Population-based study to re-evaluate optimal lymph node yield in colonic cancer.

BACKGROUND: It is well established that lymph node (LN) yield in colonic cancer resection has prognostic significance, although optimal numbers are not clear. Here, LN thresholds associated with both LN positivity and survival were evaluated in a single population-based data set. METHODS: Treatment records were linked to the Ontario Cancer Registry to identify a 25 per cent random sample of all patients with stage II/III colonic cancer between 2002 and 2008. Multivariable regression and Cox models evaluated factors associated with LN positivity and cancer-specific survival (CSS) respectively. Optimal thresholds were obtained using sequential regression analysis. RESULTS: On adjusted analysis of 5508 eligible patients, younger age (P < 0·001), left-sided tumours (P = 0·003), higher T category (P < 0·001) and greater LN yield (relative risk 0·89, 95 per cent c.i. 0·81 to 0·97; P = 0·007) were associated with a greater likelihood of LN positivity. Regression analyses with multiple thresholds suggested no substantial increase in LN positivity beyond 12-14 LNs. Cox analysis of stage II disease showed that lower LN yield was associated with a significant increase in the risk of death from cancer (CSS hazard ratio range 1·55-1·74; P < 0·001) compared with a greater LN yield, with no significant survival benefit beyond a yield of 20 LNs. Similarly, for stage III disease, a lower LN yield was associated with an increase in the risk of death from cancer (CSS hazard ratio range 1·49-2·20; P < 0·001) versus a large LN yield. In stage III disease, there was no observed LN threshold for survival benefit in the data set. CONCLUSION: There is incongruity in the optimal LN evaluation for colonic cancer. Although the historically stated threshold of 12 LNs may ensure accurate staging in colonic cancer, thresholds for optimal survival are associated with far greater yields.

Is there a sex effect in colon cancer? Disease characteristics, management, and outcomes in routine clinical practice.

INTRODUCTION: The incidence of colon cancer varies by sex. Whether women and men show differences in extent of disease, treatment, and outcomes is not well described. We used a large population-based cohort to evaluate sex differences in colon cancer. METHODS: Using the Ontario Cancer Registry, all cases of colon cancer treated with surgery in Ontario during 2002-2008 were identified. Electronic records of treatment identified use of surgery and adjuvant chemotherapy. Pathology reports for a random 25% sample of all cases were obtained, and disease characteristics, treatment, and outcomes in women and men were compared. A Cox proportional hazards model was used to identify factors associated with overall (os) and cancer-specific survival (css). RESULTS: The study population included 7249 patients who underwent resection of colon cancer; 49% (n = 3556) were women. Stage of disease and histologic grade did not vary by sex. Compared with men, women were more likely to have right-sided disease (55% vs. 44%, p ≤ 0.001). Surgical procedure and lymph node yield did not differ by sex. Adjuvant chemotherapy was delivered to 18% of patients with stage ii and 64% of patients with stage iii disease; when adjusted for patient- and disease-related factors, use of adjuvant chemotherapy was similar for women and men [relative risk: 0.99; 95% confidence interval (ci): 0.94 to 1.03]. Adjusted analyses demonstrated that os [hazard ratio (hr): 0.80; 95% ci: 0.75 to 0.86] and css (hr: 0.82; 95% ci: 0.76 to 0.90) were superior for women compared with men. CONCLUSIONS: Long-term survival after colon cancer is significantly better for women than for men, which is not explained by any substantial differences in extent of disease or treatment delivered.

Lymph node evaluation for colon cancer in routine clinical practice: a population-based study.

