Paul J. Mosca

Immunotherapy with Autologous, Human Dendritic Cells Transfected with Carcinoembryonic Antigen mRNA

Immunizations with dendritic cells (DC) transfected with RNA encoding tumor antigens induce potent tumor antigen-specific immune responses in vitro and in murine models. We performed a phase I study of patients with advanced carcinoembryonic antigen (CEA)-expressing malignancies followed by a phase II study of patients with resected hepatic metastases of colon cancer to assess safety and feasibility of administering autologous DC loaded with CEA mRNA. The immunizations were well tolerated. Of the 24 evaluable patients in the dose-escalation phase, there was 1 complete response (by tumor marker), 2 minor responses, 3 with stable disease, and 18 with progressive disease. In the phase II study, 9 of 13 patients have relapsed at a median of 122 days. Evidence of an immunologic response was demonstrated in biopsies of DC injection sites and peripheral blood of selected patients. We conclude that it is feasible and safe to administer mRNA-loaded DC to patients with advanced malignancies.

The plant amino acid mimosine may inhibit initiation at origins of replication in Chinese hamster cells.

An understanding of replication initiation in mammalian cells has been hampered by the lack of mutations and/or inhibitors that arrest cells just prior to entry into the S period. The plant amino acid mimosine has recently been suggested to inhibit cells at a regulatory step in late G1. We have examined the effects of mimosine on cell cycle traverse in the mimosine [corrected]-resistant CHO cell line CHOC 400. When administered to cultures for 14 h after reversal of a G0 block, the drug appears to arrest the population at the G1/S boundary, and upon its removal cells enter the S phase in a synchronous wave. However, when methotrexate is administered to an actively dividing asynchronous culture, cells are arrested not only at the G1/S interface but also in early and middle S phase. Most interestingly, two-dimensional gel analysis of replication intermediates in the initiation locus of the amplified dihydrofolate reductase domain suggests that mimosine may actually inhibit initiation. Thus, this drug represents a new class of inhibitors that may open a window on regulatory events occurring at individual origins of replication.

Preoperative Mobilization of Circulating Dendritic Cells by Flt3 Ligand Administration to Patients With Metastatic Colon Cancer

PURPOSE To evaluate preoperative dendritic cell (DC) mobilization and tumor infiltration after administration of Flt3 ligand (Flt3L) to patients with metastatic colon cancer. PATIENTS AND METHODS Twelve patients with colon cancer metastatic to the liver or lung received Flt3L (20 microg/kg/d subcutaneously for 14 days for one to three cycles at monthly intervals) before attempted metastasectomy. The number and phenotype of DCs mobilized into peripheral-blood mononuclear cells (PBMCs) were evaluated by flow cytometry. After surgical resection, metastatic tumor tissue was evaluated for DC infiltration. In vivo immune responses to recall antigens were measured. RESULTS After Flt3L administration, on average, the total number of leukocytes in the peripheral blood increased from 5.9 +/- 1.0 x 10(3)/mm(3) to 11.2 +/- 3.8 x 10(3)/mm(3) (mean +/- SD, P: =. 0001). The percentage of CD11c(+)CD14(-) DCs in PBMCs increased from 2.4% +/- 1.8% to 8.8% +/- 4.7% (P: =.004). Delayed-type hypersensitivity (DTH) responses to recall antigens (CANDIDA:, mumps, and tetanus) showed marginally significant increases in reactivity after Flt3L administration (P: =.06, P: =.03, and P: =.08, respectively). An increase in the number of DCs was observed at the periphery of the tumors of patients who received Flt3L compared with those of patients who had not. CONCLUSION Flt3L is capable of mobilizing DCs into the peripheral blood of patients with metastatic colon cancer and may be associated with increases in DC infiltration in the peritumoral regions. Flt3L mobilization is associated with a trend toward increased DTH responses to recall antigens in vivo. The use of Flt3L to increase circulating DCs for cancer immunotherapy should be considered.

Targeting Akt3 signaling in malignant melanoma using isoselenocyanates.

Purpose: Melanoma is the most invasive and deadly form of skin cancer. Few agents are available for treating advanced disease to enable long-term patient survival, which is driving the search for new compounds inhibiting deregulated pathways causing melanoma. Akt3 is an important target in melanomas because its activity is increased in ∼70% of tumors, decreasing apoptosis in order to promote tumorigenesis. Experimental Design: Because naturally occurring products can be effective anticancer agents, a library was screened to identify Akt3 pathway inhibitors. Isothiocyanates were identified as candidates, but low potency requiring high concentrations for therapeutic efficacy made them unsuitable. Therefore, more potent analogs called isoselenocyanates were created using the isothiocyanate backbone but increasing the alkyl chain length and replacing sulfur with selenium. Efficacy was measured on cultured cells and tumors by quantifying proliferation, apoptosis, toxicity, and Akt3 pathway inhibition. Results: Isoselenocyanates significantly decreased Akt3 signaling in cultured melanoma cells and tumors. Compounds having 4 to 6 carbon alkyl side chains with selenium substituted for sulfur, called ISC-4 and ISC-6, respectively, decreased tumor development by ∼60% compared with the corresponding isothiocyanates, which had no effect. No changes in animal body weight or in blood parameters indicative of liver-, kidney-, or cardiac-related toxicity were observed with isoselenocyanates. Mechanistically, isoselenocyanates ISC-4 and ISC-6 decreased melanoma tumorigenesis by causing an ∼3-fold increase in apoptosis. Conclusions: Synthetic isoselenocyanates are therapeutically effective for inhibiting melanoma tumor development by targeting Akt3 signaling to increase apoptosis in melanoma cells with negligible associated systemic toxicity.

