Antonio Mussa

IL-7 Up-Regulates TNF-α-Dependent Osteoclastogenesis in Patients Affected by Solid Tumor

Background Interleukin-7 (IL-7) is a potent regulator of lymphocyte development, which has also significant effects on bone; in fact it is a potent osteoclastogenic factor. Some human solid tumors produce high IL-7 levels, suggesting a potential IL-7 role on tumor development and progression. Methodology We studied 50 male patients affected by solid tumors, and their blood samples were collected at tumor diagnosis. PBMCs were isolated and cultured with/without IL-7 to study its influence on osteoclastogenesis. Serum and cell culture supernatant IL-7 levels were measured by ELISA. The quantitative analysis of IL-7 expression on T and B cells was performed by Real-Time PCR. Principal Findings Serum IL-7 levels were highest in osteolytic cancer patients, followed by cancer patients without bone lesions, and then healthy controls. We showed the IL-7 production in PBMC cultures and particularly in monocyte and B cell co-cultures. A quantitative analysis of IL-7 expression in T and B cells confirmed that B cells had a high IL-7 expression. In all cell culture conditions, IL-7 significantly increased osteoclastogenesis and an anti-IL-7 antibody inhibited it. We demonstrated that IL-7 supports OC formation by inducing the TNF-α production and low RANKL levels, which synergize in promoting osteoclastogenesis. Conclusions We demonstrated the presence of high serum IL-7 levels in patients with bone metastasis, suggesting the use of serum IL-7 level as a clinical marker of disease progression and of bone involvement. Moreover, we showed the capability of IL-7 to stimulate spontaneous osteoclastogenesis of bone metastatic patients and to induce osteoclastogenesis in cancer patients without bone involvement. These findings add further details to the disclosure of the mechanisms controlling bone metastasis in solid tumors.

Brain drain and health workforce distortions in Mozambique.

Introduction Trained human resources are fundamental for well-functioning health systems, and the lack of health workers undermines public sector capacity to meet population health needs. While external brain drain from low and middle-income countries is well described, there is little understanding of the degree of internal brain drain, and how increases in health sector funding through global health initiatives may contribute to the outflow of health workers from the public sector to donor agencies, non-governmental organisations (NGOs), and the private sector. Methods An observational study was conducted to estimate the degree of internal and external brain drain among Mozambican nationals qualifying from domestic and foreign medical schools between 1980–2006. Data were collected 26-months apart in 2008 and 2010, and included current employment status, employer, geographic location of employment, and main work duties. Results Of 723 qualifying physicians between 1980–2006, 95.9% (693) were working full-time, including 71.1% (493) as clinicians, 20.5% (142) as health system managers, and 6.9% (48) as researchers/professors. 25.5% (181) of the sample had left the public sector, of which 62.4% (113) continued working in-country and 37.6% (68) emigrated from Mozambique. Of those cases of internal migration, 66.4% (75) worked for NGOs, 21.2% (24) for donor agencies, and 12.4% (14) in the private sector. Annual incidence of physician migration was estimated to be 3.7%, predominately to work in the growing NGO sector. An estimated 36.3% (41/113) of internal migration cases had previously held senior-level management positions in the public sector. Discussion Internal migration is an important contributor to capital flight from the public sector, accounting for more cases of physician loss than external migration in Mozambique. Given the urgent need to strengthen public sector health systems, frank reflection by donors and NGOs is needed to assess how hiring practices may undermine the very systems they seek to strengthen.