BACKGROUND: Guidelines recommend that 12 or more lymph nodes (lns) be evaluated during surgical resection of colon cancer. Here, we report ln yield and its association with survival in routine practice. METHODS: Electronic records of treatment were linked to the population-based Ontario Cancer Registry to identify all patients with colon cancer treated during 2002-2008. The study population (n = 5508) included a 25% random sample of patients with stage ii or iii disease. Modified Poisson regression was used to identify factors associated with ln yield; Cox models were used to explore the association between ln yield and overall (os) and cancer-specific survival (css). RESULTS: During 2002-2008, median ln yield increased to 17 from 11 nodes (p < 0.001), and the proportion of patients with 12 or more nodes evaluated increased to 86% from 45% (p < 0.001). Lymph node positivity did not change over time (to 53% from 54%, p = 0.357). Greater ln yield was associated with younger age (p < 0.001), less comorbidity (p = 0.004), higher socioeconomic status (p = 0.001), right-sided tumours (p < 0.001), and higher hospital volume (p < 0.001). In adjusted analyses, a ln yield of less than 12 nodes was associated with inferior os and css for stages ii and iii disease [stage ii os hazard ratio (hr): 1.36; 95% confidence interval (ci): 1.19 to 1.56; stage ii css hr: 1.52; 95% ci: 1.26 to 1.83; and stage iii os hr: 1.45; 95% ci: 1.30 to 1.61; stage iii css hr: 1.54; 95% ci: 1.36 to 1.75]. CONCLUSIONS: Despite a temporal increase in ln yield, the proportion of cases with ln positivity has not changed. Lymph node yield is associated with survival in patients with stages ii and iii colon cancer. The association between ln yield and survival is unlikely to be a result of stage migration.

Eastern Canadian Colorectal Cancer Consensus Conference 2013: emerging therapies in the treatment of pancreatic, rectal, and colorectal cancers.

The annual Eastern Canadian Colorectal Cancer Consensus Conference held in Montreal, Quebec, 17-19 October 2013, marked the 10-year anniversary of this meeting that is attended by leaders in medical, radiation, and surgical oncology. The goal of the attendees is to improve the care of patients affected by gastrointestinal malignancies. Topics discussed during the conference included pancreatic cancer, rectal cancer, and metastatic colorectal cancer.

Effectiveness of Trastuzumab in Routine Clinical Practice: A Population-based Study of Patients with HER-2-positive Oesophageal, Gastroesophageal and Gastric Cancer.

AIMS: In the pivotal Trastuzumab for Gastric Cancer (ToGA) trial, trastuzumab improved median survival in patients with advanced HER-2-positive gastric and gastroesophageal cancer from 11.1 to 13.8 months; however, its effectiveness in routine clinical practice has not been evaluated. Our objective was to evaluate the uptake and outcomes of trastuzumab in a population-based cohort of patients with oesophageal, gastroesophageal and gastric cancer in Ontario, Canada. MATERIALS AND METHODS: The Ontario Cancer Registry and linked treatment records were used to identify all patients with oesophageal, gastroesophageal and gastric cancer treated with trastuzumab during 2012-2017. Outcomes were analysed from the time of first trastuzumab cycle and included a primary outcome (survival) and secondary outcomes (uptake, delivery, 30-day hospital admission and 30-day mortality). Trends over the study period and survival were evaluated. RESULTS: In total, 476 patients with oesophageal, gastroesophageal and gastric cancer received trastuzumab during the study period. The mean age was 62 years, 78% (370/476) were male, and 65% (312/476) had gastric cancer. The annual number of patients receiving trastuzumab increased over the study period (53 in 2012 and 101 in 2017). The median number of cycles of trastuzumab delivered was six. Thirty-day hospital admission and mortality rates were 17% and 4%, respectively. The median overall survival was 282 days (9.3 months). CONCLUSIONS: The median survival of patients treated with trastuzumab for advanced oesophageal, gastroesophageal and gastric cancer in routine practice is substantially less than that observed in the pivotal clinical trial. Studies of comparative effectiveness using real-world data offer insight into outcomes achieved in routine practice.

Chemotherapy for resected colorectal cancer pulmonary metastases: Utilization and outcomes in routine clinical practice.

