Experimental evidence on promotion of electric and improved biomass cookstoves.

Improved cookstoves (ICS) can deliver "triple wins" by improving household health, local environments, and global climate. Yet their potential is in doubt because of low and slow diffusion, likely because of constraints imposed by differences in culture, geography, institutions, and missing markets. We offer insights about this challenge based on a multiyear, multiphase study with nearly 1,000 households in the Indian Himalayas. In phase I, we combined desk reviews, simulations, and focus groups to diagnose barriers to ICS adoption. In phase II, we implemented a set of pilots to simulate a mature market and designed an intervention that upgraded the supply chain (combining marketing and home delivery), provided rebates and financing to lower income and liquidity constraints, and allowed households a choice among ICS. In phase III, we used findings from these pilots to implement a field experiment to rigorously test whether this combination of upgraded supply and demand promotion stimulates adoption. The experiment showed that, compared with zero purchase in control villages, over half of intervention households bought an ICS, although demand was highly price-sensitive. Demand was at least twice as high for electric stoves relative to biomass ICS. Even among households that received a negligible price discount, the upgraded supply chain alone induced a 28 percentage-point increase in ICS ownership. Although the bundled intervention is resource-intensive, the full costs are lower than the social benefits of ICS promotion. Our findings suggest that market analysis, robust supply chains, and price discounts are critical for ICS diffusion.

Factors associated with HIV-testing and acceptance of an offer of home-based testing by men in rural Zambia.

The objective of this study is to describe HIV-testing among men in rural Lusaka Province, Zambia, using a population-based survey for a cluster-randomized trial. Households (N = 120) were randomly selected from each of the 42 clusters, defined as a health facility catchment area. Individuals aged 15-60 years were invited to complete questionnaires regarding demographics and HIV-testing history. Men testing in the last year were defined as recent-testers. After questionnaire completion adults were offered home-based rapid HIV-testing. Of the 2,828 men, 53 % reported ever-testing and 25 % recently-testing. Factors independently associated with ever- and recent-testing included age 20+ years, secondary/higher education, being married or widowed, a history of TB-treatment and higher socioeconomic position. 53 % of never-testers and 57 % of men who did not report a recent-test accepted home-based HIV-testing. Current HIV-testing approaches are inadequate in this high prevalence setting. Alternative strategies, including self-testing, mobile- or workplace-testing, may be required to complement facility-based services.

Frequency of HIV-testing and factors associated with multiple lifetime HIV-testing among a rural population of Zambian men.

BACKGROUND: Across sub-Saharan Africa, men's levels of HIV-testing remain inadequate relative to women's. Men are less likely to access anti-retroviral therapy and experience higher levels of morbidity and mortality once initiated on treatment. More frequent HIV-testing by men at continued risk of HIV-infection is required to facilitate earlier diagnosis. This study explored the frequency of HIV-testing among a rural population of men and the factors associated with more frequent HIV-testing. METHODS: We conducted a secondary analysis of a population-based survey in three rural district in Zambia, from February-November, 2013. Households (N = 300) in randomly selected squares from 42 study sites, defined as a health facility and its catchment area, were invited to participate. Individuals in eligible households were invited to complete questionnaires regarding demographics and HIV-testing behaviours. Men were defined as multiple HIV-testers if they reported more than one lifetime test. Upon questionnaire completion, individuals were offered rapid home-based HIV-testing. RESULTS: Of the 2376 men, more than half (61%) reported having ever-tested for HIV. The median number of lifetime tests was 2 (interquartile range = 1-3). Just over half (n = 834; 57%) of ever-testers were defined as multiple-testers. Relative to never-testers, multiple-testers had higher levels of education and were more likely to report an occupation. Among the 719 men linked to a spouse, multiple-testing was higher among men whose spouse reported ever-testing (adjusted prevalence ratio = 3.02 95% CI: 1.37-4.66). Multiple-testing was higher in study sites where anti-retroviral therapy was available at the health facility on the day of a health facility audit. Among ever-testers, education and occupation were positively associated with multiple-testing relative to reporting one lifetime HIV-test. Almost half (49%) of ever-testers accepted the offer of home-based HIV-testing. DISCUSSION: Reported HIV-testing increased among this population of men since a 2011/12 survey. Yet, only 35% of all men reported multiple lifetime HIV-tests. The factors associated with multiple HIV-testing were similar to factors associated with ever-testing for HIV. Men living with HIV were less likely to report multiple HIV-tests and employment and education were associated with multiple-testing. The offer of home-based HIV-testing increased the frequency of HIV-testing among men. CONCLUSION: Although men's levels of ever-testing for HIV have increased, strategies need to increase the lifetime frequency of HIV-testing among men at continued risk of HIV-infection.

