furthermore, antibodies for identifying subsets of lymphocytes and monocytes (CD3, CD4, CD8, CD20, and CD68) were used. Histologic examination showed teratomas with the presence of all 3 embryonic layers in variable proportions in 23 cases, while 7 teratomas were composed only of epidermis without appendages or other tissues. Immunohistochemistry identified LCs among the suprabasal layers of epidermis in the same sites at which melanocytes were seen in the basal layer. CD1-positive LCs sometimes appeared to cross the basal membrane and penetrate the subepidermal tissue (related to their known migratory ability), and they were associated there with T-cell line lymphocytes (CD3 positive). These findings were commonly observed in teratomas that included all 3 embryonic layers and neural tissues. Notably, LCs and melanocytes were undetectable in the 7 teratomas composed of epidermis only. Our observations of LCs in ovarian teratomas led us to consider these cells to be derived from neural cells, possibly related to Schwann cells, in accord with the original description by Langerhans. In fact, LCs are always associated with melanocytes, which are universally considered to be derived from the neural crest, as are Schwann cells and peripheral nerves. Finally, we propose that LCs may be part of a cytoimmunologic system related to the T-cell compartment, with a stem cell derived from the neural crest.

Serine protease HtrA1 modulates chemotherapy-induced cytotoxicity.

Resistance to chemotherapy presents a serious challenge in the successful treatment of various cancers and is mainly responsible for mortality associated with disseminated cancers. Here we show that expression of HtrA1, which is frequently downregulated in ovarian cancer, influences tumor response to chemotherapy by modulating chemotherapy-induced cytotoxicity. Downregulation of HtrA1 attenuated cisplatin- and paclitaxel-induced cytotoxicity, while forced expression of HtrA1 enhanced cisplatin- and paclitaxel-induced cytotoxicity. HtrA1 expression was upregulated by both cisplatin and paclitaxel treatment. This upregulation resulted in limited autoproteolysis and activation of HtrA1. Active HtrA1 induces cell death in a serine protease-dependent manner. The potential role of HtrA1 as a predictive factor of clinical response to chemotherapy was assessed in both ovarian and gastric cancer patients receiving cisplatin-based regimens. Patients with ovarian or gastric tumors expressing higher levels of HtrA1 showed a higher response rate compared with those with lower levels of HtrA1 expression. These findings uncover what we believe to be a novel pathway by which serine protease HtrA1 mediates paclitaxel- and cisplatin-induced cytotoxicity and suggest that loss of HtrA1 in ovarian and gastric cancers may contribute to in vivo chemoresistance.

[Argyria: new physio-pathological and therapeutic hypothesis]. / Argirìa: nuove ipotesi fisio-patologiche e terapeutiche.

The authors describe a case of sistemic argyria. They suggest a common physiopatologic mechanism with Wilson disease and they consider the utilization of penicillamine as a ligand for the silver, since there are not other available valid treatments.

Prognostic role of thymidylate synthase polymorphisms in gastric cancer patients treated with surgery and adjuvant chemotherapy.

PURPOSE: To investigate the prognostic role of thymidylate synthase (TS) polymorphisms in gastric cancer patients treated with radical surgery and fluorouracil-based adjuvant chemotherapy. EXPERIMENTAL DESIGN: Ninety gastric cancer cases were identified among 187 patients previously enrolled in prospective case-control studies for disease susceptibility. Patients were genotyped for a G/C nucleotide change within a triple 28 bp variable number of tandem repeat sequence in the TS 5'-untranslated region (5'-UTR) and a 6 bp deletion in the TS 3'-untranslated region (3'-UTR). According to available functional data, patients with 5'-UTR 2R/2R, 2R/3C, 3C/3C genotypes were classified as low TS producers (5'-UTRlow) and patients with 5'-UTR 3G/3G, 3G/3C, 2R/3G genotypes as high TS producers (5'UTRhigh). Patients with 3'-UTR del6/del6 and del6/ins6 genotypes were classified as low TS producers (3'-UTRlow) and patients with 3'-UTR ins6/ins6 genotype as high TS producers (3'-UTRhigh). The prognostic analysis was based on 5'-UTR/3'-UTR combined genotypes. RESULTS: Ten patients (11%) were 5'-UTRhigh/3'-UTRhigh, 36 patients were 5'-UTRhigh/3'-UTRlow, 19 patients were 5'-UTRlow/3'-UTRhigh, and 25 patients were 5'-UTRlow/3'-UTRlow. 5'-UTRlow/3'-UTRlow patients showed the best outcome and the threshold of statistical significance was achieved in the comparison of disease-free survival and overall survival with 5'-UTRhigh/3'-UTRlow patients and 5'-UTRhigh/3'-UTRhigh patients. The presence of at least one high TS expression genotype showed independent adverse prognostic role in multivariate analysis. CONCLUSIONS: The prognostic role of TS polymorphisms in gastric cancer deserves further investigation because the adverse effect of high TS expression genotypes may be a relevant information to improve adjuvant chemotherapeutic strategies.

