Corazon dela Rosa

Concurrent Trastuzumab and HER2/neu-Specific Vaccination in Patients With Metastatic Breast Cancer

PURPOSE The primary objectives of this phase I/II study were to evaluate the safety and immunogenicity of combination therapy consisting of concurrent trastuzumab and human epidermal growth factor receptor 2 (HER2)/neu-specific vaccination in patients with HER2/neu-overexpressing metastatic breast cancer. PATIENTS AND METHODS Twenty-two patients with stage IV HER2/neu-positive breast cancer receiving trastuzumab therapy were vaccinated with an HER2/neu T-helper peptide-based vaccine. Toxicity was graded according to National Cancer Institute criteria, and antigen specific T-cell immunity was assessed by interferon gamma enzyme-linked immunosorbent spot assay. Data on progression-free and overall survival were collected. RESULTS Concurrent trastuzumab and HER2/neu vaccinations were well tolerated, with 15% of patients experiencing an asymptomatic decline in left ventricular ejection fraction below the normal range during combination therapy. Although many patients had pre-existing immunity specific for HER2/neu and other breast cancer antigens while treated with trastuzumab alone, that immunity could be significantly boosted and maintained with vaccination. Epitope spreading within HER2/neu and to additional tumor-related proteins was stimulated by immunization, and the magnitude of the T-cell response generated was significantly inversely correlated with serum transforming growth factor beta levels. At a median follow-up of 36 months from the first vaccine, the median overall survival in the study population has not been reached. CONCLUSION Combination therapy with trastuzumab and a HER2/neu vaccine is associated with minimal toxicity and results in prolonged, robust, antigen-specific immune responses in treated patients.

Assessment of Hepatitis C Viremia Using Molecular Amplification Technologies: Correlations and Clinical Implications

