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Background A proportion of candidates for high-dose chemotherapy with autologous PBPC support (HDC-PBPCS) will not provide an adequate PBPC yield from their first mobilization. The value of re-mobilization and the best regimen for re-mobilization in these patients is unclear. Methods: In 23 patients who failed to provide ≥ 3 × 10[sup 6] CD34[sup +] cells/kg after their first mobilization, PBPC were re-mobilized using a regimen of simultaneous administration of G-CSF and GM-CSF (10 μg/kg/day each) with leukaphereses (LP) starting Day 4 or 5 of CSF administration. Yields of WBC/kg, MNC/kg and CD34[sup +] cells/kg/L of processed blood total CD34[sup +] were compared between the first and second mobilization in each patient. The ability of the combined yield from the two mobilizations to achieve the desired threshold PBPC yield and the tolerability of the re-mobilization were determined. Results: The re-mobilization regimen was well-tolerated and no patient discontinued the regimen because of toxicity. Median collected WBC/kg/L (1.37 × 10[sup 7] versus 2.62 × 10[sup 7], p = 0.0065), MNC/kg/L (0.77 × 10[sup 7] versus 1.97 × 10[sup 7], p = 0.0003), CD34[sup +]cells/kg/L (1.64 × 10[sup 7] versus 4.18 × 10[sup 7], p = 0.001) were significantly higher after the second mobilization (G-CSF/GM-CSF combination). Percentage of CD34[sup +] cells in the leukapheresis was also significantly higher after the second mobilization (median 0.104% versus 0.195%, p = 0.036). Twelve of 22 patients achieved the target PBPC dose (> 3 × 10[sup 6]/CD34[sup +] cells/kg) after two mobilizations (six patients achieved the target from the second mobilization alone). A further eight underwent HDC-PBPCS without achieving the target PBPC dose. These patients experienced a significant delay in neutrophil and platelet engraftment when compared with those patients achieving the target dose. Discussion: This study demonstrates that the combination of G-CSF and GM-CSF is an effective and tolerable method for re-mobilization of PBPC in patients who fail to provide an adequate yield from their first mobilization. [ABSTRACT FROM AUTHOR]

The GM.CD40L vaccine, which recruits and activates dendritic cells, migrates to lymph nodes, activating T cells and leading to systemic tumor cell killing. When combined with the CCL21 chemokine, which recruits T cells and enhances T-cell responses, additive effects have been demonstrated in non-small cell lung cancer mouse models. Here, we compared GM.CD40L versus GM.CD40L plus CCL21 (GM.CD40L.CCL21) in lung adenocarcinoma patients with ≥ 1 line of treatment. In this phase I/II randomized trial (NCT01433172), patients received intradermal vaccines every 14 days (3 doses) and then monthly (3 doses). A two-stage minimax design was used. During phase I, no dose-limiting toxicities were shown in three patients who received GM.CD40L.CCL21. During phase II, of evaluable patients, 5/33 patients (15.2%) randomized for GM.DCD40L (p = .023) and 3/32 patients (9.4%) randomized for GM.DCD40L.CCL21 (p = .20) showed 6-month progression-free survival. Median overall survival was 9.3 versus 9.5 months with GM.DCD40L versus GM.DCD40L.CCL21 (95% CI 0.70-2.25; p = .44). For GM.CD40L versus GM.CD40L.CCL21, the most common treatment-related adverse events (TRAEs) were grade 1/2 injection site reaction (51.4% versus 61.1%) and grade 1/2 fatigue (35.1% versus 47.2%). Grade 1 immune-mediated TRAEs were isolated to skin. No patients showed evidence of pseudo-progression or immune-related TRAEs of grade 1 or greater of pneumonitis, endocrinopathy, or colitis, and none discontinued treatment due to toxicity. Although we found no significant associations between vaccine immunogenicity and outcomes, in limited biopsies, one patient treated with GMCD40L.CCL21 displayed abundant tumor-infiltrating lymphocytes. This possible effectiveness warrants further investigation of GM.CD40L in combination approaches.

Local radiotherapy plus intratumoral syngeneic dendritic cell injection can mediate apoptosis/cell death and immunological tumor eradication in murine models. A novel method of coordinated intraprostatic, autologous dendritic cell injection together with radiation therapy was prospectively evaluated in five HLA-A2(+) subjects with high-risk, localized prostate cancer, using androgen suppression, 45 Gy external beam radiation therapy in 25 fractions over 5 weeks, dendritic cell injections after fractions 5, 15 and 25 and then interstitial radioactive seed placement. Serial prostate biopsies before and during treatment showed increased apoptotic cells and parenchymal distribution of CD8(+) cells. CD8(+) T-cell responses to test peptides were assessed using an enzyme-linked immunosorbent spot IFN-γ production assay, demonstrating some prostate cancer-specific protein-derived peptides associated with increased titer. In conclusion, the technique was feasible and well-tolerated and specific immune responses were observable. Future trials could further test the utility of this approach and improve on temporal coordination of intratumoral dendritic cell introduction with particular timelines of therapy-induced apoptosis.