Predictive and prognostic role of E-cadherin protein expression in patients with advanced gastric carcinomas treated with palliative chemotherapy.

Loss of E-cadherin expression has been related with an adverse outcome in patients with resected gastric cancer. More recently, experimental models with cancer cell lines showed that chemosensitivity may be affected by the E-cadherin expression status. We investigated whether E-cadherin expression is correlated with the response to chemotherapy and the survival of patients with advanced gastric cancer. Consecutive patients with advanced gastric cancer who underwent palliative chemotherapy were considered eligible for study entry. Measurable disease, complete follow-up information and availability of tumor specimens for immunohistochemistry were mandatory inclusion criteria. In 70 assessable patients, 30 patients had locoregional disease and 40 patients had visceral metastases. Chemotherapy consisted of cisplatin/fluorouracil/folinic acid in 33 patients and cisplatin/fluorouracil/epirubicin/folinic acid in 37 patients. There were 13 patients with complete response, 20 with partial response, 20 with stable disease and 17 patients progressed. Thirty-eight patients had > 80% E-cadherin-positive cancer cells (positive E-cadherin expression); 15 cases showed 25-70% (reduced E-cadherin expression), and in the remaining 17 cases E-cadherin expression was < 10% (negative E-cadherin expression). The response to chemotherapy was unrelated to the E-cadherin expression status. Conversely, survival in the 32 patients with reduced/negative E-cadherin expression (25 weeks) was significantly worse than that observed in the 38 patients with preserved E-cadherin expression (36 weeks) (p < 0.01). E-cadherin expression retained its independent prognostic role in the multivariate analysis. E-cadherin expression may give prognostic information in patients with advanced gastric cancer, but it does not seem to possess a predictive role in vivo. Some of the mechanisms inducing E-cadherin downregulation, like hypermethylation, may be potentially reversible, and they deserve further investigation as the target of novel therapeutic strategies.

Prognostic relevance of histological grade and its components in node-negative breast cancer patients.

Available results highlight the lack of good level of evidence studies on the pure prognostic value of histological grade. In the present study, the prognostic relevance of histological grade and of its three components, tubule formation, nuclear pleomorphism and mitotic count, was analyzed in a series of 372 patients with node-negative breast cancer treated with locoregional therapy alone until early relapse. Histological grade was determined blindly by two observers and discordance between evaluations was resolved after joint review using a multihead microscope. No relation was observed between histological grade and any of its three components and disease-free survival. Conversely, a significant relation was observed between histological grade and distant metastasis-free survival (at 6 years, 94, 86 and 76% for grades 1, 2 and 3, respectively, P=0.013) as well as overall survival (98, 90 and 86%, P=0.001). A breakdown analysis as a function of the three components showed that neither tubule formation nor nuclear pleomorphism was associated with prognosis, and only mitotic count strongly influenced both distant metastasis-free survival (91, 82 and 74%, P=0.014) and overall survival (97, 87 and 85%, P=0.011). Histological grade suffers from a much higher subjectivity than any other microscopic evaluation of biomarkers as it is the sum of three different morphological features. Within the Italian Network for Quality Assessment of Tumor Biomarkers program we observed that histological grade is an independent prognostic variable, but also that this role is ascribable only to the number of mitotic figures. In conclusion, due to the ever smaller size of diagnosed breast cancers, resulting in less cancer tissue for biofunctional and molecular analysis, mitotic count evaluated under strict quality control conditions seems to be an accurate and feasible prognostic variable.

