David E. Modrak

Sphingolipid targets in cancer therapy.

Considerable progress has been made recently in our understanding of the role of ceramide in the induction of apoptotic cell death. Ceramide is produced by cancer cells in response to exposure to radiation and most chemotherapeutics and is an intracellular second messenger that activates enzymes, leading to apoptosis. Because of its central role in apoptosis, pharmacologic manipulation of intracellular ceramide levels should result in attenuation or enhancement of drug resistance. This may be achieved through direct application of sphingolipids or by the inhibition/activation of the enzymes that either produce or use ceramide. In addition, attention should be given to the subcellular location of ceramide generation, because this has been shown to affect the biological activity of sphingolipids. This review summarizes the sphingolipid biosynthetic pathway, as it relates to the identification of important targets for drug discovery, and the development of novel agents capable of enhancing chemotherapy. [Mol Cancer Ther 2006;5(2):200–8]

New MUC1 Serum Immunoassay Differentiates Pancreatic Cancer From Pancreatitis

PURPOSE To evaluate a new immunoassay for identification and quantitation of MUC1 in the sera of patients with pancreatic cancer or pancreatitis. The sensitivity and specificity of the assay are examined and compared to results from a CA19-9 immunoassay. METHODS An in vitro enzyme immunoassay was established with monoclonal antibody PAM4 as the capture reagent, and a polyclonal anti-MUC1 antibody as the probe. Patient sera were obtained from healthy, adult patients with acute and chronic pancreatitis, and those with pancreatic and other forms of cancer, and were measured for PAM4-reactive MUC1. RESULTS At a cutoff of 10.2 units/mL, 41 (77%) of 53 pancreatic cancer patients, none of the healthy individuals (n = 43), and only four (5%) of 87 patients with pancreatitis were positive above this value. Among nonpancreatic cancers investigated, colorectal cancers gave the highest percentage of positives (14%; five of 36). Overall, the sensitivity and specificity of the immunoassay for pancreatic cancer were 77% and 95%, respectively. Receiver operator characteristic analyses for discrimination of pancreatic cancer from pancreatitis provided an area under the curve of 0.89 (95% CI, 0.82 to 0.93), with a specificity of 95.4% and a positive likelihood ratio of 16.8. A direct pair-wise comparison of PAM4 and CA19-9 immunoassays for discrimination of pancreatic cancer and pancreatitis resulted in a significant difference (P < .003), with the PAM4 immunoassay demonstrating superior sensitivity and specificity. CONCLUSION The high sensitivity and specificity observed suggest that the PAM4-based immunoassay of circulating MUC1 may be useful in the diagnosis of pancreatic cancer.

PAM4-Reactive MUC1 Is a Biomarker for Early Pancreatic Adenocarcinoma

Purpose: The anti-MUC1 monoclonal antibody (MAb), PAM4, has a high specificity for pancreatic adenocarcinoma compared with other cancers, normal tissues, or pancreatitis. In order to assess its role in early pancreatic cancer development, we examined the expression of the PAM4-reactive MUC1 in the noninvasive precursor lesions, pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN). Experimental Design: Tissue microarrays prepared from formalin-fixed, paraffin-embedded specimens were assessed by immunohistology for expression of the PAM4-reactive, non–variable number of tandem repeats (VNTR), MUC1 epitope, and the VNTR epitope bound by the MA5 MAb. Results: The PAM4-reactive MUC1 epitope was not detected in normal pancreas but was expressed in 87% (48 of 55) of invasive pancreatic adenocarcinomas, including early stage 1 disease: PAM4 labeled 94% (44 of 47) of the earliest PanIN lesions, PanIN-1A and 1B, along with 91% (10 of 11) of PanIN-2, 40% (2 of 5) of PanIN-3, and 86% (31 of 36) of intraductal papillary mucinous neoplasia lesions. A mostly diffuse pattern of labeling was observed. A second, unrelated, anti-MUC1 MAb, MA5, showed considerably less sensitivity with early PanIN-1 lesions; only 61% (25 of 41) were positive and the labeling did not differentiate normal pancreas from PanINs. Conclusions: The results suggest that expression of the PAM4-reactive antigen may represent an early event in the development of invasive pancreatic adenocarcinoma, and is unrelated to the VNTR peptide core epitopes of MUC1. Detection of this biomarker using immunohistology, in vitro immunoassays, and in vivo antibody–based imaging may provide new opportunities for the early detection and improved diagnosis of pancreatic cancer.