BACKGROUND: The role of chemotherapy in the setting of resected colorectal cancer pulmonary metastases (CRCPM) is not well defined. Here we describe utilization of peri-operative chemotherapy and outcomes among patients with resected CRCPM in the general population. METHODS: All cases of CRCPM who underwent resection from 2002 to 2009 were identified using the Ontario Cancer Registry (OCR). Electronic treatment records identified peri-operative chemotherapy delivered within 16 weeks before or after pulmonary metastasectomy (PM). Modified Poisson regression was used to evaluate factors associated with chemotherapy delivery. Cox proportional models were used to explore the association between post-operative chemotherapy and cancer-specific (CSS) and overall survival (OS). RESULTS: The study population included 420 patients. Thirty-six percent of patients (151/420) received peri-operative chemotherapy. Among these patients, 75% (113/151) received post-operative chemotherapy. Factors that were independently associated with use of post-operative chemotherapy included higher socioeconomic status (SES) and no prior adjuvant chemotherapy (p < 0.01). In adjusted analyses post-operative chemotherapy was not associated with improved CSS (HR 0.99, 95% CI 0.67-1.47) or OS (HR 0.93 95% CI 0.66-1.31). In exploratory analyses, among those patients who did not receive previous adjuvant therapy for the primary colorectal cancer, post-operative chemotherapy following lung metastasectomy was associated with HR 0.50 (95% CI 0.27-0.95) for OS and HR 0.59 (95% CI 0.27-1.27) for CSS. CONCLUSION: One third of patients with resected CRCPM in routine practice receive peri-operative chemotherapy. A randomized controlled trial is warranted to evaluate whether chemotherapy following resection of CRCPM is associated with improved survival.

Regional lymph node involvement in patients undergoing liver resection for colorectal cancer metastases.

BACKGROUND: While the indications for surgery among patients with colorectal cancer liver metastases (CRCLM) are expanding, the role of surgery in patients with hepatic lymph node involvement remains controversial. We report management and outcomes in a population-based cohort of patients undergoing hepatectomy with concomitant hepatic lymphadenectomy for CRCLM. METHODS: All cases of hepatectomy for CRCLM in the Canadian Province of Ontario from 2002 to 2009 were identified using the population-based Ontario Cancer Registry and linked electronic records of treatment. Pathology reports were used to identify concomitant lymphadenectomy with liver resection as well as extent of disease and surgical procedure. RESULTS: Among 1310 patients who underwent resection for CRCLM, 103 (8%) underwent simultaneous regional lymphadenectomy. Seventy-one percent of cases with lymphadenectomy (70/103) had a major liver resection (≥3 segments). Of the 103 lymphadenectomy cases, 80 (78%) were hepatic pedicle, 16 (16%) were celiac and 7 (7%) were para-aortic. The mean number of nodes removed was 2.2 (range 1-15). Ninety-day mortality was 6%. Twenty-nine percent (30/103) of cases had positive nodes. Unadjusted overall survival at 5 years for positive vs negative nodes was 21% vs 42% (p = 0.003); cancer-specific survival was 10% vs 43% (p < 0.001). In adjusted analyses, hepatic node involvement was associated with inferior OS (HR 2.19, p = 0.010) and CSS (HR 3.07, p = 0.002). CONCLUSIONS: Patients with resected CRC liver metastases with regional lymph node involvement have inferior survival compared to patients with negative nodes. Despite this poor prognostic factor, a small proportion of cases with involved nodes will achieve long-term survival.

Chemotherapy delivery for resected colorectal cancer liver metastases: Management and outcomes in routine clinical practice.