Prognostic analysis of E-cadherin gene promoter hypermethylation in patients with surgically resected, node-positive, diffuse gastric cancer.

PURPOSE: Recent investigations have demonstrated that hypermethylation is a frequent mechanism for silencing tumor suppressor genes. This is a potentially reversible epigenetic change, and it is the target of a novel class of anticancer compounds with demethylating activity. Better understanding of the clinical implications of hypermethylation will allow the optimal planning of future trials with demethylating drugs. In this perspective, we investigated whether hypermethylation in the CDH1 promoter region is correlated with poor prognosis of patients with surgically resected, node-positive, diffuse gastric cancer. EXPERIMENTAL DESIGN: Consecutive cases of diffuse gastric cancer were considered eligible for study entry. Additional inclusion criteria were radical surgery with a minimum of D1 lymphadenectomy, complete follow-up information, and availability of tumor specimens for methylation-specific PCR and immunohistochemistry analyses. RESULTS: CDH1 promoter hypermethylation was found in 40 of 73 cases (54%), and it was significantly associated with worse prognosis. In patients with and without hypermethylation, the 5-year event-free survival rate was 30% and 62%, respectively, and the 5-year overall survival rate was 35% and 67%, respectively. CDH1 promoter hypermethylation retained its prognostic role for disease-free survival (P < 0.001) and overall survival (P < 0.001) in multivariate analysis. Immunohistochemistry showed a significant association between CDH1 methylation and E-cadherin expression (P < 0.001). CONCLUSIONS: This study shows adverse prognostic effect of CDH1 promoter hypermethylation in patients with diffuse gastric cancer. This form of cancer, and other types with frequent hypermethylation and silencing of critical tumor suppressor genes, would make appropriate targets for the testing of novel compounds with demethylating activity.

Vascular endothelial growth factor and p53 expressions in liver and abdominal metastases from colon cancer.

OBJECTIVE: To determine the relationship between p53 overexpression and vascular endothelial growth factor (VEGF) upregulation in liver and abdominal metastases from colon cancer. The analysis in the two metastatic sites was carried out to evaluate the potential role of microenvironment in the molecular regulation of VEGF. METHODS: Bioptic specimens of liver and abdominal metastases from colon carcinomas were examined by immunohistochemistry for p53 and VEGF expressions. Consecutive cases with assessable tumor tissue were selected. RESULTS: The study population consisted of 24 cases having liver metastases and 34 cases having abdominal metastases. Abdominal metastases showed a higher number of VEGF-positive cases and a higher intensity of VEGF immunoreactivity than liver metastases did (p = 0.01). The combined analysis of p53 and VEGF showed a strong association between the two markers in the 24 liver metastases; 9 cases were VEGF positive/p53 positive and 15 cases were VEGF negative/p53 negative. This relationship was not found in the 34 abdominal metastases, which showed concordance between the two markers in 9 VEGF-positive/p53-positive cases only. CONCLUSIONS: Microenvironment factors like hypoxia may have a predominant role in inducing VEGF expression and they can override the molecular control of p53 on VEGF.

Effects of iron overload and hepatitis C virus positivity in determining progression of liver fibrosis in thalassemia following bone marrow transplantation.

To identify the role of iron overload in the natural history of liver fibrosis, we reviewed serial hepatic biopsy specimens taken annually from patients cured of thalassemia major by bone marrow transplantation. The patients underwent transplantation between 1983 and 1989 and did not receive any chelation or antiviral therapy. Two hundred eleven patients (mean age, 8.7 +/- 4 years) were evaluated for a median follow-up of 64 months (interquartile range, 43-98 months) by a median number of 5 (interquartile range, 3-6) biopsy samples per patient. Hepatic iron concentration was stratified by tertiles (lower, 0.5-5.6 mg/g; medium, 5.7-12.7 mg/g; upper, 12.8-40.6 mg/g dry weight). Forty-six (22%) patients showed signs of liver fibrosis progression; the median time to progression was 51 months (interquartile range, 36-83 months). In a multivariate Cox proportional hazard model, the risk for fibrosis progression correlated to medium hepatic iron content (hazard rate, 1.9; 95% confidence interval [CI], 0.74-5.0), high hepatic iron content (hazard rate, 8.7; 95% CI, 3.6-21.0) and hepatitis C virus (HCV) infection (hazard rate, 3.1; 95% CI, 1.5-6.5). A striking increase in the risk for progression was found in the presence of both risk factors. None of the HCV-negative patients with hepatic iron content lower than 16 mg/g dry weight showed fibrosis progression, whereas all the HCV-positive patients with hepatic iron concentration greater than 22 mg/g dry weight had fibrosis progression in a minimum follow-up of 4 years. Thus, iron overload and HCV infection are independent risk factors for liver fibrosis progression, and their concomitant presence results in a striking increase in risk.