Comparison of Positron Emission Tomography Scanning and Sentinel Node Biopsy in the Detection of Inguinal Node Metastases in Patients With Anal Cancer

BACKGROUND Inguinal lymph node metastases in patients with anal cancer are an independent prognostic factor for local failure and overall mortality. Inguinal lymph node status can be adequately assessed with sentinel node biopsy, and the radiotherapy strategy can subsequently be changed. We compared this technique vs. dedicated 18F-fluorodeoxyglucose positron emission tomography (PET) to determine which was the better tool for staging inguinal lymph nodes. METHODS AND MATERIALS In our department, 27 patients (9 men and 18 women) underwent both inguinal sentinel node biopsy and PET-CT. PET-CT was performed before treatment and then at 1 and 3 months after treatment. RESULTS PET-CT scans detected no inguinal metastases in 20 of 27 patients and metastases in the remaining 7. Histologic analysis of the sentinel lymph node detected metastases in only three patients (four PET-CT false positives). HIV status was not found to influence the results. None of the patients negative at sentinel node biopsy developed metastases during the follow-up period. PET-CT had a sensitivity of 100%, with a negative predictive value of 100%. Owing to the high number of false positives, PET-CT specificity was 83%, and positive predictive value was 43%. CONCLUSIONS In this series of patients with anal cancer, inguinal sentinel node biopsy was superior to PET-CT for staging inguinal lymph nodes.

Expression of Phosphatidylethanolamine N-Methyltransferase in Human Hepatocellular Carcinomas

Objective: Hepatic phosphatidylethanolamine is converted into phosphatidylcholine by the enzyme phosphatidylethanolamine N-methyltransferase (PEMT) when the dietary choline supply is inadequate. Our previous reports implicated PEMT in the regulation of hepatocyte growth and transformation. In the present study, we analyzed PEMT activity, Pempt gene status and its mRNA expression in 29 human hepatocellular carcinomas (HCC). Methods: The status of the Pempt gene and PEMT2 mRNA expression were evaluated with Southern and Northern blotting, respectively, in HCC and the noninvolved liver. PEMT activity was assessed by biochemical assay. Cell proliferation markers were defined by immunohistochemical or histoautoradiographic methods. Results: PEMT activity was lower in HCC than in the noninvolved liver and it was negligible in 62% of the tumors. No deletions or mutations of the Pempt gene were found and PEMT2 mRNA expression was absent or reduced in HCC compared with peritumoral liver tissue. PEMT2 mRNA expression was inversely related to tumor proliferation and to histologic grade. Patients whose HCC did not express PEMT2 mRNA showed poorer outcomes for cancer-related survival than those with PEMT2-positive HCC. Conclusions: The present findings suggest that (1) clones lacking PEMT2 expression may have been selected during liver tumorigenesis and progression, and (2) PEMT2 expression seems to be associated with clinical progression.

In vivo migration of labeled autologous natural killer cells to liver metastases in patients with colon carcinoma

BackgroundBesides being the effectors of native anti-tumor cytotoxicity, NK cells participate in T-lymphocyte responses by promoting the maturation of dendritic cells (DC). Adherent NK (A-NK) cells constitute a subset of IL-2-stimulated NK cells which show increased expression of integrins and the ability to adhere to solid surface and to migrate, infiltrate, and destroy cancer. A critical issue in therapy of metastatic disease is the optimization of NK cell migration to tumor tissues and their persistence therein. This study compares localization to liver metastases of autologous A-NK cells administered via the systemic (intravenous, i.v.) versus locoregional (intraarterial, i.a.) routes.Patients and methodsA-NK cells expanded ex-vivo with IL-2 and labeled with 111In-oxine were injected i.a. in the liver of three colon carcinoma patients. After 30 days, each patient had a new preparation of 111In-A-NK cells injected i.v. Migration of these cells to various organs was evaluated by SPET and their differential localization to normal and neoplastic liver was demonstrated after i.v. injection of 99mTc-phytate.ResultsA-NK cells expressed a donor-dependent CD56+CD16+CD3- (NK) or CD56+CD16+CD3+ (NKT) phenotype. When injected i.v., these cells localized to the lung before being visible in the spleen and liver. By contrast, localization of i.a. injected A-NK cells was virtually confined to the spleen and liver. Binding of A-NK cells to liver neoplastic tissues was observed only after i.a. injections.ConclusionThis unique study design demonstrates that A-NK cells adoptively transferred to the liver via the intraarterial route have preferential access and substantial accumulation to the tumor site.