We report a single center phase II trial of sequential vaccination followed with vaccine plus interleukin-2 (IL-2). Vaccination consisted of autologous cells cultured from primary tumor or resected metastasis, transduced to express B7.1 surface molecule and then irradiated. The vaccine would hypothetically costimulate tumor-reactive T cells before IL-2 exposure. Treatment plan was 3 subcutaneous vaccine injections at 4-week intervals and subcutaneous IL-2 treatment for 6 weeks starting at week 7. Sixty-six patients enrolled, of whom 39 received at least 1 vaccine; most observed toxicity was attributable to IL-2 not vaccine; best responses were 3% pathologic complete response, 5% partial response, 64% stable disease, and 28% disease progression. Median survival was 21.8 months (95% confidence interval 17.8 to 29.6). Significant postvaccination increases in IFN-gamma responses to autologous tumor were observed in 2/26 cases. Eighty-one percent of posttreatment subdermal delayed-type hypersensitivity tests (using nontransduced, irradiated autologous tumor cells) had biopsies demonstrating injection site lymphocytic infiltration. Post hoc comparison of the median survival of subjects whose biopsies had lymphocytic infiltration appears longer than in the 19% noninfiltrated (28.4 vs. 17.8 mo, P=0.045, two-sided log-rank test). The single arm design precludes conclusive comparison of objective response rates (not different here) or median survival (longer here) versus those of historical series using similar IL-2 schedules alone. Better outcomes could be logically associated to vaccine response (detectable lymphocytic infiltrates) or to random events that a single arm study design cannot address. This vaccine approach may merit further clinical development.

We have developed an approach for identifying primitive mobilized peripheral blood cells (PBSC) that express high levels of aldehyde dehydrogenase (ALDH). PBSC were stained with a fluorescent ALDH substrate, termed BODIPY trade mark -aminoacetaldehyde (BAAA), and then analysed using flow cytometry. A population of cells with a low side scatter (SSC) and a high level of BAAA staining, termed the SSCloALDHbr population, was readily discriminated and comprised a mean of 3 +/- 5% of leukapheresis samples. A mean of 73 +/- 11% of the SSCloALDHbr population expressed CD34 and 56 +/- 25% of all the mobilized CD34+ cells resided within the SSCloALDHbr population. The SSCloALDHbr population was largely depleted of cells with mature phenotypes and enriched for cells with immature phenotypes. Sorted SSCloALDHbr and SSCloALDHbr CD34+ PBSC were enriched for progenitors with the ability to (1) generate colony-forming units (CFU) and long-term culture (LTC)-derived CFU, (2) expand in primary and secondary LTC, and (3) generate multiple cell lineages. In 21 cancer patients who had undergone autologous PBSC transplantation, the number of infused SSCloALDHbr cells/kg highly correlated with the time to neutrophil and platelet engraftment (P < 0.015 and P < 0.003 respectively). In summary, peripheral blood SSCloALDHbr cells have the phenotypic and functional properties of primitive haematopoietic cells and their number correlates with engraftment following autologous transplantation.

The SteriCell cell processing instrument is a good choice for a stem cell processing laboratory that is of sufficient size that they cannot share an apheresis machine with the blood bank. It is a laboratory instrument, with no facility for patient connection. Because of its minimal size and weight, it is easily stored in a cramped laboratory. Its automated programs are appropriate for processing of bone marrow and peripheral blood stem cells, and it is quite easy to learn how to use (in our laboratory, most individuals have been completely facile with the SteriCell after fewer than six processings). Based on reported results from other instruments, the SteriCell provides cell yields that are comparable to competing instruments. Service (provided by Haemonetics) has been satisfactory, and support from Terumo has been excellent. We can recommend this instrument to any other laboratory.

We have measured the presence of granulocyte-macrophage colony forming cells (CFU-GM) and CD34+ cells in blood and bone marrow. We have compared these hematopoietic cell assays using regression analysis. We have found that under the limited and specific case of blood cells from an individual recovering from myelo-suppressive chemotherapy, the fraction of cells that is CD34 positive is predictive of the number of granulocyte-macrophage colonies (CFU-GM) which will grow. This result is in agreement with published data. We have found, however, that in bone marrow aspirates, or in the blood of individuals recovering from cyclophosphamide chemotherapy and receiving either granulocyte-macrophage colony stimulating factor (G-CSF) or folinic acid (FA) therapy, there is poor correlation between CD34+ cell fraction and CFU-GM. Accordingly, the use of CD34+ fraction cannot be relied upon to substitute for the CFU-GM assay in assessing the hematopoietic cell content of blood or bone marrow samples.