Synergistic interaction between sphingomyelin and gemcitabine potentiates ceramide-mediated apoptosis in pancreatic cancer

We have examined the mechanism by which sphingomyelin (SM) enhances chemotherapy in human pancreatic cancer cells, focusing on the correlation between ceramide metabolism and apoptosis. Dose response curves for gemcitabine in the absence or presence of 0.2 mg/mL SM provided IC50 values of 78.3 ± 13.7 and 13.0 ± 3.0 nmol/L, respectively. The cytotoxic effect of the combined treatment was synergistic (combination index = 0.36). Using annexin-V staining, the percentage of apoptotic cells was 3.6 ± 2.6% for the untreated cells, 6.5 ± 3.8% for the 0.2 mg/mL SM-treated cells, and 19.9 ± 12.9% for the 100 nmol/L gemcitabine-treated cells, but increased significantly to 42.1 ± 12.7% with the combined treatment (P < 0.001, compared with gemcitabine-treated group). The percentage of cells losing mitochondrial membrane potential followed a similar trend. The ceramide content of untreated and gemcitabine-treated cells was not significantly different (0.46 ± 0.29 and 0.59 ± 0.34 pmol ceramide/nmole PO4). However, when 0.2 mg/mL SM was added, ceramide levels were 1.09 ± 0.42 and 1.58 ± 0.55 pmol ceramide/nmol PO4, for the SM alone and SM with gemcitabine-treated cells, respectively (P = 0.038). Acidic SMase was activated by exposure to gemcitabine but not SM, whereas the activities of neutral SMase and glycosylceramide synthase did not change with either gemcitabine or SM. The data are consistent with gemcitabine-induced activation of acidic SMase and indicate that the addition of SM can yield increased production of ceramide, mitochondrial depolarization, apoptosis, and cell death. Because SM by itself is relatively nontoxic, addition of this lipid to agents that induce apoptosis may prove useful to enhance apoptosis and increase cytotoxicity in cancer cells.

Combined 90Yttrium-DOTA-Labeled PAM4 antibody radioimmunotherapy and gemcitabine radiosensitization for the treatment of a human pancreatic cancer xenograft

We have examined the application of 90Y‐DOTA‐cPAM4, anti‐MUC1 IgG, in combination with the front‐line drug gemcitabine as a potential therapeutic for pancreatic cancer. Athymic nude mice bearing CaPan1 human pancreatic cancer xenografts were administered 2 mg of gemcitabine on days 0, 3, 6, 9 and 12 with concurrent 90Y‐DOTA‐cPAM4 (100 μCi) provided on day 0. A second group of mice received a second cycle of treatment 5 weeks after the start of the first cycle. Control groups of mice included those that received either treatment arm alone, the combined modality treatment employing a nontargeting control antibody (hLL2, anti‐B‐cell lymphoma) and a final group that was left untreated. Gemcitabine administered as a single agent provided no antitumor effect. A single cycle of the combined 90Y‐DOTA‐cPAM4 and gemcitabine treatment provided greater inhibition of tumor growth than was observed for any of the other treatment procedures. Tumor growth was delayed for a period of 7 weeks. Two cycles of gemcitabine with concomitant 90Y‐DOTA‐cPAM4 yielded significant tumor regression and increased median survival to 21 weeks vs. 12 weeks for mice receiving a single cycle of therapy (p<0.024). Median tumor volume doubling‐times were 18 weeks in mice treated with 2‐cycles of therapy vs. 7 weeks in mice given only 1‐cycle (p<0.001), and 3.5 weeks for the group that received 2‐cycles of gemcitabine concomitant with equitoxic nontargeting 90Y‐DOTA‐hLL2 (p<0.001). These data suggest that addition of 90Y‐DOTA‐cPAM4 RAIT to a gemcitabine treatment regimen may provide enhanced antitumor efficacy for the treatment of pancreatic cancer.

