Cheryl F. Murphy

Establishing human prostate cancer cell xenografts in bone: Induction of osteoblastic reaction by prostate‐specific antigen‐producing tumors in athymic and SCID/bg mice using LNCaP and lineage‐derived metastatic sublines

LNCaP lineage‐derived human prostate cancer cell lines C4‐2 and C4‐2B4 acquire androgen independence and osseous metastatic potential in vivo. Using C4‐2 and C4‐2B4 the goals of the current investigation were 1) to establish an ideal bone xenograft model for prostate cancer cells in intact athymic or SCID/bg mice using an intraosseous route of tumor cell administration and 2) to compare prostate cancer metastasis by administering cells either through intravenous (i.v.) or intracardiac administration in athymic or SCID/bg mice. Subsequent to tumor cell administration, prostate cancer growth in the skeleton was assessed by radiographic bone density, serum prostate‐specific antigen (PSA) levels, presence of hematogenous prostate cancer cells and histopathologic evaluation of tumor specimens in the lymph node and skeleton. Our results show that whereas LNCaP cells injected intracardially failed to develop metastasis, C4‐2 cells injected similarly had the highest metastatic capability in SCID/bg mice. Retroperitoneal and mediastinal lymph node metastases were noted in 3/7 animals, whereas 2/7 animals developed osteoblastic spine metastases. Intracardiac injection of C4‐2 in athymic hosts produced spinal metastases in 1/5 animals at 8–12 weeks post‐injection; PC‐3 injected intracardially also metastasized to the bone but yielded osteolytic responses. Intravenous injection of either LNCaP or C4‐2 failed to establish tumor colonies. Intrailiac injection of C4‐2 but not LNCaP nor C4‐2B4 cells in athymic mice established rapidly growing tumors in 4/8 animals at 2–7 weeks after inoculation. Intrafemoral injection of C4‐2 (9/16) and C4‐2B4 (5/18) but not LNCaP (0/13) cells resulted in the development of osteoblastic bone lesions in athymic mice (mean: 6 weeks, range: 3–12 weeks). In SCID/bg mice, intrafemoral injection of LNCaP (6/8), C4‐2 (8/8) and C4‐2B4 (8/8) cells formed PSA‐producing, osteoblastic tumors in the bone marrow space within 3–5 weeks after tumor cell inoculation. A stepwise increase of serum PSA was detected in all animals. Reverse transcription‐polymerase chain reaction (RT‐PCR) to detect hematogenously disseminated prostate cancer cells could not be correlated to either serum PSA level or histological evidence of tumor cells in the marrow space. We have thus established a PSA‐producing and osteoblastic human prostate cancer xenograft model in mice. Int. J. Cancer 77:887–894, 1998.© 1998 Wiley‐Liss, Inc.

Immunologic and clinical outcomes of a randomized phase II trial of two multipeptide vaccines for melanoma in the adjuvant setting.

Purpose: Human melanoma cells express shared antigens recognized by CD8+ T lymphocytes, the most common of which are melanocytic differentiation proteins and cancer-testis antigens. However, peptide vaccines for melanoma usually target only one or two MHC class I–associated peptide antigens. Because melanomas commonly evade immune recognition by selective antigen loss, optimization of melanoma vaccines may require development of more complex multipeptide vaccines. Experimental Design: In a prospective randomized clinical trial, we have evaluated the safety and immunogenicity of a vaccine containing a mixture of 12 peptides from melanocytic differentiation proteins and cancer-testis antigens, designed for human leukocyte antigen types that represent 80% of the melanoma patient population. This was compared with a four-peptide vaccine with only melanocytic differentiation peptides. Immune responses were assessed in peripheral blood and in vaccine-draining lymph nodes. Results: These data show that (a) the 12-peptide mixture is immunogenic in all treated patients; (b) immunogenicity of individual peptides is maintained despite competition with additional peptides for binding to MHC molecules; (c) a broader and more robust immune response is induced by vaccination with the more complex 12-peptide mixture; and (d) clinical outcome in this peptide vaccine trial correlates with immune responses measured in the peripheral blood lymphocytes. Conclusions: These data support continued investigation of complex multipeptide vaccines for melanoma.

MAGE-A1-, MAGE-A10-, and gp100-Derived Peptides Are Immunogenic When Combined with Granulocyte-Macrophage Colony-Stimulating Factor and Montanide ISA-51 Adjuvant and Administered as Part of a Multipeptide Vaccine for Melanoma

Twelve peptides derived from melanocyte differentiation proteins and cancer-testis Ags were combined and administered in a single mixture to patients with resected stage IIB, III, or IV melanoma. Five of the 12 peptides included in this mixture had not previously been evaluated for their immunogenicity in vivo following vaccination. We report in this study that at least three of these five peptides (MAGE-A196–104, MAGE-A10254–262, and gp100614–622) are immunogenic when administered with GM-CSF in Montanide ISA-51 adjuvant. T cells secreting IFN-γ in response to peptide-pulsed target cells were detected in peripheral blood and in the sentinel immunized node, the node draining a vaccine site, after three weekly injections. The magnitude of response typically reached a maximum after two vaccines, and though sometimes diminished thereafter, those responses typically were still detectable 6 wks after the last vaccines. Most importantly, tumor cell lines expressing the appropriate HLA-A restriction element and MAGE-A1, MAGE-A10, or gp100 proteins were lysed by corresponding CTL. This report supports the continued use of the MAGE-A196–104, MAGE-A10254–262, and gp100614–622 epitopes in peptide-based melanoma vaccines and thus expands the list of immunogenic peptide Ags available for human use. Cancer-testis Ags are expressed in multiple types of cancer; thus the MAGE-A196–104 and MAGE-A10254–262 peptides may be considered for inclusion in vaccines against cancers of other histologic types, in addition to melanoma.