BACKGROUND: International guidelines recommend peri-operative chemotherapy for patients with resectable colorectal cancer liver metastases (CRCLM). Chemotherapy delivery in routine practice is not well described. METHODS: All cases of CRC who underwent resection of LM in 2002-2009 were identified using the population-based Ontario Cancer Registry. Electronic treatment records identified chemotherapy delivered within 16 weeks before or after hepatectomy. All pathology reports were reviewed to describe extent of LM. Modified Poisson regression was used to evaluate factors associated with chemotherapy delivery. Cox proportional hazards model and propensity score analysis were used to explore the association between post-operative chemotherapy and cancer-specific (CSS) and overall (OS) survival. RESULTS: We identified 1310 patients. Sixty-two percent of cases (815/1310) received peri-operative chemotherapy; 25% (200/815) pre-operative, 45% (366/815) post-operative, and 31% (249/815) pre- and post-operative. Utilization of chemotherapy increased over time from 51% in 2002 (57/112) to 73% in 2009 (157/216, p < 0.001). Fifty-four percent of patients received FOLFOX, 41% FOLFIRI, and 10% 5-FU monotherapy. Factors that were independently associated with greater utilization of post-operative chemotherapy included younger age (p < 0.001), female sex (p = 0.050), shorter disease-free interval (p = 0.006), and no prior adjuvant chemotherapy (p < 0.001). Utilization of chemotherapy varied substantially across geographic regions (from 24% to 71%, p = 0.001). Post-operative chemotherapy was associated with improved CSS (HR 0.58, 95%CI 0.44-0.76) and OS (HR 0.49, 95%CI 0.38-0.61); results were consistent in propensity score analysis. CONCLUSION: Utilization of chemotherapy for resected CRCLM in routine practice has evolved with emerging evidence. Post-operative chemotherapy is associated with improved survival in the general population.

Association between the time to surgery and survival among patients with colon cancer: A population-based study.

Factors associated with time-to-surgery (TTS) and survival in colon cancer has not been well studied. Cancer Care Ontario recommends surgery within 42 days of diagnosis and that 90% of patients meet this benchmark. We describe factors associated with TTS and survival in routine clinical practice. METHODS: Retrospective population-based cohort study of patients receiving elective colonic resection after diagnosis of colon cancer in Ontario, Canada from 2002 to 2008 followed until 2012. Factors associated with TTS were identified using multivariate log-binomial and Quantile regression at 42 days and 90th percentiles. The association between TTS and cancer-specific (CSS) and overall survival (OS) were examined using multivariate Cox regression. RESULTS: 4326 patients; median age 71 years and 52% male. Median TTS was 24 days (IQR 14-37); at the 90th percentile 56 days. Factors associated with TTS ≥ 42 days and >90th percentile included older age, co-morbid illness, surgeon volume, and stage I disease (P < 0.05 for all). In patients whose TTS was either at 42 days or 90th percentile, those ≥80 years old waited two weeks longer than those <60 years, individuals with co-morbid illness waited 10 days longer than without co-morbidity, and patients with stage I disease waited 10 days longer than those with stage IV disease (P < 0.05 for all). Delay in TTS > 42 days or >90th percentile was not associated with OS or CSS. CONCLUSION: Age, co-morbidity, and stage of cancer are associated with TTS. There was no association between TTS and CSS or OS.

Adjuvant Chemotherapy for Stage II Colon Cancer: Practice Patterns and Effectiveness in the General Population.

AIMS: Although guidelines do not recommend adjuvant chemotherapy (ACT) for stage II colon cancer, many state that ACT may be considered in high-risk disease. Here we describe practice patterns and outcomes associated with ACT in the general population. MATERIALS AND METHODS: All cases of colon cancer diagnosed in Ontario 2002-2008 were identified using the Ontario Cancer Registry, which was linked to electronic treatment records. Pathology reports were obtained for a 25% random sample of cases. High-risk disease was defined as: T4 tumours, <12 lymph nodes, poorly differentiated histology, lymphovascular invasion. Modified Poisson regression was used to evaluate factors associated with ACT. The Cox proportional hazards model was used to explore the association between ACT and cancer-specific (CSS) and overall survival. RESULTS: The study population included 2488 patients with stage II colon cancer; 1175 (47%) with high-risk disease. ACT was delivered to 18% of all patients and 24% of patients with high-risk disease. ACT rates were higher among younger patients (51% age 20-49 years versus 16% age 70-79, P < 0.001) and varied considerably across geographic regions (range 10-39%, P < 0.001). Among all patients with stage II colon cancer, ACT was not associated with improved CSS (hazard ratio 1.41, 95% confidence interval 1.09-1.82) or overall survival (hazard ratio 1.16, 95% confidence interval 0.94-1.42). Stratified survival analysis for patients with high-risk disease did not show benefit to ACT (CSS hazard ratio 1.14, 95% confidence interval 0.84-1.55; overall survival hazard ratio 1.02, 95% confidence interval 0.79-1.31). CONCLUSION: ACT use varies across age groups and geographic regions. ACT is not associated with improved survival among patients with stage II colon cancer including those with high-risk disease.