Treatment outcomes of multidrug-resistant tuberculosis patients in Gauteng, South Africa.

PURPOSE: Multidrug-resistant tuberculosis (MDR-TB) is associated with lengthy treatment, expensive and potentially toxic regimens, and high rates of treatment failure and death. This study describes the outcomes of 351 MDR-TB patients who started treatment between 2004 and 2007 at the provincial MDR-TB referral hospital in Johannesburg, South Africa, and investigates risk factors associated with death. METHODS: The study involved the assessment of factors associated with treatment outcomes using a retrospective review of patient records, drug-susceptibility data and spoligotyping of isolates. RESULTS: Treatment success (completion/cure) was recorded in 158 (48.8 %) patients, while 65 (20 %) died, 93 (28.7 %) defaulted, 8 (2.5 %) failed treatment, 11(3.1 %) were transferred out to other health facilities and 16 (4.6 %) had no recorded final outcome. The proportion of successful treatment increased significantly over time. Univariable and multivariable analysis (P = 0.05) identified the year of MDR-TB diagnosis and spoligotype-defined families as factors associated with treatment outcome. No associations were found between treatment outcome and human immunodeficiency virus (HIV) status, previous TB and additional MDR resistance to streptomycin or ethambutol. Molecular typing of the strains revealed a diverse group of spoligotypes, with Beijing, LAM4 and H3 making up the largest groups. CONCLUSIONS: This is the first published study to investigate treatment outcomes at this facility and to find a link between genotype and treatment outcome, suggesting that genotype determination could potentially serve as a prognostic factor.

Heteroclitic immunization induces tumor immunity.

In tumor transplantation models in mice, cytotoxic T lymphocytes (CTLs) are typically the primary effector cells. CTLs recognize major histocompatibility complex (MHC) class I-associated peptides expressed by tumors, leading to tumor rejection. Peptides presented by cancer cells can originate from viral proteins, normal self-proteins regulated during differentiation, or altered proteins derived from genetic alterations. However, many tumor peptides recognized by CTLs are poor immunogens, unable to induce activation and differentiation of effector CTLs. We used MHC binding motifs and the knowledge of class I:peptide:TCR structure to design heteroclitic CTL vaccines that exploit the expression of poorly immunogenic tumor peptides. The in vivo potency of this approach was demonstrated using viral and self-(differentiation) antigens as models. First, a synthetic variant of a viral antigen was expressed as a tumor antigen, and heteroclitic immunization with peptides and DNA was used to protect against tumor challenge and elicit regression of 3-d tumors. Second, a peptide from a relevant self-antigen of the tyrosinase family expressed by melanoma cells was used to design a heteroclitic peptide vaccine that successfully induced tumor protection. These results establish the in vivo applicability of heteroclitic immunization against tumors, including immunity to poorly immunogenic self-proteins.

Coupling and uncoupling of tumor immunity and autoimmunity.

