Eastern Canadian Colorectal Cancer Consensus Conference: setting the limits of resectable disease.

The annual Eastern Canadian Colorectal Cancer Consensus Conference was held in Montreal, Quebec, October 22-24, 2009. Health care professionals involved in the care of patients with colorectal cancer participated in presentation and discussion sessions for the purposes of developing the recommendations presented here. This consensus statement addresses current issues in the management colorectal cancer, such as the management of hepatic and pulmonary metastases, the role of monoclonal antibodies to the epidermal growth factor receptor, and the benefits and safety of chemotherapy in elderly patients. The management of gastrointestinal neuroendocrine tumours and gastric cancer are also discussed.

Implication of unfavorable histology, MYCN amplification and diploidy for stage I and II neuroblastomas.

BACKGROUND: Surgery is the first line treatment for low-grade neuroblastomas. In stage I tumors, the presence of MYCN amplification is rarely detected and the Shimada histology is not always taken into consideration when deciding on the treatment. This study concerns the significance of these two factors in the evolution of children with low-grade neuroblastomas. METHODS: We analyzed the assessment and follow-up of children with low-grade neuroblastomas (stages I and II) with or without MYCN amplification, with either a favorable or unfavorable histology and with or without tumor cell diploidy. Favorable histology was defined as stroma-poor tumors with more than 5 % differentiating neuroblasts and a mitosis karyorrhexis index (MKI) of less than 100/5000 cells. RESULTS: From 1995 to 2006, out of 114 neuroblastomas, nine (7.9 %) were stage I and 21 (18.4 %) stage II. Of these 30 patients, 27 underwent surgery alone and three received chemotherapy after surgery. The combination of MYCN amplification, unfavorable histology and diploidy was noted in one patient who developed metastases within two months. MYCN amplification alone was noted in two cases who are still tumor-free after two years. Unfavorable histology alone was noted in four patients, of whom one suffered a recurrence of the tumor (previously stage I) and three are tumor-free after six years. Tumor cell diploidy alone was present in 11 patients whose evolution is satisfactory. CONCLUSION: Because MYCN amplification and unfavorable histology are rare in early stage neuroblastomas, these tumors may be misclassified if they are not investigated further. It seems that no single clinical or biological feature can be considered a significant factor in establishing a prognosis or determining whether additional treatment is required.

Results of an unrelated transplant search strategy using partially HLA-mismatched cord blood as an immediate alternative to HLA-matched bone marrow.

Cord blood (CB) is an alternative to other sources of stem cells for transplantation. However, the impact of including CB in the initial strategy of unrelated graft search in a cohort of patients has been the object of limited analysis. Here, we report the results of such a strategy in 91 consecutive children. Absence of mismatch was required for adult donors, and up to two mismatches were allowed for CB grafts, with a nucleated cell dose over 2.5 x 10(7) cells/kg. A graft was found for 84 of the 85 children who remained available for a 3-month search. In all, 64 patients were transplanted, 36 with CB and 28 with bone marrow (BM). Primary graft failure, acute grade II-IV and extensive chronic graft-versus-host disease occurred in five, five and zero CB, and in three, one and two BM patients, respectively. The 3-year survival was 59% in CB and 57% in BM patients. Accepting CB as a source of stem cells offers a graft to almost every child in need of an unrelated transplantation, with a probability of survival similar to that of unrelated BM transplantation.

Unrelated bone marrow transplantation for leukocyte adhesion deficiency.

The severe form of leukocyte adhesion deficiency type I (LAD-I) usually leads to death early in life. Allogeneic haematopoietic transplantation is the only cure. Unrelated transplantation has been reported only once. We describe three children with LAD-I transplanted with T cell non-depleted bone marrow from unrelated HLA-matched donors. All patients engrafted, one of them at second transplant. One patient developed grade I and one grade II acute GVHD. Two patients are alive, one of them with a decrease in CD11/CD18 expression. Early referral for HLA-matched unrelated BMT is a reasonable option for patients with LAD-I lacking an HLA-matched related donor.

Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to moloney murine leukemia virus-based vectors.

The low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34(+) cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin(-) bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.

Superior transduction of mouse hematopoietic stem cells with 10A1 and VSV-G pseudotyped retrovirus vectors.

The inefficient transduction of human hematopoietic stem cells (HSC) with amphotropic retroviral vectors has been an obstacle to gene therapy for hematopoietic diseases. We have previously reported low levels of amphotropic retrovirus receptor (Pit-2) mRNA and higher levels of gibbon ape leukemia virus (GALV) or 10A1 retrovirus receptor (Pit-1) mRNA in mouse and human HSC. The vesicular stomatitis virus (VSV-G) uses an abundant membrane phospholipid as a receptor. We hypothesized that transduction of HSC requires relatively high levels of retrovirus receptor molecules. Because mouse HSC can be efficiently transduced by ecotropic virus through the abundant ecotropic receptor, the mouse is an ideal model to compare receptor levels and transduction. We have developed a cotransduction assay where ecotropic retrovirus transduction is a positive internal control for downstream steps in retrovirus transduction. A comparison of mouse HSC transduction with amphotropic, 10A1, and VSV-G envelopes showed that the level of amphotropic and 10A1 receptor mRNA in HSC correlated with the frequency of transduction. Transduction with VSV-G vectors was similar to that with 10A1 vectors. We conclude that the level of retrovirus receptor on HSC is critical for HSC transduction and that GALV or VSV-G vectors would be better for human HSC transduction.

Alternative viral envelopes for oncoretroviruses to increase gene transfer into hematopoietic stem cells.

The hematopoietic stem cell is the target for gene therapy of human blood disease. Low retroviral receptors for the commonly used vectors and quiescence of hematopoietic stem cells are believed to be major obstacles to the success of gene therapy. The development of new stem cell assays has allowed better understanding of the biology and phenotype of hematopoietic stem cells, leading to selection of highly enriched populations of hematopoietic stem cells. Quantitation of retrovirus receptors on these enriched populations of hematopoietic stem cells has resulted in the identification of subpopulations of cells expressing high levels of retrovirus receptors. New promising retrovirus envelopes are being developed. In this review, we discuss those issues that may help to resolve the problem of low gene transfer efficiency into human hematopoietic stem cells.

Amphotropic retrovirus transduction of hematopoietic stem cells.

Mice treated with cytokines for 5 days have large numbers of hematopoietic stem cells (HSCs) in their peripheral blood and bone marrow at 1 and 14 days after the last injection. We fractionated the HSCs from the bone marrow of these mice using elutriation at flow rates of 25, 30 and 35 ml/min. The subpopulations of HSCs from cytokine-treated mice show a 3- to 8-fold higher level of mRNA encoding the amphotropic retrovirus receptor (amphoR) compared with the corresponding HSC subpopulation from untreated mouse bone marrow. In an earlier study with mouse HSCs we showed a direct correlation between high levels of amphoR mRNA and efficient retrovirus transduction. We have now utilized our gene transfer protocol to assay amphotropic retrovirus transduction efficiency using HSCs from the bone marrow of mice treated with granulocyte-colony stimulating factor/stem cell factor (G-CSF/SCF). To extend these findings to a more clinically relevant protocol we analyzed the amphoR mRNA levels in HSCs from human cord blood and adult bone marrow. The amphoR mRNA level in HSCs from human bone marrow and fresh cord blood was detectable at an extremely low level compared with the HSC population in cryopreserved cord blood samples. The 12- to 22-fold increase in amphoR mRNA in HSCs from cryopreserved cord blood renders these HSCs likely candidates for high efficiency, gene transfer.