Sequential, cycling maintenance therapy for post transplant multiple myeloma.

High-dose chemotherapy with autologous stem cell transplantation in patients with newly diagnosed multiple myeloma can prolong survival but is not curative. Maintenance therapy post transplant may prolong the disease-free interval and impact overall survival. We have conducted a phase II pilot study of 28 post transplant myeloma patients treated with a sequential, cycling maintenance regimen. The regimen was designed to include a variety of active myeloma agents chosen for ease of administration to enhance patient compliance and scheduled sequentially to minimize toxicity. The 12-month cycling schedule included dexamethasone (months 1-3); melphalan and prednisone (months 4, 5); cyclophosphamide and prednisone (months 6, 7); alpha-interferon (months 8-10); followed by a drug holiday (months 11, 12). The regimen was generally well tolerated with five patients developing reversible grade III-IV toxicity (diabetes-induced hyperglycemia in four, neutropenia in one). There was one toxic death on study due to non-neutropenic pneumonia and sepsis. Median event-free survival from transplant was 36.9 months (95% CI 23.6 - upper limit not yet reached) with median overall survival not yet reached at a median follow-up of 44 months. This concept of cycling, sequential maintenance with various agents, perhaps including newer biological, targeted agents, warrants further investigation in multiple myeloma.

Surgical resection and peri-operative chemotherapy for colorectal cancer liver metastases: A population-based study.

BACKGROUND: Most literature describing surgery for colorectal cancer (CRC) liver metastases (LM) comes from high volume centres. Here, we report management and outcomes achieved in routine clinical practice. METHODS: All cases of CRC in Ontario who underwent resection of LM in 1994-2009 were identified using the population-based Ontario Cancer Registry. Electronic treatment records identified chemotherapy delivery. Temporal trends are described for 3 periods: 1994-1999, 2000-2004, 2005-2009. We describe volume of resected CRCLM as a ratio of incident cases per CRCLM resection. Overall (OS) and cancer-specific survival (CSS) are measured from time of LM resection. RESULTS: 2717 patients underwent resection of CRCLM. Between 1994 and 2009 there was a 78% increase in case volume; from one resection for every 48 incident cases to one resection for every 27 incident cases, p < 0.001. Use of peri-operative chemotherapy increased over study periods from 44% (306/700), to 52% (429/830), to 65% (777/1187, p < 0.001). Chemotherapy utilization rates varied across geographic regions (range 43%-69%, p < 0.001). Post-operative mortality rates at 30 and 90 days were 2.5% and 4.3% respectively. Five year OS during the study periods was 36% (95% CI 32-39%), 40% (95% CI 36-43%), and 46% (95% CI 43-49%) (p < 0.001); CSS was 38% (95% CI 35-42%), 42% (95% CI 38-45%), 49% (95% CI 44-53%) (p < 0.001). The temporal improvement in OS/CSS persisted on adjusted analyses. CONCLUSIONS: Outcomes of patients with resected CRCLM in routine practice is comparable to those reported from high volume centres. Survival improved over the study period despite a greater proportion of patients with CRC undergoing liver resection.

Adenovector engineered interleukin-2 expressing autologous plasma cell vaccination after high-dose chemotherapy for multiple myeloma--a phase 1 study.

Eight multiple myeloma patients participated in a phase I trial evaluating the feasibility and safety of subcutaneous vaccination with adenovirus engineered, autologous plasma cells after high-dose therapy. Plasma cells were concentrated from bone marrow harvests by negative selection and high gradient magnetic separation. The mean plasma cell yield was 2.61 x 10(8). Transgene expression measured 48 h after plasma cell infection with an IL-2 expressing adenovirus averaged 2.95 ng/ml/10(6) cells. Vaccine production was successful for 88% of patients. Two months after high-dose therapy, six patients received from one to five injections of 3.5-9.0 x 10(7) cells/vaccine. Vaccines were well tolerated with only minor systemic symptoms reported. Injection with tumor cells induced a local inflammatory response consisting predominantly of CD8+ and/or TIA-1+ T-lymphocytes. Myeloma specific anti-tumor responses, assessed by interferon-gamma (IFN-gamma) release and cytotoxic T cell killing of autologous tumor cells, were not enhanced after vaccination in one evaluable patient. Clinical response, manifested as a decrease in serum paraprotein, was not observed in the one patient who had measurable disease at the time of vaccination. These results demonstrate that the generation of adenovector modified plasma cell vaccines is technically feasible and can be safely administered post-transplant. Further studies of immunlogic and clinical efficacy are required.