Intra-arterial continuous infusion for treatment of pancreatic and biliary tract cancer

SummaryBackground. Systemic chemotherapy does not satisfactorily improve the poor prognosis of pancreas and biliary tract cancer unresectable or metastatic to the liver. Intra-arterial infusion of antineoplastic agents can give higher concentrations to the tumor and slighter concentrations to the whole body, with a potential of efficacy and lower toxicity, due to the hepatic clearance. Methods. Based on a safe and ambulatorial technique of transcutaneous arterial port implantation, this study was designed to evaluate feasibility and toxicity of 5-fluorouracil (5-FU) intra-arterial continuous infusion combined with systemic gemcitabine with dose escalation. Seventeen patients affected by pancreatic (14) or biliary tract (3) cancer received up to six cycles of treatment. Treatment consisted of intravenous gemcitabine on d 1 and 8 and intra-arterial 5-FU continuous infusion on d 1–14 every 21 d. Dose-escalation levels were 900 and 1000 mg/m2 for gemcitabine and 8, 10, 12, 15, and 17 mg/kg/d for 5-FU. Consecutive cohorts of three patients were planned at each dose level. Results. Gastrointestinal toxicity (vomiting and diarrhea [3rd–4th degree] and gastritis), constituted the dose-limiting toxicity, with a maximum-tolerated dose of 1000 mg/m2 for gemcitabine and 15 mg/kg/d for 5-FU. Hematological toxicity was present in a minority of patients. No patient had acute or later complications such as arterial thrombosis related to the implanted arterial port, sclerosis cholangitis, or chemical cholecistitis.Conclusion. 5-Fluorouracil intra-arterial continuous infusion, combined with systemic gemcitabine, seems to be a feasible and safe regimen that could give interesting results in pancreatic cancer.

YKL-40 expression in anal carcinoma predicts shorter overall and disease-free survival.

Sir: Anal cancer incidence has increased considerably in recent years, especially in human immunodeficiency virus (HIV)+ patients. Its treatment has changed over the past two decades, shifting from radical surgery toward sphincter-conserving treatment based on radiotherapy (RT) and chemotherapy (CM). Conservative approaches are highly effective, but treatment failure is still relatively common, with recent studies reporting a 5-year disease-free survival rate of 56%. Locally recurrent anal cancer reduces patient survival and seriously impairs quality of life, since RT may increase wound complications (e.g. dehiscence, infection and persistent anal sinus). Searching for a new, reliable marker of radioresistance for anal cancer, our attention was drawn to YKL40, a secreted protein whose function is still poorly understood. Physiologically, it may have a role in cell migration and connective tissue modelling 2–4 and be involved in inflammatory response. Elevated YKL-40 serum levels have been described in more aggressive colonic, ovarian and breast carcinomas and in squamous cell carcinoma of the head and neck. In glioblastomas, YKL-40 expression has also been found to be associated with radioresistance and poorer prognosis. These premises prompted us to investigate a possible prognostic and predictive role of YKL-40 immunohistochemical staining in anal cancer. We collected a series of 34 anal cancer biopsy specimens from a cohort of patients treated by the same protocol of CM–RT between January 2003 and December 2006, with a mean follow-up from the initial diagnosis of 30 months (median 24). At the time of diagnosis, one tumour was stage I (3%), 21 were stage II (61.6%), 12 were stage III (35.4%) and none was stage IV. Histologically, all tumours were squamous carcinomas, 10 of which were keratinizing, 13 nonkeratinizing and 11 basaloid carcinomas. Co-infection with HIV was detected in 8 ⁄ 34 cases, regardless of the tumour histological subtype. Loco-regional failure occurred in 12 ⁄ 34 (35.2%) patients, after a mean follow-up of 23.6 months (median 19.5 months): six patients underwent abdominoperianal resection, four of whom had died and two were alive with disease. No statistical differences were noticed in age, gender, histological type or tumour grade, HIV status or tumour stage among the patients who developed recurrences, probably due to the small number of cases studied. Immunohistochemical analysis with antibodies raised against Ki67, p53, p63, epidermal growth factor receptor and YKL-40 was performed in 31 of 34 cases. Higher levels of p53 nuclear stain were significantly associated with poorer CM–RT response and were found to influence diseasefree survival (P < 0.02). Overexpression of YKL-40 was noted in 15 cases and was an independent predictor of Cumulative proportion surviving (Kaplan-Meier) YKL-40 and OS