A mouse-human phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer

A first-in-human phase 1 clinical trial of the PEGylated protease-activated fluorescent probe, LUM015, enables tumor imaging at a safe and tolerable dose in humans. Protease probe tested in humans Cancer cells secrete more of the protease cathepsin than healthy cells, partly as a way to enzymatically remodel their surroundings for tumor growth and metastasis. Whitley et al. developed an imaging probe that could be activated in the presence of these cathepsins, thus allowing surgeons to distinguish tumor margins intraoperatively. Their probe, called LUM015, was able to signal the presence of cancer in vivo in a mouse sarcoma model, and in a so-called “co-clinical trial” in 15 patients, it was safe and cleaved as expected in different types of tumor tissues. With favorable biodistribution and pharmacokinetics also demonstrated, protease-activated probes are now poised for further adaptation to tumor resections, signaling the presence of residual cancer. Local recurrence is a common cause of treatment failure for patients with solid tumors. Intraoperative detection of microscopic residual cancer in the tumor bed could be used to decrease the risk of a positive surgical margin, reduce rates of reexcision, and tailor adjuvant therapy. We used a protease-activated fluorescent imaging probe, LUM015, to detect cancer in vivo in a mouse model of soft tissue sarcoma (STS) and ex vivo in a first-in-human phase 1 clinical trial. In mice, intravenous injection of LUM015 labeled tumor cells, and residual fluorescence within the tumor bed predicted local recurrence. In 15 patients with STS or breast cancer, intravenous injection of LUM015 before surgery was well tolerated. Imaging of resected human tissues showed that fluorescence from tumor was significantly higher than fluorescence from normal tissues. LUM015 biodistribution, pharmacokinetic profiles, and metabolism were similar in mouse and human subjects. Tissue concentrations of LUM015 and its metabolites, including fluorescently labeled lysine, demonstrated that LUM015 is selectively distributed to tumors where it is activated by proteases. Experiments in mice with a constitutively active PEGylated fluorescent imaging probe support a model where tumor-selective probe distribution is a determinant of increased fluorescence in cancer. These co-clinical studies suggest that the tumor specificity of protease-activated imaging probes, such as LUM015, is dependent on both biodistribution and enzyme activity. Our first-in-human data support future clinical trials of LUM015 and other protease-sensitive probes.

Histopathologic characteristics, recurrence patterns, and survival of 129 patients with desmoplastic melanoma

BackgroundDesmoplastic melanoma (DM) has been associated with higher local recurrence rates than other types of cutaneous melanoma. Current controversies regarding locoregional treatment strategies warrant further investigation.MethodsRetrospective review of a prospectively maintained melanoma database identified 129 patients with DM out of >12,500 melanoma patients referred for treatment from 1980 to 2003. Clinical and histopathologic characteristics, recurrence, and survival were analyzed.ResultsThe median follow-up was 4.0 years. Of the 129 patients identified, 82 (63.6%) were male, and the median age was 55.2 years. American Joint Committee on Cancer staging was I, II, and III in 25.6%, 68.0%, and 6.4% of patients, respectively, and the mean tumor thickness was 4.42 mm. Overall survival was 76% at 5 years and 64% at 10 years; median survival was 13.0 years. A total of 51 patients (39.5%) experienced disease recurrence, with a median time to recurrence of 1.3 years. The first recurrence was local in 18 patients (14.0%), nodal in 18 patients (14.0%), and distant in 15 patients (11.6%), with median survivals of 6.7, 7.8, and 1.8 years, respectively. Statistically significant predictors of recurrence were a final positive margin status and stage, and predictors of overall survival were patient age and stage.ConclusionsCompared with other types of melanoma, DMs do demonstrate a tendency toward local recurrence, thus suggesting that narrower excision margins may not be appropriate in this population. Scrutiny of final surgical margins is critical to the local management of DM. In addition, the potential for regional nodal involvement must be considered at the time of diagnosis and during surveillance for disease recurrence.

Immunoregulatory T cells in cancer immunotherapy.