Ceramide Regulates Gemcitabine-Induced Senescence and Apoptosis in Human Pancreatic Cancer Cell Lines

Bioactive sphingolipids are potent intracellular signaling molecules having profound effects on cell death, growth, and differentiation. Pharmacologic manipulation of sphingolipid levels could have a significant effect on the induction of apoptosis by anticancer agents, and thus, improve treatment efficacy. We observed that gemcitabine cannot completely kill AsPc1 and Panc1 human pancreatic cancer cells in culture; even at high concentrations of gemcitabine, 30% to 40% of the cells remain viable. By adding sphingomyelin to the culture medium, gemcitabine-induced cell death increased synergistically to >90%. Panc1 cells that survived high concentrations of gemcitabine had an increase in β-galactosidase activity, a marker of senescence. The inclusion of sphingomyelin with gemcitabine reduced β-galactosidase activity, as compared with cells treated with gemcitabine alone. Expression of p21waf1/cip1 in both cell lines exposed to sphingomyelin, gemcitabine, and gemcitabine + sphingomyelin varied relative to the untreated group. C8-ceramide induced both cell death and senescence in a dose-dependent manner. These results indicate that gemcitabine induces senescence in pancreatic cancer cells and that sphingomyelin-enhanced chemosensitivity is achieved through reducing the induction of senescence by redirecting the cell to enter the apoptotic pathway. Ceramide levels seem to be critical to this decision, with cell cycle progression being uninhibited at low ceramide levels, senescence induced at moderate levels, and apoptosis initiated at high levels. Our results provide further evidence that targeting the sphingolipid metabolism is a means of enhancing the efficacy of chemotherapeutic agents. (Mol Cancer Res 2009;7(6):890–6)

Improved Targeting of Pancreatic Cancer: Experimental Studies of a New Bispecific Antibody, Pretargeting Enhancement System for Immunoscintigraphy

Purpose: The early detection and diagnosis of pancreatic cancer remains a major clinical challenge in which imaging procedures have a central role. The purpose of this study was to evaluate a pretargeting method with a bispecific PAM4 (bsPAM4; anti-MUC1) antibody for radioimmunoscintigraphy of experimental human pancreatic cancer. Experimental Design: A bispecific F(ab′)2 antibody was generated from chimeric PAM4 Fab′ and murine 734 (anti-indium-diethylenetriaminepentaacetic acid) Fab′ fragments and then used in conjunction with 2 peptide haptens (111In-IMP-156 and 99mTc-IMP-192). Biodistribution studies and radioimmunoscintigraphic imaging properties of the radiolabeled bsPAM4, and pretargeted, radiolabeled peptides were examined in the CaPan1 human pancreatic tumor grown as s.c. xenografts in athymic nude mice. Tumor uptake and tumor:nontumor ratios were compared with a nontargeting irrelevant anti-CD20, bispecific rituximab, radiolabeled peptides alone, and with directly labeled PAM4. Results: Biodistribution results indicated significantly greater tumor uptake of radiolabeled peptides at 3 h after injection when pretargeting was performed with bsPAM4 as compared with the bispecific rituximab [20.2 ± 5.5 percentage of injected dose per gram of tissue (%ID/g) versus 0.9 ± 0.1%ID/g, respectively, for 111In-IMP-156, and 16.8 ± 4.8%ID/g versus 1.1 ± 0.2%ID/g, respectively, for 99mTc-IMP-192]. Similar results were obtained at the 24-h time point. Tumor:nontumor ratios were >30 for all of the tissues except the kidneys, where a ratio of 7.8 ± 2.8 was observed. By immunoscintigraphy, tumors could be visualized as early as 30 min after injection of the radiolabeled peptide. Conclusions: These studies demonstrate the feasibility of using the pretargeted, bispecific antibody technology for nuclear imaging of pancreatic cancer. The advantage of pretargeted bsPAM4 antibody as an imaging platform is the high specificity for pancreatic cancer as compared with the physicochemical parameters identified by current imaging technologies.