Adoptive transfer of genetically modified human hematopoietic stem cells into preimmune canine fetuses.

To develop a surrogate model system for assaying gene transfer into human hematopoietic stem cells (HSCs) with in vivo repopulating potential, we injected human marrow cells transduced with a reporter retroviral vector in long-term marrow cultures (LTMCs), into the yolk sacs of preimmune canine fetuses. Of eight mid-gestation fetuses injected through the exteriorized uterine wall and under ultrasound guidance, seven were born alive. One puppy died in the neonatal period accidentally. The remaining six puppies are all healthy at 31 months of age. There was no evidence for graft-versus-host disease or any untoward effects of in utero adoptive transfer of transduced human LTMC cells. All puppies were chimeras. Human cells, detected by fluorescence in situ hybridization, were present in blood, declining from 38% to 0.05% between 10 and 44 weeks after birth. Corresponding numbers for marrow were from 20% to 0.05%. Human cells were also detected in assays of hematopoietic cell progenitors and in stimulated blood cultures. All six puppies were positive for the presence of proviral DNA at various time-points after birth. In three dogs, provirus was detected up to 41 weeks after birth in blood or marrow, and in one dog up to 49 weeks in blood. These data support the further development of this large-animal model system for studies of human hematopoiesis.

Engraftment of gene-marked hematopoietic progenitors in myeloma patients after transplant of autologous long-term marrow cultures.

We conducted a phase I hematopoietic stem cell (HSC) gene-marking trial in patients undergoing autologous blood or marrow stem cell transplant for the treatment of multiple myeloma. Between 500 and 1000 ml of bone marrow was harvested from each of 14 myeloma patients and 1 syngeneic donor. A mean of 3.3x10(9) cells per patient were plated in 20 to 50 long-term marrow culture (LTMC) flasks and maintained for 3 weeks. LTMCs were exposed on days 8 and 15 to clinical-grade neo(r)-containing retrovirus supernatant (G1Na). A mean of 8.23x10(8) day-21 LTMC cells containing 5.2x10(4) gene-marked granulocyte-macrophage progenitor cells (CFU-GM) were infused along with an unmanipulated peripheral blood stem cell graft into each patient after myeloablative therapy. Proviral DNA was detected in 71% of 68 tested blood and bone marrow samples and 150 of 2936 (5.1%) CFU-GM derived from patient bone marrow samples after transplant. The proportion of proviral DNA-positive CFU-GM declined from a mean of 9.8% at 3 months to a mean of 2.3% at 24 months postinfusion. Southern blots of 26 marrow and blood samples were negative. Semiquantitative PCR analysis indicated that gene transfer was achieved in 0.01-1% of total bone marrow and blood mononuclear cells (MNCs). Proviral DNA was also observed in EBV-transformed B lymphocytes, in CD34+ -enriched bone marrow cells, and in CFUs derived from the latter progenitors. Gene-modified cells were detected by PCR in peripheral blood and bone marrow for 24 months after infusion of LTMC cells. Sensitivity and specificity of the PCR assays were independently validated in four laboratories. Our data confirm that HSCs may be successfully transduced in stromal based culture systems. The major obstacle to therapeutic application of this approach remains the overall low level of genetically modified cells among the total hematopoietic cell pool in vivo.

Gene therapy for canine alpha-L-iduronidase deficiency: in utero adoptive transfer of genetically corrected hematopoietic progenitors results in engraftment but not amelioration of disease.