Self-antigens, in the form of differentiation antigens, are commonly recognized by the immune system on melanoma and other cancers. We have shown previously that active immunization of mice against the melanocyte differentiation antigen, a tyrosinase-related protein (TRP) gp75(TRP-1) (the brown locus protein) expressed by melanomas, could induce tumor immunity and autoimmunity manifested as depigmentation. In this system, tumor immunity and autoimmunity were mediated by autoantibodies. Here, we characterize immunity against another tyrosinase family glycoprotein TRP-2 (the slaty locus protein), using the same mouse model and method of immunization. As observed previously for gp75(TRP-1), immunity was induced by DNA immunization against a xenogeneic form of TRP-2, but not against the syngeneic gene, and depended on CD4(+) cells. Immunization against TRP-2 induced autoantibodies and autoreactive cytotoxic T cells. In contrast to immunization against gp75(TRP-1), both tumor immunity and autoimmunity required CD8(+) T cells, but not antibodies. Only autoimmunity required perforin, whereas tumor immunity proceeded in the absence of perforin. Thus, immunity induced against two closely related autoantigens that are highly conserved throughout vertebrate evolution involved qualitatively different mechanisms, i.e., antibody versus CD8(+) T cell. However, both pathways led to tumor immunity and identical phenotypic manifestations of autoimmunity.

Correlates of aldosterone-induced increases in Cai2+ and Isc suggest that Cai2+ is the second messenger for stimulation of apical membrane conductance.

Studies were performed on monolayers of cultured A6 cells, grown on permeable filters, to determine the second messenger system involved in the aldosterone-induced increase in electrogenic sodium transport. Addition of aldosterone (1 microM) to the solution bathing the basal surface of cells caused both an increase in Isc and threefold transient rise in intracellular calcium Cai2+ after a delay of approximately 60 min. Because both events were inhibited by actinomycin D and cyclohexamide, they appeared to require transcriptional and translational processes. Addition of BAPTA to the bathing media to chelate Cai2+ reduced Isc and the delayed Cai2+ transient; 50 microM BAPTA inhibited Isc and the rise in Cai2+ by greater than 80%. Further studies suggested that the action of aldosterone to increase Isc may be dependent on a calcium/calmodulin-dependent protein kinase, because W-7 and trifluoperazine reduced the aldosterone-induced Isc in a dose-dependent manner. Taken together, these observations suggest that calcium is a second messenger for the action of aldosterone on sodium transport, and suggest, for the first time, that agonists which bind to intracellular receptors can utilize, via delayed processes dependent on de novo transcription and translation, intracellular second messenger systems to regulate target cell function.

Tumor immunity and autoimmunity induced by immunization with homologous DNA.

The immune system can recognize self antigens expressed by cancer cells. Differentiation antigens are prototypes of these self antigens, being expressed by cancer cells and their normal cell counterparts. The tyrosinase family proteins are well characterized differentiation antigens recognized by antibodies and T cells of patients with melanoma. However, immune tolerance may prevent immunity directed against these antigens. Immunity to the brown locus protein, gp75/ tyrosinase-related protein-1, was investigated in a syngeneic mouse model. C57BL/6 mice, which are tolerant to gp75, generated autoantibodies against gp75 after immunization with DNA encoding human gp75 but not syngeneic mouse gp75. Priming with human gp75 DNA broke tolerance to mouse gp75. Immunity against mouse gp75 provided significant tumor protection. Manifestations of autoimmunity were observed, characterized by coat depigmentation. Rejection of tumor challenge required CD4(+) and NK1.1(+) cells and Fc receptor gamma-chain, but depigmentation did not require these components. Thus, immunization with homologous DNA broke tolerance against mouse gp75, possibly by providing help from CD4(+) T cells. Mechanisms required for tumor protection were not necessary for autoimmunity, demonstrating that tumor immunity can be uncoupled from autoimmune manifestations.

Black patients sustain vision loss while White and South Asian patients gain vision following delamination or segmentation surgery for tractional complications associated with proliferative diabetic retinopathy.