An in vitro model to optimize dose scheduling of multimodal radioimmunotherapy and chemotherapy: Effects of p53 expression

Several reports have appeared on the use of combined radioimmunotherapy (RAIT) and chemotherapy. The choice of drug to use with RAIT and how to space the two treatments has not been completely addressed. Because every patients cancer presents with a specific molecular phenotype, we hypothesized that it may be necessary to tailor therapy based on specific gene expression. We addressed how the form of expression of a single gene, the p53 tumor suppressor, would impact the choice of agents, as well as sequence and spacing of agents. p53 regulates cell cycle arrest to allow for DNA repair after therapy‐induced small DNA damage or induction of apoptosis if damage is great and has been shown to affect chemo‐ and radiosensitivity of cancer cells. We established 3 stable p53 transfectants of the SKOV‐3 p53null parental line (p53wt, p53143mut or p53273mut). p53 expression was confirmed using flow cytometry, using the DO1 pan‐p53 Ab and the PAb240 anti‐p53mut Ab. The colorimetric MTT assay was then used to measure dose‐dependent growth inhibition from single modality chemotherapy (doxorubicin, carboplatin, paclitaxel or topotecan) or radioimmunotherapy (90Y‐RS‐7 IgG anti‐EGP1). The % survival vs. log [drug] were plotted to obtain the IC50. We then used a matrix design in which we varied the sequence of the first and second modality of treatment and the spacing between the 2 treatments to determine the most synergistic and antagonistic combinations for the parental SKOV‐3 and each of the 3 transfectants. The IC50 for each therapeutic agent varied as a function of the form of p53 expressed. For example, of the 4 lines, the p53wt transfectant was the most resistant to topotecan and the 143mut was the most resistant to carboplatin. The 273mut was quite sensitive to both doxorubicin and paclitaxel, whereas the p53null and wt were not. For multimodal treatments, most combinations of RAIT and chemotherapy resulted in a 30–40% growth inhibition (GI) and were either additive or moderately antagonistic. The 3 best (>60% GI) and 3 worst (<25% GI) combinations were identified and were unique to the parental p53null and to the 3 transfectants. Certain combinations showed clear synergy and others were antagonistic, with the first treatment modality blocking the growth inhibitory effects of the second treatment modality. The form of p53 expressed affects chemosensitivity and radiosensitivity and will influence optimal multimodal therapy with RAIT and chemotherapy and the dose‐schedule (sequential with RAIT first or with drug first) when more than 1 agent is used.

Abstract 4622: PAM4-antigen levels in the serum of patients with pancreatic carcinoma: Early detection of disease and correlation with responses to radioimmunotherapy

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: A serum-based enzyme immunoassay (EIA) employing MAb-PAM4 demonstrates high specificity and sensitivity for detection of advanced pancreatic carcinoma (PC). We are currently exploring use of the immunoassay as a means to detect early-stage PC, as well as detection of nascent disease progression or relapse during routine post-treatment follow-up. We now also report initial results that provide correlation of serum PAM4-antigen levels with responses to radiolabeled PAM4 therapy. Methods: The PAM4-based EIA was employed for detection and quantitation of antigen in the serum of pancreatic carcinoma patients with known stage of disease (N=68). An additional 11 patients with stage-4 disease who participated in an ongoing phase-1b clinical trial to evaluate the combination of 90Y-hPAM4 IgG, (clivatuzumab tetrexate) radioimmunotherapy with low-dose, radiosensitizing, gemcitabine (Gem) were also examined. Serum specimens from this latter group were collected at baseline prior to treatment, at the end of the 4-week treatment cycle, and again at 4 weeks post-treatment (week 8 from baseline), when a CT scan was performed to determine tumor response. Results: Overall, the sensitivity of the immunoassay for detection of PC was 81%, with a 5% false-positive rate for the healthy volunteers (N=19). When examined by stage of disease, sensitivity rates were 91%, 86% and 62% for stages 3/4 advanced disease, stage-2, and stage-1 PC, respectively. For those patients who had treatment with 90Y-hPAM4 + Gem, a decrease in antigen levels greater than 40%, observed at the end of the treatment cycle (4 weeks), with sustained decline in the antigen levels at the 8-week post-treatment evaluation, provided presumptive evidence of tumor response, either stable disease (SD, N=2) or partial response (PR, N=5) by CT RECIST criteria. None of the patients with progressive disease (N=4) had a sustainable decrease in serum levels of PAM4-antigen, whereas all of the responders did. The median decrease in serum PAM4-mucin levels at the 8-week follow-up evaluation timepoint was 54% (range: 47% - 98% decrease), with a median tumor response (only SD and PR responders) being a decrease in tumor size of 37% (range: 46% decrease to 4% increase), from a baseline median primary tumor size of 10.0 cm (range 2.6 - 12.1 cm). Conclusions: These results suggest that the PAM4-based immunoassay should be further evaluated for use in the detection of early pancreatic carcinoma, as well as to evaluate tumor response to therapy. (Supported in part by grant CA096924 from the NIH.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4622.