Canine alpha-L-iduronidase (iduronidase) deficiency is a model of the human lysosomal storage disorder mucopolysaccharidosis type I (MPS I). We used this canine model to evaluate the therapeutic potential of hematopoietic stem cell (HSC) gene therapy for enzyme deficiencies. In previous studies, iduronidase-deficient dogs infused with autologous marrow cells genetically modified to express iduronidase had long-term engraftment with provirally marked cells, but there was no evidence of proviral iduronidase expression or clinical improvement. The presence of humoral and cellular immune responses against iduronidase apparently abrogated the therapeutic potential of HSC gene therapy in these experiments. To evaluate HSC gene therapy for canine MPS I in the absence of a confounding immune response, we have now performed in utero adoptive transfer of iduronidase-transduced MPS I marrow cells into preimmune fetal pups. In three separate experiments, 17 midgestation fetal pups were injected with 0.5-1.5 x 10(7) normal or MPS I allogeneic long-term marrow culture (LTMC) cells transduced with neo(r)- or iduronidase-containing retroviral vectors. Nine normal and three MPS I pups survived the neonatal period and demonstrated engraftment of provirally marked progenitors at levels of up to 12% for up to 12 months. However, the proportion of provirally marked circulating leukocytes was approximately 1%. Neither iduronidase enzyme nor proviral-specific transcripts were detected in blood or marrow leukocytes of any MPS I dog. Humoral immune responses to iduronidase were not detected in neonates, even after "boosting" with autologous iduronidase-transduced LTMC cells. All MPS I dogs died at 8-11 months of age from complications of MPS I disease with no evidence of amelioration of MPS I disease. Our results suggest that iduronidase-transduced primitive hematopoietic progenitors can engraft in fetal recipients, contribute to hematopoiesis, and induce immunologic nonresponsiveness to iduronidase in MPS I dogs. However, the therapeutic potential of HSC gene transfer in this model of iduronidase deficiency appears to be limited by poor maintenance of proviral iduronidase gene expression and relatively low levels of genetically corrected circulating leukocytes.

Preclinical assessment of human hematopoietic progenitor cell transduction in long-term marrow cultures.

Long-term marrow cultures (LTMCs) were established from 27 human marrows. Hematopoietic cells were subjected to multiple rounds of exposure to retroviral vectors during 3 weeks of culture. Seven different retroviral vectors were evaluated. LTMCs were assessed for viability, replication-competent retrovirus, progenitors capable of proliferating in immune-deficient mice, and gene transfer. The average number of adherent cells and committed granulocyte-macrophage progenitors (CFU-GM) recovered from LTMCs was 28% and 11% of the input totals, respectively. There was no evidence by marker rescue assay or polymerase chain reaction (PCR) of replication-competent virus production during LTMC. No toxicity to cellular proliferation due to the transduction procedure was observed. The adherent layers of LTMCs exposed to retroviral vectors were positive for proviral DNA by PCR and by Southern blot analysis. Fifty-three percent of 1,427 individual CFU-GM from transduced LTMC adherent layers were positive for vector-derived DNA. For neocontaining vectors, the average G418 resistance was 28% of 1,393 LTMC-derived CFU-GM. Forty percent of 187 tissues from 30 immune-deficient mice injected with human LTMC cells were positive for human DNA 4-5 weeks after adoptive transfer. These studies indicate that multiple exposures of human LTMCs to retroviral vectors result in consistent and reproducible LTMC viability and gene transfer into committed progenitors. Our results further support the use of transduced LTMC cells in clinical trials of hematopoietic stem cell gene transfer.

Stem cell function and engraftment is not affected by "in vivo purging" with rituximab for autologous stem cell treatment for patients with low-grade non-Hodgkin's lymphoma.