PurposeThis retrospective comparative case series aims to determine whether patient ethnicity (White versus South Asian versus Black) is related to the outcome of surgical treatment for traction complications of severe proliferative diabetic retinopathy (PDR).SettingMoorfields Eye Hospital London, UK.MethodsAll patients who underwent vitrectomy with, delamination and/or segmentation for PDR over a 5-year period (2009-2014) were reviewed retrospectively. Patients were divided into White, South Asian or Black groups, and their age, gender, HbA1C and type of diabetes were recorded. A total of 484 patients (253 White, 117 South Asian, 114 Black) were included. Twenty-one patients were excluded due to inadequate documentation.OutcomesLogMAR Visual acuity (converted from Snellen) (VA), was recorded pre-operatively and ~6 months post surgery (range 5-8 months). Surgical outcome was classified according to the type and duration of tamponade required post-operatively.ResultsPre-operative VA and HbA1C values were similar across all three ethnic groups (P=0.64 and 0.569, respectively). Change in VA (mean±SD) was 0.41±0.78, 0.14±0.76 and -0.26±0.57 in White, South Asian and Black patient groups respectively (P<0.001). Multiple regression analysis showed that post-op VA was significantly related to race and pre-op VA only (both P<0.001). The Black patient group were more likely to require silicone oil tamponade (P<0.001) and long-term retention of silicone oil (P<0.001) than the White and South Asian patient groups.ConclusionsThis study demonstrates that Black patients on average lose vision following delamination surgery for traction complications of PDR while White and South Asian patients gain vision. The same group is also at higher risk of retaining silicone more than 6 months after surgery. This difference remains even when corrected for glycaemic control. The higher risk of visual loss and long-term retention of silicone oil in black patients requires further investigation. If these results are confirmed, surgeons should consider their patients' ethnicity before proceeding with surgical treatment of diabetic tractional detachment.

Characterization of molecular abnormalities in human fibroblastic neoplasms: a model for genotype-phenotype association in soft tissue tumors.

Desmoid tumors and fibrosarcomas (FS) are part of a wide spectrum of disordered fibroblastic growth that display striking clinical and phenotypic differences. This study was designed to characterize molecular abnormalities that are associated with these differences and to determine their clinical relevance. A cohort of 24 desmoid tumors and 25 low-grade (LG) and 14 high-grade (HG) FS that were clinically and pathologically well characterized was analyzed for alterations in expression of Ki-67, Bcl-2, retinoblastoma gene product (pRB), and p53 by immunohistochemistry. LG-FS and HG-FS showed abnormal expression of Ki-67 (32 versus 86%), Bcl-2 (48 versus 57%), and pRB (56 versus 93%). In contrast, desmoid tumors showed a normal phenotype with these markers. p53 overexpression was identified in 20% of LG-FS and in 29% of HG-FS cases but only in 4% of desmoid tumors. There was an increasing trend in the proportion of abnormal expression of Ki-67, Bcl-2, pRB, and p53 with the increase of tumor aggressiveness from desmoid tumors to LG-FS to HG-FS. The molecular differences between tumor entities were highly statistically significant (P < 0.01). Significant associations between abnormal expression of pRB and recurrence-free survival of LG-FS patients (P = 0.05) and between Ki-67 overexpression and recurrence-free survival for tumors of >5 cm were observed (P = 0.02). The demonstrated differences of molecular alterations in HG-FS, LG-FS, and desmoids appear to be related to biological aggressiveness of such tumors, and they might be useful to differentiate between histologically similar cases of desmoid tumors and LG-FS. pRB and Ki-67 status may be useful to predict recurrence in certain subsets of patients.

Influence of biologic factors and anatomic site in completely resected liposarcoma.