A Multipeptide Vaccine is Safe and Elicits T-cell Responses in Participants With Advanced Stage Ovarian Cancer

Nine participants with epithelial ovarian, fallopian tube, or primary peritoneal carcinoma, who were human leukocyte antigen (HLA)-A1+, HLA-A2+, or HLA-A3+, were eligible to enroll in a phase 1 study designed to assess the safety and immunogenicity of a peptide-based vaccine. Participants received 5 class I major histocompatibility complex-restricted synthetic peptides derived from multiple ovarian cancer-associated proteins plus a class II major histocompatibility complex-restricted synthetic helper peptide derived from tetanus toxoid protein. The vaccines were administered with granulocyte macrophage-colony stimulating factor in Montanide ISA-51 adjuvant over a 7-week period. All vaccine-related toxicities were grade 1 to 2, the most common being injection site reaction (grade 2, 100%), fatigue (grade 1, 78%), and headache (grade 1, 67%). Lymphocytes from the peripheral blood and a node draining a secondary vaccine site (sentinel immunized node) were harvested during the course of vaccination and T-cell responses to the peptides were evaluated using an enzyme-linked immunosorbent spot assay. CD8+ T-cell responses were detected in 1 participant ex vivo and in 8 of 9 participants (89%) after in vitro stimulation. All 4 HLA-A2 and HLA-A3–restricted peptides were immunogenic. This includes 2 peptides, folate binding protein (FBP191−199) and Her-2/neu754−762, which had not previously been evaluated in vaccines in humans. Responding T cells required over 200 nM for half-maximal reactivity. These data support continued investigation of these peptides as immunogens for patients with ovarian cancer but, owing to low potency, also suggest a need for additional immunomodulation in combination with vaccines to increase the magnitude and to improve the quality of the T-cell responses.

Immunogenicity for CD8+ and CD4+ T cells of 2 formulations of an incomplete freund's adjuvant for multipeptide melanoma vaccines.

An incomplete Freunds adjuvant (IFA) commonly used in experimental cancer vaccines has recently been reformulated. Oleic acid used in the surfactant was purified from a vegetable source (olives, IFA-VG) rather than an animal source (beef tallow, IFA-AN). To provide an insight into the adjuvant properties of the new formulation, we reviewed T-cell responses, by enzyme-linked immunospot assay, to multipeptide vaccines in 2 sequential clinical trials that spanned this transition of adjuvants. Analyses included 194 patients who received either IFA-AN or IFA-VG for all vaccines, and a subset of 93 patients best matched by study arm for vaccine antigens (12 melanoma peptides restricted by major histocompatibility complex class I, 12MP; plus a tetanus helper peptide, tet) administered with IFA but without granulocyte macrophage-colony stimulating factor. Inflammation was observed at vaccine sites clinically for almost all patients, even including ulceration in a subset with each IFA formulation. CD8+ T-cell response rates to the 12 melanoma peptides were 53% [95% confidence interval (CI), 44%, 61%)] for IFA-AN and 46% [95% CI, 32%, 59%)] for IFA-VG. In the 93 patient subset, these rates were 73% [95% CI, 61%, 83%)] and 70% [95% CI, 47%, 87%)], respectively. CD4+ T-cell responses to tetanus helper peptide were identified in 94% [95% CI, 86%, 98%)] and 96% [95% CI, 78%, 100%)], respectively. Responses to individual human leukocyte antigen (HLA)-A1, A2, and DR associated peptides were largely preserved, but reactivity trended lower for some HLA-A3 associated peptides. Despite the necessarily retrospective nature of the analysis and limitations of multiple comparisons, our summary data support the use of IFA-VG as an adjuvant with multipeptide vaccines in melanoma patients.

Entamoeba histolytica: Cytopathogenicity and lectin activity of avirulent mutants

Three clones of Entamoeba histolytica (L-6, C93, C919) were isolated by mutagenesis with ethyl methanesulfonate from the axenic strain HM1:IMSS and were studied for adherence, cytolytic, and soluble galactose inhibitable lectin activity. Avirulent clones adhered to and killed fewer Chinese hamster ovary cells than HM1:IMSS (P less than 0.01). However, only C919 was deficient in adherence to red blood cells. Galactose (1.0 g) completely inhibited adherence of all the mutants to Chinese hamster ovary cells; however, adherence to erythrocytes was only partially inhibitable by galactose. Avirulent mutants were more susceptible to being killed by human neutrophils in vitro (P less than 0.01 compared to HM1:IMSS). Soluble protein preparations from all the avirulent mutants were markedly less mitogenic for human lymphocytes and had lower lectin activity for Chinese hamster ovary cells compared to the HM1:IMSS wild type (P less than 0.01 for each activity with each mutant). Indirect immunofluorescence with a monoclonal antibody (F-14) that recognizes the Gal/GalNAc lectin was positive for L-6 and C919. These findings utilizing avirulent mutants of E. histolytica further support a role for the amebic galactose inhibitable lectin in the in vivo pathogenesis of amebiasis.