The chimeric anti-CD20 monoclonal antibody rituximab (Rituxan; IDEC Pharmaceuticals, San Diego, CA, and Genentech, Inc, San Francisco, CA) has recently been approved by the US Food and Drug Administration as single-agent treatment of relapsed/refractory low-grade or follicular non-Hodgkin's lymphoma. Initial results from the pivotal clinical trial revealed that response rates to rituximab were higher in patients who previously had high-dose therapy and autologous stem cell transplantation. We have initiated a clinical trial that combines the use of rituximab with high-dose chemotherapy followed by autologous stem cell transplantation for patients with chemosensitive relapsed follicular small cleaved or mantle cell lymphoma. A unique feature of this study is that in addition to eight maintenance infusions of rituximab after autologous stem cell transplantation, patients also received rituximab 375 mg/m2 2 days before a granulocyte colony-stimulating factor-mobilized stem cell collection as "in vivo purge." We report on preliminary results demonstrating the safety and efficacy of the in vivo purge on 10 patients undergoing stem cell mobilization, nine of whom have already undergone transplantation. The peripheral blood CD34+ counts were 14.92 and 20 x 10(6)/L on day 4 and day 5, respectively, of the stem cell mobilization with granulocyte colony-stimulating factor. This compares with 11.7 and 11.8 x 10(6)/L, respectively, for the control population. The median CD34 stem cell yield in the graft collection was 3.7 x 10(6)/kg in patients receiving rituximab in vivo purge compared with 3.1 x 10(6)/kg in the control population. The target stem cell collection was successfully collected in six of 10 patients in a 1-day single large-volume leukapheresis collection, while two patients required 2 days and the last two patients required 3 days. Functional assays revealed the stem cell colony-forming unit-granulocyte monocyte and burst-forming unit-erythrocyte to be 55 and 44 colonies per plate, respectively, for the patients receiving the in vivo rituximab purge. This compares favorably with 37 and 38.5 colonies per plate, respectively, for the control population. Neutrophil engraftment took a median of 11 days for both cohorts; platelet independence was achieved in 8 days compared with 10 days for the control population. The median number of platelet transfusions was two for patients receiving rituximab and 2.5 for the control group. Assessment of serum cytokines immediately before the rituximab infusion during the stem cell mobilization and immediately after revealed a twofold to sevenfold increase in interleukin-1beta, tumor necrosis factor-alpha, and interleukin-6. The polymerase chain reaction analysis for minimal residual disease in stem cell collections and in peripheral blood and bone marrow samples of these patients will help to determine the efficacy of rituximab in vivo purge on disease progression.

Combination therapy with anti-ICOS and cyclosporine enhances cardiac but not islet allograft survival.

The blockade of costimulatory signals is a powerful strategy to prevent allograft rejection and facilitate transplantation tolerance. In recent years, a series of novel costimulatory molecules have been identified, including an inducible costimulatory molecule (ICOS). To date, little has been uncovered regarding the therapeutic potential of blocking ICOS signaling in the setting of transplantation. In a fully MHC-mismatched mouse model, we studied the effect of blocking ICOS signaling using a specific monoclonal antibody (anti-ICOS mAb) in combination with cyclosporine on cardiac and islet allograft survival. We demonstrated that combined treatment with anti-ICOS mAb and cyclosporine can induce long-term graft acceptance in cardiac but not islet allografts, suggesting that the type of transplanted tissue significantly influences the immunologic patterns of graft acceptance or rejection in this model.

Asymptomatic bacteriuria in antenatal patients in ilorin, Nigeria.

OBJECTIVE: To determine the prevalence of asymptomatic bacteriuria, bacteriology and sensitivity pattern in Ilorin using the gold standard of urine culture. METHODS: A prospective study was carried out from 1st July to 31st October 2007, at the University of Ilorin Teaching Hospital (UITH) on 125 consenting asymptomatic pregnant women. A structured proforma was used to collect information from the women and a midstream urine specimen collected for bacteriological culture. RESULTS: Of the 125 pregnant women, 50 had bacteriuria on urine culture giving a prevalence of 40%. The mean age of the women was 28.5 years with a standard deviation of 4.95. The age ranged between 14 and 40 years. Staphylococcus aureus was the commonest pathogen isolated (72%), followed by Proteus spp (14%). Most of the organisms showed good sensitivity to Nitrofurantoin and gentamicin. CONCLUSION: The prevalence of asymptomatic bacteriuria in Ilorin is high and routine urine culture is advocated for all pregnant women at booking.