PURPOSE: Soft tissue sarcoma (STS) encompasses a group of neoplasms that are anatomically and biologically diverse. Retroperitoneal/visceral (RP/V) tumors have a poorer prognosis than extremity/trunk (E/T) lesions, and this has been attributed to frequent presentation with tumors of large size and multiorgan involvement that precludes complete resection. The worse prognosis that is associated with RP/V tumors has also been thought to be histopathologically dependent and not necessarily related to anatomic site. The aim of this study was to determine the role of anatomic site and biologic features in prognosis and outcome in patients after complete resection by examining a large cohort of STS patients with a single histopathology, ie, liposarcoma. METHODS: All patients who were treated for liposarcoma from July 1, 1982, through July 1, 1998, were included. Univariate analyses were performed using log-rank test and Kaplan-Meier estimates, and multivariate analyses were performed using Cox regression. The three end points examined were local recurrence (LR), distant recurrence, and disease-specific survival (DSS). RESULTS: Seven hundred twenty patients with liposarcoma were evaluated, and of these, 460 had completely resected primary or completely resected locally recurrent disease. Breakdown of anatomic site was 65% E/T (n = 301) and 35% RP/V (n = 159). The median follow-up period for patients who underwent complete resection was 42 months (range, 1 to 194 months). We found that RP/V site is a poor prognosticator that is independent of patient sex and age; tumor size, grade, and margin; and recurrent presentation. Sixty-nine percent of patients with RP/V tumors who died had local disease only and no distant metastasis at the time of death. CONCLUSION: In liposarcoma, tumor location exerts as strong an influence on prognosis as biology. In contrast to extremity liposarcoma, LR without distant metastasis often results in death for patients with RP/V tumors. For these patients, local control accomplished by complete surgical resection +/- adjuvant radiation therapy should impact strongly on DSS.

Association of local recurrence with subsequent survival in extremity soft tissue sarcoma.

PURPOSE: The aim of this study was to analyze local recurrence in a large cohort of prospectively followed patients with primary extremity soft tissue sarcoma. In particular, we analyzed the correlation of local recurrence with subsequent metastasis and disease-specific survival. PATIENTS AND METHODS: Patients who underwent treatment for primary extremity soft tissue sarcoma from July 1982 through July 1995 at Memorial Sloan-Kettering Cancer Center were the subject of this study. Local recurrence, distant metastasis, and disease-specific survival were used as end points of the study. The influence of local recurrence on subsequent distant metastasis and disease-specific survival were examined using the Cox proportional hazards model. RESULTS: We treated 911 patients, of whom 297 (33%) developed recurrent disease. Local recurrence occurred in 116 patients (13%), metastasis in 167 (18%), and synchronous local recurrence and metastasis in 13 (2%). Of 116 patients who developed local recurrence, 38 subsequently developed metastasis and 34 died of disease. Metastasis after local recurrence was predicted in patients with initial high-grade (P = .005; risk = 3.5) or deep (P = .02; risk = 2.9) tumors. Tumor mortality after local recurrence was predicted in patients with initial high-grade (P = .007; risk = 3.7) or large (> 5 cm; P = .01; risk = 3.2) primary tumors. DISCUSSION: These findings suggest that there is a strong association of local recurrence with the development of subsequent metastasis and tumor mortality, and that local recurrence is a poor prognostic factor. It would seem prudent to consider patients who develop local recurrence and have high-grade tumors as being at high risk for systemic disease and therefore eligible for investigational adjuvant systemic therapy.

Synovial sarcoma: a multivariate analysis of prognostic factors in 112 patients with primary localized tumors of the extremity.