Abstract 5341: Sensitivity of NHL to killing by anti-HLA-DR and anti-CD74 mAbs is increased by interferon-gamma

Background: HLA-DR and CD74 are similarly, but not identically, expressed and induced by interferons on a variety of cells. Expression of both antigens on hematological malignancies led to their development as targets for antibody-based therapy. The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab (Immunomedics Inc, Morris Plains, NJ), is in clinical evaluation for therapy of NHL, multiple myeloma (MM), and CLL after preclinical evidence of activity in these tumor types. A humanized anti-HLA-DR mAb, hL243γ4P (IMMU-114, Immunomedics) has demonstrated anti-tumor activity in vitro and in vivo, and clinical evaluation is planned. In addition to expression in hematologic cancers, these antigens are expressed on the surface of other tumor types, including melanoma and renal cell carcinoma, and in the cytoplasm of others, including pancreatic and colonic carcinomas, and glioblastomas (GBM). Methods: We examined whether the ability of anti-HLA-DR and anti-CD74 mAbs to kill cancer cells can be increased by using IFNγ as an inducer of antigen expression. Using a panel of diverse cancer cell lines (including NHL, MM, GBM, and pancreatic and colonic carcinomas), we examined IFNγ-induced changes in surface and cytoplasmic HLA-DR and CD74 expression. Sensitivity of malignant cells to milatuzumab and hL243γ4P was assessed with and without INFγ by cytotoxicity assays. Results: Without IFNγ surface expression of HLA-DR and CD74 were present on 2/2 NHL, 2/2 MM, and only weakly positive on 2/2 GBM cell lines. Surface CD74 and HLA-DR were weak or undetectable on 4/4 colon and 4/4 pancreatic carcinomas. Cytoplasmic CD74 and HLA-DR were seen in NHL, MM, GBM, and 1/4 colon and 1/4 pancreatic (CD74 only) carcinomas. Two-day incubation with IFNγ increased surface and cytoplasmic expression of both HLA-DR and CD74 in all the NHL and GBM, and 3/4 pancreatic cancer lines, but not MM cell lines. In all 4 colon lines, IFNγ increased cytoplasmic expression of both antigens, and surface expression of HLA-DR in 3/4 and CD74 in 2/4. Upregulation of HLA-DR and CD74 ranged from 23-3700%. Increased killing by both hL243γ4P (58%) and milatuzumab (33%) was seen in vitro after INFγ exposure in WSU-FSCCL NHL cells. No cell killing was observed using these mAbs in vitro on U118 (GBM), Capan-1 (pancreatic carcinoma), or LoVo (colon carcinoma), despite upregulation of the antigens in these cell lines. A CD74-transfected version of the U118 GBM cell line has been prepared for comparison of milatuzumab sensitivity based on antigen density only. Conclusions: Cell surface and cytoplasmic expression of CD74 and HLA-DR are increased on cell lines from a variety of cancer types after INFγ exposure. This increased expression correlates with increased toxicity of anti-HLA-DR and anti-CD74 mAbs in a NHL cell line, and is under evaluation in other cancer types. These studies could prove useful in predicting the potential benefit of combined INFγ and mAb therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5341.