Many of the tumour antigens targeted by active immunisation strategies are in fact self-antigens. Successful anticancer immunotherapy will therefore require not only potent methods of T cell activation, but also successful interference with mechanisms of immune tolerance that have evolved to prevent tissue destruction by autoreactive T cells. In addition to thymic deletion, anergy and skewing of T cell cytokine expression, a role for immunoregulatory T cells in the maintenance of self-tolerance has been suggested. Suppression of autoreactive T cells by regulatory T cells has been suggested to occur by both cytokine and cell-contact-dependent mechanisms. In murine models, suppression of auto-reactive T cells mediated by cell contact has been attributed to a population of spontaneously occurring CD4+CD25+ T cells. Cells with similar phenotype and function have been found in healthy humans. In murine models, these cells behave as regulatory T cells, counteracting autoimmune and inflammatory reactions, and have a role in tolerance and in peripheral T cell homeostasis. Of interest for cancer immunotherapy is the fact that depleting these cells results in the induction of antitumour immune responses, particularly after tumour specific vaccination. One hypothesis is that depleting these CD4+CD25+ counter-regulatory T cells in humans with cancer will enhance the efficacy of anticancer immunisations.

Mimosine Targets Serine Hydroxymethyltransferase

The plant amino acid, mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells (Mosca, P. J., Dijkwel, P. A., and Hamlin, J. L.(1992) Mol. Cell. Biol. 12, 4375-4383). Mimosine appears to prevent the formation of replication forks at early-firing origins when delivered to mammalian cells approaching the G1/S boundary, and blocks DNA replication when added to S phase cells after a lag of ∼2.5 h. We have shown previously that [3H]mimosine can be specifically photo-cross-linked both in vivo and in vitro to a 50-kDa polypeptide (p50) in Chinese hamster ovary (CHO) cells. In the present study, six tryptic peptides (58 residues total) from p50 were sequenced by tandem mass spectrometry and their sequences were found to be at least 77.5% identical and 96.5% similar to sequences in rabbit mitochondrial serine hydroxymethyltransferase (mSHMT). This assignment was verified by precipitating the [3H]mimosine-p50 complex with a polyclonal antibody to rabbit cSHMT. The 50-kDa cross-linked product was almost undetectable in a mimosine-resistant CHO cell line and in a CHO gly− cell line that lacks mitochondrial, but not cytosolic, SHMT activity. The gly− cell line is still sensitive to mimosine, suggesting that the drug may inhibit both the mitochondrial and the cytosolic forms. SHMT is involved in the penultimate step of thymidylate biosynthesis in mammalian cells and, as such, is a potential target for chemotherapy in the treatment of cancer.

Dendritic cell vaccines.

Dendritic cells are antigen-presenting cells that have been shown to stimulate tumor antigen-specific T cell responses in preclinical studies. Consequently, there has been intense interest in developing dendritic cell based cancer vaccines. A variety of methods for generating dendritic cells, loading them with tumor antigens, and administering them to patients have been described. In recent years, a number of early phase clinical trials have been performed and have demonstrated the safety and feasibility of dendritic cell immunotherapies. A number of these trials have generated valuable preliminary data regarding the clinical and immunologic response to DC-based immunotherapy. The emphasis of dendritic cell immunotherapy research is increasingly shifting toward the development of strategies to increase the potency of dendritic cell vaccine preparations.

Circulating Tumor Cells in Melanoma Patients

Circulating tumor cells (CTCs) are of recognized importance for diagnosis and prognosis of cancer patients. With melanoma, most studies do not show any clear relationship between CTC levels and stage of disease. Here, CTCs were enriched (∼400X) from blood of melanoma patients using a simple centrifugation device (OncoQuick), and 4 melanocyte target RNAs (TYR, MLANA, MITF, and MIF) were quantified using QPCR. Approximately one-third of melanoma patients had elevated MIF and MLANA transcripts (p<0.0001 and p<0.001, respectively) compared with healthy controls. In contrast, healthy controls had uniformly higher levels of TYR and MITF than melanoma patients (p<0.0001). There was a marked shift of leukocytes into the CTC-enriched fractions (a 430% increase in RNA recovery, p<0.001), and no relationship between CTC levels and stage of disease was found. CTCs were captured on microfabricated filters and cultured. Captured melanoma CTCs were large cells, and consisted of 2 subpopulations, based on immunoreactivity. One subpopulation (∼50%) stained for both pan-cytokeratin (KRT) markers and the common leukocyte marker CD-45, whereas the second subpopulation stained for only KRT. Since similar cells are described in many cancers, we also examined blood from colorectal and pancreatic cancer patients. We observed analogous results, with most captured CTCs staining for both CD-45/KRT markers (and for the monocyte differentiation marker CD-14). Our results suggest that immature melanocyte-related cells (expressing TYR and MITF RNA) may circulate in healthy controls, although they are not readily detectable without considerable enrichment. Further, as early-stage melanomas develop, immature melanocyte migration into the blood is somehow curtailed, whereas a significant proportion of patients develop elevated CTC levels (based on MIF and MLANA RNAs). The nature of the captured CTCs is consistent with literature describing leukocyte/macrophage-tumor cell fusion hybrids, and their role in metastatic progression.