Hepatitis C virus, a positive-strand RNA virus classified within the Flaviviridae family, is an important cause of chronic hepatitis. Hepatic complications associated with chronic HCV infection include chronic active hepatitis in approximately 60% of cases, bridging fibrosis and cirrhosis in at least 20%, end-stage liver disease, and possibly hepatocellular carcinoma [1-5]. Several extrahepatic syndromes are also associated with HCV infection, including essential mixed cryoglobulinemia and membranoproliferative glomerulonephritis [6-9]. Serologic tests that detect antibodies to HCV structural and nonstructural antigens are useful screening assays, and the prevalence of HCV infection in the United States is currently estimated to be between 1% and 2% of the general population. Sensitive qualitative tests for detection of viral RNA in serum by reverse transcription polymerase chain reaction (PCR) have been developed and shown to be useful for diagnosing active hepatitis C [10]. In one cross-sectional study of seropositive blood donors and clinic patients, more than 90% of persons with antibodies to HCV also had evidence of HCV viremia, as evidenced by the detection of the viral RNA genome in serum by reverse transcription PCR testing [11]. Accurate quantitative methods for measuring viral nucleic acid levels in patient serum have recently been developed. Several recent studies have indicated that the level of HCV viremia correlates with the clinical stage of disease; patients with advanced stages of liver disease such as severe chronic active hepatitis, cirrhosis, and endstage liver disease had higher serum levels of HCV RNA than patients with mild HCV infections. Moreover, the HCV viremia titer may predict a subsequent response to antiviral therapy [11-15]. Two different technologies have been used to assess HCV viremia: the branched-DNA (bDNA) assay [13, 16] and quantitative PCR [11, 12]. The bDNA assay uses a novel approach to viral detection called signal amplification technology. Briefly, viral nucleic acids in a clinical specimen (for example, plasma or serum) are first solubilized by the addition of denaturing reagents and are then hybridized to microtiter plates through the use of virus-specific capture probes. The bound viral nucleic acid is then reacted with virus-specific extender probes followed by bDNA polymers. The bDNA polymers contain multiple repetitive binding sites for oligonucleotide-linked enzymes that catalyze the activation of chemiluminescent substrates; thus, signal amplification is achieved without amplification of viral nucleic acid. Quantitative PCR relies on amplification of viral nucleic acids in the presence of a specific competitor molecule. In this assay, the ratio of amplification products derived from the native viral RNA and the competitive template are compared. In the quantitative competitive reverse transcription PCR assay, known amounts of synthetic internal control HCV RNA are titrated directly into clinical specimens and are then coextracted with the wild-type HCV RNA before analysis by competitive reverse transcription PCR [11]. Adding the competitor RNA directly into the clinical specimen allows the assay to be internally controlled for variations in efficiency of RNA extraction, complementary DNA synthesis, and PCR because the competitor is present during all steps. Quantitative PCR assays that use synthetic competitor templates as internal controls have been shown to be highly accurate for measuring various viral nucleic acids in clinical specimens. The current study compared the bDNA assay with a quantitative competitive reverse transcription PCR assay (quantitative PCR) for detecting and measuring HCV RNA levels in serum specimens in various patient populations. Methods Clinical Specimens We selected for analysis 400 specimens from four different sources: prospective blood donors at the Puget Sound Blood Center (Seattle, Washington) or Irwin Memorial Blood Centers (San Francisco, California), patients attending hepatology clinics at either the University of Washington Medical Center or Harborview Medical Center (Seattle), patients receiving long-term renal dialysis at the Northwest Kidney Center (Seattle), and HCV-infected liver transplant recipients followed at the University of Washington. All serum samples were collected and processed within 4 hours of venipuncture to optimize detection of viral RNA [17] and were analyzed by laboratory personnel blinded to the clinical data. All specimens were tested for HCV antibodies by second-generation enzyme immunoassay (EIA2; Abbott Laboratories, North Chicago, Illinois) and by second-generation recombinant immunoblot assay (RIBA-II; Ortho Diagnostics, Raritan, New Jersey) when results of the enzyme immunoassay were positive. All 400 specimens were initially screened for HCV RNA by reverse transcription PCR combined with a specific radioactive probe hybridization, which is a highly sensitive and specific qualitative (nonquantitative) screening assay for HCV RNA [18, 19]. The analytical sensitivity of our reverse transcription PCR assay is less than 10 molecules of purified synthetic HCV RNA [19]; a clinical specificity of greater than 99% has been shown in previous studies and confirmed by ongoing proficiency testing approved by the College of American Pathology. We selected 101 of the 400 specimens as negative controls because they were negative for antibody to HCV by enzyme immunoassay and for HCV RNA by reverse transcription PCR; 299 specimens were selected as viremic specimens because they were positive for both antibody to HCV and HCV RNA by the screening reverse transcription PCR assay. To evaluate the bDNA and quantitative PCR assays for detecting and measuring HCV RNA levels in patients followed prospectively, serum specimens were obtained at monthly intervals from 19 consecutive HCV-infected patients; 18 of these were treated for 6 months with recombinant interferon-, 3 million U three times per week, and 1 was treated with an escalating dose regimen of interferon for 48 months. Patients were classified as having a complete virologic response to interferon if their serum tested negative for HCV RNA by reverse transcription PCR at the end of therapy; we chose this virologic end point because reverse transcription PCR is the most sensitive test available for detection of HCV viremia. Branched-DNA Assay The bDNA assay was done as recommended by the manufacturer (Chiron Corp.; Emeryville, California); the design and principles of the assay are discussed above and in a previous report [16]. Quantitation of HCV RNA (expressed as equivalents per mL) was calculated by comparing relative luminescence with that obtained from an HCV RNA standard curve. All samples were assayed in duplicate, and the mean value of the duplicate tests was used for data analysis. Quantitative Polymerase Chain Reaction Known quantities of synthetic internal control HCV RNA were directly titrated into clinical specimens before nucleic acid extraction. Viral and internal control RNAs were coextracted from serum through the guanidinium thiocyanate method and were reverse transcribed; complementary DNA was amplified by PCR using primers derived from the highly conserved 5-noncoding region of the HCV genome. Polymerase chain reaction amplification products were detected by either agarose gel electrophoresis plus ethidium bromide staining or Southern blot analysis in which a radiolabeled internal probe was used. The RNA copy number was deduced by comparing the PCR amplification product band intensity with the intensity of the internal control bands. The linear range of quantitative PCR ranged from 103 to 1010 RNA molecules per mL (3 to 10 logs of HCV RNA per mL) by comparison with end-point dilution analysis of HCV RNA [11]. Results Linearity of Hepatitis C Virus RNA Quantitation in Vitro We assessed the linearity of HCV RNA quantitation in vitro using a serum sample obtained from an immunosuppressed patient with previously determined high-titer HCV viremia: The undiluted serum HCV RNA titer was 9.5 logs per mL by quantitative PCR. Eighteen 0.5-log dilutions of the high-titer serum samples into noninfected human sera were prepared, and HCV RNA was tested in duplicate by bDNA assay, quantitative PCR, and the qualitative screening assay, reverse transcription PCR (Figure 1). Dilutions 1 through 15 all tested positive for HCV RNA by reverse transcription PCR, whereas dilutions 16 through 18 tested negative. In contrast, only dilutions 1 through 6 were positive by bDNA assay; dilution 7 was indeterminate; and dilutions 8 to 18 were negative. The bDNA assay showed a nearly perfect slope within the linear range of the assay, from 6.0 logs to 8.5 logs when expressed as molecules per mL, or 5.5 logs to 7.9 logs when expressed as equivalents per mL as defined by the manufacturer (Figure 1). The bDNA assay went off scale at HCV RNA levels greater than 7.9 log-equivalents per mL. Figure 1. Analysis of hepatitis C virus (HCV) RNA in serial dilutions of high-titer patient serum by two different quantitative assays, branched-DNA (bDNA) and quantitative polymerase chain reaction (Q-PCR), and a highly sensitive qualitative assay for HCV RNA, reverse transcription PCR (RT-PCR). open circles closed circles The quantitative PCR assay gave linear results on the serially diluted serum specimen, from 4-log molecules per mL to 9.5-log molecules per mL. The reverse transcription PCR end point was estimated to be 100 molecules per mL or approximately 35 log-equivalents per mL; thus, reverse transcription PCR was approximately 4 orders of magnitude more sensitive than bDNA assay for detecting HCV RNA in vitro. The linear range of HCV RNA quantitation by quantitative PCR exceeded that of the bDNA assay by at least 3 orders of magnitude, although quantitative PCR varied slightly more than the bDNA assay. Within the linear range of the bDNA assay, the in vitro correlation between