PURPOSE: Synovial sarcoma is a high-grade tumor that is associated with poor prognosis. Previous studies analyzing prognostic factors are limited because of inclusion of heterogeneous cohorts of patients with nonextremity and recurrent tumors. The objective of this study was to determine independent prognostic factors of primary synovial sarcoma localized to the extremity. PATIENTS AND METHODS: Between July 1, 1982, and June 30, 1996, 112 patients underwent surgical resection for cure at our institution and then were followed-up prospectively. Clinical and pathologic factors examined for prognostic value included age, sex, tumor site and location, depth, size, microscopic status of surgical margins, invasion of bone or neurovascular structures, and monophasic or biphasic histology. The end points analyzed were the time to first local recurrence that was not preceded by a distant recurrence, time to any distant recurrence, and time to disease-related mortality. These end points were modeled using the method of Kaplan and Meier and analyzed by the log-rank test and Cox regression. RESULTS: The median duration of follow-up among survivors in this cohort of 112 patients was 72 months. The 5-year local-recurrence, distant-recurrence, and mortality rates were 12%, 39%, and 25%, respectively. Tumor size > or = 5 cm (P =.001; relative risk [RR] = 2. 7; 95% confidence interval [CI], 1.5 to 5.2) and the presence of bone or neurovascular invasion (P =.04; RR = 2.3; 95% CI, 1.0 to 5. 3) were independent adverse predictors of distant recurrence. Tumor size > or= 5 cm (P =.003; RR = 2.3; 95% CI, 1.4 to 6.3) and the presence of bone or neurovascular invasion (P =.03; RR = 2.7; 95% CI, 1.0 to 6.5) were also independent adverse predictors of mortality. CONCLUSION: The natural history of primary synovial sarcoma of the extremity is related to tumor size and invasion of bone and neurovascular structures.

Prognostic factors associated with long-term survival for retroperitoneal sarcoma: implications for management.

PURPOSE: Retroperitoneal soft tissue sarcomas are rare tumors. Studies characterizing long-term follow-up and patterns of recurrence are limited. The purpose of this analysis is to identify patterns of recurrence and prognostic factors associated with long-term survival after resection of retroperitoneal soft tissue sarcomas. METHODS: Between July 1, 1982, and June 30, 1990, 198 adult patients were identified from our prospective soft tissue sarcoma database carrying the diagnosis of retroperitoneal soft tissue sarcoma who were eligible for > or = 5 years of follow-up. Of these, 48 patients (25%) were documented to be alive > or = 5 years from the time of operation. Statistical analysis was by log-rank or Wilcoxon test for univariate analysis. Multivariate analysis was by the Cox model. RESULTS: The recurrence rate during the follow-up period was approximately 5% per year from the time of initial operation. Of the patients who were disease-free for > or = 5 years from initial surgery, 40% recurred by 10 years. Radiation therapy was the only factor significant (P = .02) for a reduction in the risk of local recurrence. Age < or = 50 years and high-grade tumors were significant factors (P = .003 and .009, respectively) for an increased risk of distant metastasis. Incomplete gross resection was the only factor significant for an increased risk of tumor mortality (P = .003). CONCLUSION: Complete surgical resection at the time of primary presentation is likely to afford the best chance for long-term survival. With long-term follow-up, it is clear that recurrence will continue to occur, and a 5-year disease-free interval is not a cure. Patients with an incomplete initial resection, age less than 50 years, and high-grade tumors are candidates for investigational adjuvant therapy.

Vaccination with a bivalent G(M2) and G(D2) ganglioside conjugate vaccine: a trial comparing doses of G(D2)-keyhole limpet hemocyanin.

Immunization with GMK vaccine (G(M2) ganglioside conjugated to keyhole limpet hemocyanin mixed with QS-21 adjuvant) induces anti-G(M2) antibodies in close to 100% of patients. We found previously that anti-G(D2) antibodies could be induced in some patients using G(D2)-keyhole limpet hemocyanin + QS-21 (GDK). In this trial, we wished: (a) to determine whether immunization with both GMK and GDK vaccines could induce antibodies against both G(M2) and G(D2); and (b) to determine the optimal dose of GDK. Thirty-one patients with melanoma or sarcoma who had no evidence of disease after complete surgical resection were immunized with both GMK (30 microg of G(M2)) and GDK on weeks 1, 2, 3, 4, 12, 24, and 36. Patients were assigned to one of five GDK dose levels (3, 10, 30, 70, or 130 microg of G(D2)). Anti-G(M2) IgM or IgG were induced in 97% of patients. The dose of GDK did not affect the anti-G(M2) response, although at the highest GDK dose level, 3 of 7 patients did not make anti-G(M2) IgG. GDK was less immunogenic; overall 45% of patients developed either IgM or IgG against G(D2). At GDK doses of 30 or 70 microg, 8 of 11 patients (73%) made either IgM or IgG anti-G(D2) antibodies. We conclude that both GMK and GDK vaccines can induce antibodies against G(M2) and G(D2) in a majority of patients and are safe. The optimal dose of GDK appears to be either 30 or 70 microg when administered with GMK vaccine.