Reversibility of cirrhosis in patients cured of thalassemia by bone marrow transplantation.

BACKGROUND: Cirrhosis is a well-known complication of thalassemia major. In this context, it is a consequence of iron overload and hepatitis C virus infection and generally seems to be irreversible. OBJECTIVE: To determine whether cirrhosis in thalassemia major can be reversible. DESIGN: Retrospective study. SETTING: Bone Marrow Transplantation Unit and Pathology Service, Pesaro Hospital, Pesaro, Italy. PATIENTS: 6 patients who developed liver cirrhosis before or after their thalassemia was cured by bone marrow transplantation (age at transplantation, 11 to 25 years). After diagnosis of cirrhosis, the patients received iron depletion and antiviral therapies. MEASUREMENTS: Each liver biopsy specimen was coded. A liver pathologist and a member of the transplantation center with expertise in hepatopathology graded the specimens by using the Ishak staging and grading systems. Neither knew the patient's identity or the sequence of biopsy with regard to the time of treatment. RESULTS: After the patients received iron depletion and antiviral therapies, liver biopsy specimens showed impressive reduction in liver iron stores. In 4 patients, iron removal was complete. Serum aminotransferase levels decreased in all patients and normalized in 5; histologic inflammatory activity decreased in all patients and disappeared in 2. Follow-up biopsies showed regression of incomplete or definite cirrhosis in all patients; 3 patients had presented with portal fibrosis without bridging, and the others had portal fibrosis and portal-to-portal bridging. Several biopsies and the presence of many portal spaces confirmed the diagnosis of cirrhosis; follow-up biopsies confirmed regression of cirrhosis. CONCLUSION: In some patients in whom bone marrow transplantation has cured thalassemia, cirrhosis may be reversible after iron removal treatment.

Color ultrasound-guided fine needle cutting biopsy for the characterization of diffuse liver damage in critical bone marrow transplanted patients.

BACKGROUND AND OBJECTIVES: The optimal method for liver biopsy in patients with simultaneous bone marrow and liver impairment has not yet been established. New approaches (e.g. imaging-guided methods) for this procedure are needed. In spite of coagulopathy, immunosuppression, anemia or ascites, we histologically characterized liver damage in a series of bone marrow transplanted patients using color-Doppler ultrasonography, which permits very keen visualization (and assessment) of hepatic parenchyma and vessels, and a fine needle for percutaneous biopsy. DESIGN AND METHODS: We performed percutaneous liver biopsy using a Menghini-type automatic very fine cutting needle (1.2 mm, 18G) under color ultrasound guidance in 16 bone marrow transplanted adult patients consecutively seen in our units from 1998 to 2001. The patients had clinically defined diffuse serious liver damage; liver biopsy was performed between 3 and 10 months after allogeneic (n= 11) or autologous (n= 5) transplantation. RESULTS: Fifteen patients tolerated the procedure well and had no discomfort, while one patient developed intrahepatic hemorrhage. All liver biopsies were suitable for histologic examination and informative, revealing the specific etiology of liver damage: graft-versus-host disease in six patients, drug toxicity in five, hepatitis C virus acute reactivation in two, and in one each vanishing bile duct syndrome, nodular regenerative hyperplasia and hemochromatosis. Biopsy detected potentially injurious concomitant factors, e.g., occult intrahepatic hepatitis B virus infection and reactivation. Histology radically changed the presumptive clinical diagnosis in 10 of the 16 patients and led to successful treatment changes in six. INTERPRETATION AND CONCLUSIONS: Percutaneous biopsy with a small cutting needle under color ultrasound guidance carries a low risk of complications and provides reliable information regarding liver histology in critically ill patients, in the early stage after bone marrow transplantation. We suggest including this imaging-guided mini-invasive procedure to the standard work-up of post-transplant liver damage.