Sentinel lymph node and breast cancer staging: final results of the Turin Multicenter Study.

Aim of the study Validation of the sentinel node (SN) technique in breast cancer by means of lymphoscintigraphy. Materials and methods From December 1996 to January 1999 102 T1-T2 breast carcinoma cases were recruited in Turin. 99mTc-human serum albumin colloids were injected subdermally the day before surgery (mean activity, 5.2 ± 2.5 MBq). Scintigraphic imaging was performed after injection. After identification of the SN during surgery by a hand-held gamma probe, the SN was excised and sent for histologic examination. SN histology was compared with that of other axillary nodes. Results The SN detection rate was 86.3%; among 88 cases with an identified SN, 37 (42%) had axillary metastases; the SN was metastatic in 35 cases (sensitivity, 94.6%); in 51.3% of pN+ cases (19/37) the SN was the only metastatic site. In two of the 53 negative SNs, SN histology did not match with that of the remaining axilla (negative predictive value, 96.2%; staging accuracy, 97.7%). Conclusions Our results agree with those reported in the literature; however, except in clinical trials and experienced structures axillary lymph node dissection should not be abandoned when mandatory for prognostic purposes, considering that at present SN biopsy alone is not completely accurate for axillary staging, especially in the absence of an adequate learning period.

Low Foxp3 expression in negative sentinel lymph nodes is associated with node metastases in colorectal cancer

In a recent commentary, Sobhanu and Le Gouvello,1 take advantage of the account of Chaput et al 2 of a new population of T regulatory (Treg) lymphocytes (CD8+) to address the more general question of whether accumulation of Tregs (both CD8+ and conventional CD4+) must be considered a prognostic factor in colorectal cancer (CRC). Tregs (Foxp3+) play a pivotal role in maintaining immune system homeostasis through their ability to suppress immunological responses, including tumour immunity against tumour-associated antigens. In their interesting commentary, Sobhanu and Le Gouvello1 argue that the in vivo immunosuppressive effect of these cells in CRC still remains controversial. Actually, accordingly to the available data, we believe it reasonable to state that CD4+ Tregs do not contribute to CRC escape from host immunity. While earlier studies showed a higher density of tumour-infiltrating Tregs in advanced …

Added value of combined gene and protein expression of CK20 and CEA in non-macroscopically involved lymph nodes of colorectal cancer.

A methacarn fixation permits an approach that comprises multiple techniques. In this study the procedure is used to examine 100 mesenteric lymph nodes from patients with colon cancer by means of histology, immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR). The evaluated nodes are found to be grossly free of metastases. The combined expression of both messenger RNA (mRNA) and protein is investigated to validate the presence of structural (cytokeratin 20, or CK20) and tumor-specific (carcinoembrionic antigen, or CEA) markers. Histological analysis shows micrometastases on 4 nodes. IHC analysis identifies isolated (CK20 and CEA positive) tumor cells on 14 other nodes. In this group, none of the nodes that are positive for CK20 IHC express the related mRNA. RT-PCR confirms the CEA IHC positivity in 50% of the cases. The double CEA IHC/RT-PCR positivity would have up-staged 33% of the pN0 cases to pN1. This approach offers a technological framework for further studies that aim to validate the clinical significance of protein/mRNA expression of tumor markers in colorectal cancer sentinel lymph nodes.