Priming for T-cell-mediated rejection of established tumors by cutaneous DNA immunization.

DNA immunization has been shown to elicit both antibody and CTL responses against antigens expressed by infectious organisms. Because CTL responses have been implicated in rejection of cancer, we investigated whether DNA immunization by particle bombardment using a gene gun could induce CTL responses that were capable of rejecting tumors in mice. DNA immunization by particle bombardment using genes encoding beta-galactosidase and ovalbumin primed mice to generate CTLs in two genetic backgrounds (DBA/2 and C57BL/6 strains, respectively). DNA immunization was more potent in inducing CTLs than immunization with an optimized regimen of ovalbumin peptide plus immune adjuvant. Immunity induced by DNA immunization protected mice against s.c. challenge with tumors expressing the beta-galactosidase antigen. Tumors were rejected even when DNA immunization was started 3 or 7 days after tumor challenge as tumors were becoming established. Tumor rejection required CD8(+) T cells, confirming a role for CTLs in vivo. These studies show that DNA immunization by particle bombardment can efficiently induce CTL responses that are capable of rejecting even established tumors.

Overexpression of cyclin D1 is associated with poor prognosis in extremity soft-tissue sarcomas.

Binding of G1 cyclins to cyclin-dependent kinases leads to phosphorylation of the retinoblastoma protein and progression through G1 and S phases of the cell cycle. Overexpression of cyclins is thought to deregulate this process and has been noted in many human malignancies. This study was conducted to assess patterns of expression and potential gene amplification of the G1 cyclins in 84 patients affected with extremity soft-tissue sarcomas. Sixty cases were primary tumors, whereas the remaining 24 cases were locally recurrent lesions. There were 58 high-grade and 26 low-grade tumors. Immunohistochemical analyses were conducted using antibodies to cyclins D1, E, and A. Southern blot analysis was performed on DNA available from 53 of 84 patients with a cyclin D1-specific probe. Cyclin D1 overexpression was noted in 23 of 79 informative cases (29%), whereas cyclin E was found overexpressed in 26 of 80 cases (33%) and cyclin A overexpression was observed in 9 of 81 cases (11%). Overexpression of cyclins D1, E, or A each correlated significantly with high tumor grade (P <0.05). On multivariate analysis, neither cyclin E nor cyclin A were significant predictors of outcome. However, overexpression of cyclin D1 was significantly associated with worse overall survival for the entire group as well as in the subset of high-grade lesions (P <0.05), notwithstanding the relatively short follow-up time (mean, 2.4 years). Nevertheless, the presence of a significant association between laboratory data and outcome implies that the study is adequately powered. Furthermore, none of the cases demonstrated CCND1 gene amplification. These data support the concept that cyclin D1 overexpression determines the evolution of a particularly aggressive subset of these lesions.

The biological significance of failure at the primary site on ultimate survival in soft tissue sarcoma.

Local tumor recurrence after complete resection may be due to treatment factors or represent a manifestation of tumor biology. The association of local tumor recurrence, distant metastases, and death in patients undergoing treatment for extremity soft tissue sarcoma (STS) has been described but continues to be enigmatic. After definitive multimodality treatment for extremity STS, local tumor recurrence is associated with development of distant metastasis, and metastases are implicated in subsequent disease-specific death. The relationship is an enigma, and the causality is unclear. Conversely, for patients with retroperitoneal STS, a direct relationship between local tumor recurrence and disease-specific death has been shown. In this article, current concepts are analyzed and reviewed.