MicroRNAs induced in melanoma treated with combination targeted therapy of Temsirolimus and Bevacizumab.

BackgroundTargeted therapies directed at commonly overexpressed pathways in melanoma have clinical activity in numerous trials. Little is known about how these therapies influence microRNA (miRNA) expression, particularly with combination regimens. Knowledge of miRNAs altered with treatment may contribute to understanding mechanisms of therapeutic effects, as well as mechanisms of tumor escape from therapy. We analyzed miRNA expression in metastatic melanoma tissue samples treated with a novel combination regimen of Temsirolimus and Bevacizumab. Given the preliminary clinical activity observed with this combination regimen, we hypothesized that we would see significant changes in miRNA expression with combination treatment.MethodsUsing microarray analysis we analyzed miRNA expression levels in melanoma samples from a Cancer Therapy Evaluation Program-sponsored phase II trial of combination Temsirolimus and Bevacizumab in advanced melanoma, which elicited clinical benefit in a subset of patients. Pre-treatment and post-treatment miRNA levels were compared using paired t-tests between sample groups (patients), using a p-value < 0.01 for significance.ResultsmicroRNA expression remained unchanged with Temsirolimus alone; however, expression of 15 microRNAs was significantly upregulated (1.4 to 2.5-fold) with combination treatment, compared to pre-treatment levels. Interestingly, twelve of these fifteen miRNAs possess tumor suppressor capabilities. We identified 15 putative oncogenes as potential targets of the 12 tumor suppressor miRNAs, based on published experimental evidence. For 15 of 25 miRNA-target mRNA pairings, changes in gene expression from pre-treatment to post-combination treatment samples were inversely correlated with changes in miRNA expression, supporting a functional effect of those miRNA changes. Clustering analyses based on selected miRNAs suggest preliminary signatures characteristic of clinical response to combination treatment and of tumor BRAF mutational status.ConclusionsTo our knowledge, this is the first study analyzing miRNA expression in pre-treatment and post-treatment human metastatic melanoma tissue samples. This preliminary investigation suggests miRNAs that may be involved in the mechanism of action of combination Temsirolimus and Bevacizumab in metastatic melanoma, possibly through inhibition of oncogenic pathways, and provides the preliminary basis for further functional studies of these miRNAs.

VEGFR-2 expression in human melanoma: revised assessment.

Vascular endothelial growth factor (VEGF) is an angiogenic factor that also functions as an autocrine growth factor for VEGF receptor (VEGFR)‐2+ melanomas. In multiple studies, VEGFR‐2 was detected by immunostaining in 78–89% of human melanoma cells, suggesting that most patients with melanoma would benefit from anti‐VEGF therapy. Here, we evaluated 167 human melanoma specimens in a tissue microarray to verify the presence of VEGFR‐2, but found disparities in staining with commercial antibodies A‐3 and 55B11. Antibody A‐3 stained melanoma cells in 79% of specimens, consistent with published results; however, we noted extensive nonspecific staining of other cells such as smooth muscle and histiocytes. In contrast, antibody 55B11 stained melanoma cells in only 7% (95% confidence interval: 3.3–11.5) of specimens. As an internal positive control for VEGFR‐2 detection, vascular endothelial cells were stained with antibody 55B11 in all specimens. We compared VEGFR‐2+ and VEGFR‐2− melanoma cell lines by immunoblotting and immunohistochemistry after small interfering RNA (siRNA) knockdown and transient overexpression of VEGFR‐2 to validate antibody specificity. Immunoblotting revealed that A‐3 primarily cross‐reacted with several proteins in both cell lines and these were unaffected by siRNA knockdown of VEGFR‐2. In contrast, 55B11 staining of VEGFR‐2+ cells was mostly eliminated by siRNA knockdown of VEGFR‐2 and increased in VEGFR‐2− melanoma cell lines following transfection to express ectopic VEGFR‐2. Our results show that relatively few melanoma cells (<10%) express detectable levels of VEGFR‐2, and therefore, the majority of patients with melanoma are unlikely to benefit from antiproliferative effects of anti‐VEGF therapy.

Immunologic hierarchy and promiscuity of melanoma helper peptides

Background Melanoma vaccines have been designed to expand specific CD8+ T-cells, but melanoma-reactive helper T-cells also can have antitumor activity. We previously observed clinical activity of a vaccine incorporating 6 melanoma helper peptides (6MHP), and found associations between CD4+ T cell response and survival. With the present study, in the spirit of personalized cancer immunotherapy, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides, and