Prediction of pathological response to neoadjuvant treatment in rectal cancer with a two-protein immunohistochemical score derived from stromal gene-profiling.

BACKGROUND: Preoperative chemoradiotherapy followed by surgical mesorectal resection is the standard of care for locally advanced rectal carcinomas. Yet, predicting that patients will respond to treatment remains an unmet clinical challenge. EXPERIMENTAL DESIGN: Using laser-capture microdissection we isolated RNA from stroma and tumour glands from prospective pre-treatment samples (n = 15). Transcriptomic profiles were obtained hybridising PrimeView Affymetrix arrays. We modelled a carcinoma-associated fibroblast-specific genes filtering data using GSE39396. RESULTS: The analysis of differentially expressed genes of stroma/tumour glands from responder and non-responder patients shows that most changes were associated with the stromal compartment; codifying mainly for extracellular matrix and ribosomal components. We built a carcinoma-associated fibroblast (CAF) specific classifier with genes showing changes in expression according to the tumour regression grade (FN1, COL3A1, COL1A1, MMP2 and IGFBP5). We assessed these five genes at the protein level by means of immunohistochemical staining in a patient's cohort (n = 38). For predictive purposes we used a leave-one-out cross-validated model with a positive predictive value (PPV) of 83.3%. Random Forest identified FN1 and COL3A1 as the best predictors. Rebuilding the leave-one-out cross-validated regression model improved the classification performance with a PPV of 93.3%. An independent cohort was used for classifier validation (n = 36), achieving a PPV of 88.2%. In a multivariate analysis, the two-protein classifier proved to be the only independent predictor of response. CONCLUSION: We developed a two-protein immunohistochemical classifier that performs well at predicting the non-response to neoadjuvant treatment in rectal cancer.

PRL-3 is essentially overexpressed in primary colorectal tumours and associates with tumour aggressiveness.

Phosphatase PRL-3 has been involved in different types of cancer, especially in metastases from colorectal carcinoma (CRC). In this study, we explored both isoforms of PRL-3 as a biomarker to predict the recurrence of stage IIIB-C CRC. Overexpression of PRL-3 was investigated in primary human colorectal tumours (n=20) and hepatic metastases (n=36) xenografted in nude mice, samples characterised by absence of human non-tumoral cells, showing a high degree of expression in metastases (P=0.001). In 27 cases of matched normal colonic mucosa/primary tumour/hepatic metastases, PRL-3 overexpression occurs in primary tumours vs normal mucosa (P=0.001) and in hepatic metastases vs primary tumours (P=0.045). Besides, our results in a series of 80 stage IIIB-C CRC primary tumours showed that high levels of PRL-3 were an independent predictor of metastasis (P<0.0001; OR: 9.791) in multivariate analysis of a binary logistic regression and that PRL-3 expression tightly correlates with parameters of bad outcome. Moreover, PRL-3 expression associated with poor outcome in univariate (P<0.0001) and multivariate Cox models (hazard ratio: 3.322, 95%, confidence interval: 1.405-7.852, P=0.006). In conclusion, PRL-3 is a good marker of aggressiveness of locally advanced CRS and a promising predictor of distant metastases. Nevertheless, for prognosis purposes, it is imperative to validate the cutoff value of PRL-3 expression in a larger and consecutive series and adjuvant setting.

Heart and liver xenotransplantation under low-dose tacrolimus: graft survival after withdrawal of immunosuppression.