Effect of dose on immune response in patients vaccinated with an HER-2/neu intracellular domain protein-based vaccine

PURPOSE To evaluate the safety of an HER-2/neu intracellular domain (ICD) protein vaccine and to estimate whether vaccine dose impacts immunogenicity. PATIENTS AND METHODS Twenty-nine patients with HER-2/neu-overexpressing breast or ovarian cancer and with no evidence of disease after standard therapy received a low- (25 microg), intermediate- (150 microg), or high-dose (900 microg) HER-2/neu ICD protein vaccine. The vaccine was administered intradermally, monthly for 6 months, with granulocyte-macrophage colony-stimulating factor as an adjuvant. Toxicity and both cellular and humoral HER-2/neu-specific immunity was evaluated. RESULTS The vaccine was well tolerated. The majority of patients (89%) developed HER-2/neu ICD-specific T-cell immunity. The dose of vaccine did not predict the magnitude of the T-cell response. The majority of patients (82%) also developed HER-2/neu-specific immunoglobulin G antibody immunity. Vaccine dose did not predict magnitude or avidity of the HER-2/neu-specific humoral immune response. Time to development of detectable HER-2/neu-specific immunity, however, was significantly earlier for the high- versus low-dose vaccine group (P =.003). Over half the patients retained HER-2/neu-specific T-cell immunity 9 to 12 months after immunizations had ended. CONCLUSION The HER-2/neu ICD protein vaccine was well tolerated and effective in eliciting HER-2/neu-specific T-cell and antibody immunity in the majority of breast and ovarian cancer patients who completed the vaccine regimen. Although the dose of vaccine did not impact the magnitude of T-cell or antibody immunity elicited, patients receiving the highest dose developed HER-2/neu-specific immunity more rapidly than those who received the lowest dose.