Insect cells as HLA-restricted antigen-presenting cells for the IFN-gamma elispot assay.

Measurement of specific cellular immune responses in patients undergoing immunotherapy is difficult. Established approaches, including cytotoxicity (e.g., 51Cr release) and cytokine release assays, require in vitro culturing for several weeks or more of patients' peripheral blood mononuclear cells (PBMC) and the addition of exogenous cytokines. Therefore, the immunological response does not reflect in vivo conditions. To address these disadvantages, we have used an interferon-gamma (IFN-gamma) Elispot assay for detecting peptide-specific CD8(+) lymphocytes in PBMC. A limitation of this assay is the lack of a reproducible source of antigen-presenting cells (APCs). Currently available APCs often lead to significant background levels. It has been shown that transfected insect cells can express empty MHC class I molecules on their surface. We have transfected Drosophila melanogaster S2 cells and the Lepidopteran line Sf9 with the gene encoding human HLA-A2.1. We demonstrate that insect cells expressing a human HLA molecule effectively function as APCs in the IFN-gamma Elispot assay. Initially the feasibility of the assay was assessed using CD8(+) T cells from HLA-A2.1(+) donors with known reactivity against an HLA-A2.1-binding epitope of the influenza matrix protein. Use of insect cells as APCs abrogated background spots, increasing sensitivity. We further observed that a short-term prestimulation of PBMC with peptide-pulsed insect cells markedly enhanced the frequency of peptide-specific T cells that could be measured in the Elispot assay without increasing the background. This approach was then used to measure CD8(+) T cell reactivity to a peptide from tyrosinase, an antigen that is processed and presented by melanoma cells. Insect cells expressing human HLA molecules provide a standard APC for monitoring CD8(+) T cell responses to tumor and viral peptides during immunotherapy.

Adjuvant radiotherapy for margin-positive high-grade soft tissue sarcoma of the extremity.

PURPOSE: Adjuvant radiotherapy (RT) has been shown to improve local control in patients with soft tissue sarcoma of the extremities (STS). The specific impact of adjuvant radiation on patients with positive margins, however, has not been clearly defined. The purpose of this study was to determine if adjuvant RT improves local control in patients with high-grade STS who had positive margins of resection. METHODS AND MATERIALS: Between 8/82 and 2/97, 110 adult patients with primary high-grade STS of an extremity underwent limb sparing surgery and were found to have a histologically positive microscopic surgical margin. Ninety-one (83%) received RT and 19 (17%) had no RT. The two groups were balanced with regard to size, site, location, and tumor depth. Adjuvant RT was delivered with brachytherapy (BRT) alone in 34 patients, external beam radiotherapy (EBRT) alone in 33 patients, or BRT+EBRT in 24 patients. The BRT dose was 45 Gy when used alone and 15-20 Gy when used as a boost. The EBRT dose was 60-70 Gy when used alone and 45-50 Gy when given with BRT. The median follow-up time was 41 months (range, 3-186 months). RESULTS: The overall 5 year local control rate was 71%. This rate was significantly higher in the RT group compared to the no RT group (74% vs. 56%, respectively) (p = 0.01). On univariate analysis, lower extremity site and proximal location were also found to be predictors of improved local control (p = 0.03 and 0.03, respectively). However, only proximal location and the use of RT retained their significance as predictors of improved local control on multivariate analysis (p = 0.003 and 0.01, respectively). The overall 5-year distant relapse-free survival, disease-free survival, and overall survival rates were 54%, 44%, and 53%, respectively. No statistical differences were found in these survival rates between RT and no RT groups. CONCLUSION: Based on this study, adjuvant radiotherapy seems to improve local control in patients with high-grade STS of the extremity with positive margins. However, local recurrence still occurs in a substantial proportion of patients, mandating further need for improvement.