BACKGROUND: The hamster-to-rat xenotransplantation model is a useful model to investigate the features of extended host response to long-surviving xenografts. Early xenoantibody responses are T-cell independent and resistant to tacrolimus. Treatment with the combination of mofetil mycophenolate plus FK506 avoids acute xenograft rejection completely, but after withdrawal of immunosuppression hamster grafts are rejected by a process called late xenograft rejection (LXR). METHODS: Hamster hearts and livers were transplanted into Lewis rats. Grafted rats were treated with mofetil mycophenolate (25 mg/kg/day) for 8 days and FK506 (0.2 mg/kg/day) for 31 days. Serum IgM and IgG levels were determined by flow cytometry and interferon-gamma levels by ELISA. IgM, IgG, and C3 deposits were measured in tissue by immunofluorescence, and leukocyte infiltration was measured by immunoperoxidase staining. Results. Survival of heart and liver xenografts in the rats was 48+/-4 days and 63+/-8 days, respectively. After cessation of all immunosuppression, hearts were rejected in 18+/-4 days and livers in 33+/-8 days. Production sequences of xenoantibodies in the two organs differed substantially, especially 7 days after transplantation and at the moment of rejection. Quantification of interferon-gamma levels indicated that there were no significant changes after transplantation. Histological and immunohistochemical studies showed signs of humoral mechanism of LXR in rats undergoing heart transplantation and cellular mechanism of LXR in those that received a liver transplant. Conclusions. These observations suggest that rejection in the hamster-to-rat heart xenotransplantation model is mediated by a T cell-independent B-cell response to which a T cell-dependent B-cell response is added in LXR. In the liver xenotransplantation model, our hypothesis is that LXR is mediated by a mixed cell mechanism, involving lymphocytes CD4+ CD45RC+, macrophages, and cytotoxic T lymphocytes. In summary, we have demonstrated and compared the peculiar features of LXR in two different organs.

Lymphocyte populations in Parkinson's disease and in rat models of parkinsonism.

To assess the involvement of the immune system in Parkinson's disease we studied the phenotype of circulating lymphocytes in 30 untreated and 34 treated patients. We found a numeric decrease in helper T cells (higher in CD4(+)CD45RA(+) than in CD4(+)CD29(+)) and B cells, and a rise in activated, CD4(+)CD25(+) lymphocytes that was correlated with lymphocyte depletion. All these alterations were independent of levodopa treatment. In addition, we performed striatal dopamine depletion in rats with either MPP(+) or 6-OHDA, showing that MPP(+) but not 6-OHDA can increase CD4(+)CD25(+) lymphocytes. Thus, mechanisms other than dopamine deficit may explain the immune activation in Parkinson's disease.

Liver xenotransplantation: changes in lipid and lipoprotein concentration after long-term graft survival.

BACKGROUND/AIMS: Today, scientists devote considerable effort to the study of mechanisms of xenograft rejection, but with liver xenotransplantation (XTx) researchers face the added problem of metabolic incompatibility between species. To date, there have been few studies of molecular xenogeneic interactions, perhaps because little progress has been made in solving immunological problems. This study is an initial analysis of lipoprotein metabolism in a hamster-to-rat hepatic xenotransplantation model. METHODS: There were 6 experimental groups (n=8): (1) male Sprague-Dawley (S.D.) rats (220-280 g); (2) male Golden Syrian hamsters (100-150 g); (3) S.D. rats, "sham" operation with immunosuppression; (4) S.D. rat-to-S.D. rat alloTx; (5) S.D. rat-to-S.D. rat alloTx with immunosuppression; (6) XTx hamster G.S-to-S.D. rat with immunosuppression. Mofetil mycophenolate (25 mg/kg/d) was administered for 14 days and FK506 (0.2 mg/kg/d) for 45 days (groups 3, 5 and 6). After 24 h fasting, animals were sacrificed (day +50 postransplantation) and a complete lipoprotein profile was determined. Serum lipoproteins were subfractioned by ultracentrifugation in density gradient. RESULTS: There was a large increase in serum lipid levels in xenografted rats compared with control rats and allografted rats. Xenografted rats presented a severely altered lipoprotein profile compared with normal rats. Surprisingly, the characterisation of lipoproteins in xenografted rats displayed the same composition as donor animals. Histological study did not show signs of alteration of the hepatic architecture. CONCLUSIONS: Since the liver is the main solid organ co-ordinator of metabolic pathways, such as lipid metabolism, hepatic xenotransplantation makes changes in lipid concentrations in the recipient and also changes in lipid compositions of lipoproteins. Hepatic xenotransplantation is not a feasible solution given the organ's metabolic complexity.