Functional T Cell Responses to Tumor Antigens in Breast Cancer Patients Have a Distinct Phenotype and Cytokine Signature

The overall prevalence with which endogenous tumor Ags induce host T cell responses is unclear. Even when such responses are detected, they do not usually result in spontaneous remission of the cancer. We hypothesized that this might be associated with a predominant phenotype and/or cytokine profile of tumor-specific responses that is different from protective T cell responses to other chronic Ags, such as CMV. We detected significant T cell responses to CEA, HER-2/neu, and/or MAGE-A3 in 17 of 21 breast cancer patients naive to immunotherapy. The pattern of T cell cytokines produced in response to tumor-associated Ags (TAAs) in breast cancer patients was significantly different from that produced in response to CMV or influenza in the same patients. Specifically, there was a higher proportion of IL-2-producing CD8+ T cells, and a lower proportion of IFN-γ-producing CD4+ and/or CD8+ T cells responding to TAAs compared with CMV or influenza Ags. Finally, the phenotype of TAA-responsive CD8+ T cells in breast cancer patients was almost completely CD28+CD45RA− (memory phenotype). CMV-responsive CD8+ T cells in the same patients were broadly distributed among phenotypes, and contained a high proportion of terminal effector cells (CD27−CD28−CD45RA+) that were absent in the TAA responses. Taken together, these results suggest that TAA-responsive T cells are induced in breast cancer patients, but those T cells are phenotypically and functionally different from CMV- or influenza-responsive T cells. Immunotherapies directed against TAAs may need to alter these T cell signatures to be effective.

Level of HER-2/neu protein expression in breast cancer may affect the development of endogenous HER-2/neu-specific immunity

We questioned whether the incidence or magnitude of the humoral or cellular immune response to the self-tumor antigen HER-2/neu is influenced by the level of HER-2/neu protein overexpression as defined by immunohistochemical staining of tumors in breast cancer patients. We obtained peripheral blood from 104 women with stage II, III, and IV pathologically confirmed HER-2/neu-overexpressing breast cancer. Patients were categorized with +1 (n = 14), +2 (n = 20), or +3 (n = 70) HER-2/neu overexpression by institutional pathologic report. Circulating antibodies to HER-2/neu were evaluated using ELISA. T-cell responses to HER-2/neu were measured using an antigen-specific tritiated thymidine incorporation assay. Eighty-two percent of subjects with HER-2/neu antibodies were +3 overexpressors compared with 18% +2 overexpressors and 0% +1 overexpressors, a highly significant difference (P < 0.001), and there were significant differences in the magnitude of the HER-2/neu-specific antibodies between groups with varying HER-2/neu protein expression (P = 0.022). In addition, 65% of subjects with HER-2/neu-specific T cells were +3 overexpressors compared with 16% +2 overexpressors and 19% +1 overexpressors (P = 0.001). Data presented here indicate that endogenous HER-2/neu-specific humoral and T-cell immunity is greater in patients with +3 protein overexpression in their tumors than in patients with lower levels of HER-2/neu expression. Overexpression of a self-tumor-associated protein is a potential mechanism by which immunogenicity is enhanced and may aid in the identification of biologically relevant proteins to target for immune-based molecular cancer therapies. [Mol Cancer Ther 2008;7(3):449–54]

Insulin-like growth factor binding protein 2 is a target for the immunomodulation of breast cancer

Breast cancer is immunogenic and well suited to treatment via immunomodulation. The disease is often treated to remission and time to relapse is generally measured in years in many cases. Immune-based therapeutics, such as cancer vaccines, may be able to affect the clinical progression of micrometastatic disease. Immune targets must be identified that have the potential to inhibit tumor growth. Insulin-like growth factor-binding protein-2 (IGFBP-2) has direct effects on breast cancer proliferation via stimulation of critical signaling pathways. We questioned whether IGFBP-2 was an immune target in breast cancer. IGFBP-2-specific IgG antibody immunity was preferentially detected in breast cancer patients compared with controls (P = 0.0008). To evaluate for the presence of T-cell immunity, we identified potential pan-HLA-DR binding epitopes derived from IGFBP-2 and tested the peptides for immunogenicity. The majority of epitopes elicited peptide-specific T cells in both patients and controls and had high sequence homology to bacterial pathogens. IGFBP-2 peptide-specific T cells could respond to naturally processed and presented IGFBP-2 protein, indicating that these peptides were native epitopes of IGFBP-2. Finally, both immunization with IGFBP-2 peptides as well as adoptive transfer of IGFBP-2-competent T cells mediated an antitumor effect in a transgenic mouse model of breast cancer. This is the first report of IGFBP-2 as a human tumor antigen that may be a functional therapeutic target in breast cancer.

Tumor Antigen ^ Specific T-Cell Expansion Is Greatly Facilitated by In vivo Priming

Purpose: Adoptive T-cell therapy is a promising strategy for the treatment of patients with established tumors but is often limited to specific cancers where tumor-infiltrating lymphocytes, the source of T cells for ex vivo culture, can be obtained. In this study, we evaluated the feasibility of expanding HER-2/neu–specific T cells derived from peripheral blood ex vivo following in vivo priming with a HER-2/neu peptide vaccine. Experimental Design: Peripheral blood mononuclear cells from cytomegalovirus (CMV)–seronegative and CMV-seropositive donors as well as HER-2/neu–positive cancer patients who had or had not been vaccinated with a HER-2/neu peptide–based vaccine was used as a source of T lymphocytes. Antigen-specific T-cell lines were generated by in vitro stimulation with antigen followed by nonspecific expansion on CD3/CD28 beads. The ability to expand antigen-specific T cells was assessed using IFN-γ and granzyme B enzyme-linked immunosorbent spot. The phenotype of the resultant T-cell lines was evaluated by flow cytometry, including the presence of FOXP3-expressing CD4+ T cells. Results: The frequencies of CMV-specific T cells generated from CMV+ donors were >11-fold higher than the frequencies from CMV− donors (P = 0.001), with 22-fold increase of total number of CD3+ T cells. The frequencies of HER-2/neu–specific T cells generated from the primed patients were >25-fold higher than the frequencies from unvaccinated patients (P = 0.006), with an average of a 19-fold increase of total number of CD3+ T cells. Using peripheral blood as the source of T cells did not result in concurrent expansion of FOXP3+CD4+ regulatory T cells despite the use of interleukin-2 in in vitro culture. Both CD4+ and CD8+ HER-2/neu–specific T cells could be expanded. The extent of ex vivo expansion correlated with the magnitude of immunity achieved during immunization (P = 0.008). Conclusion: Tumor-specific T cells can be efficiently expanded from the peripheral blood ex vivo following in vivo priming with a vaccine. This approach provides an effective method to generate tumor-specific polyclonal T cells for therapeutic use that could be applied to cancer patients with any tumor type.

Pediatric AIDS-Associated Lymphocytic Interstitial Pneumonia and Pulmonary Arterio-Occlusive Disease: Role of VCAM-1/VLA-4 Adhesion Pathway and Human Herpesviruses

Because the mechanisms of lymphocyte accumulation in the lungs of children with AIDS-associated lymphocytic interstitial pneumonia (LIP) are unknown, we studied the relative contributions of known adhesion pathways in mediating lymphocyte adherence to endothelium and the potential role of human herpesviruses in the expansion of these lesions. LIP was characterized by lymphoid hyperplasia of the bronchus-associated lymphoid tissue (BALT) and infiltration of the pulmonary interstitium with CD8(+) T lymphocytes. In some individuals there was expansion of the alveolar septae with dense aggregates of B lymphocytes, many containing the Epstein-Barr viral (EBV) genome. Patients with concurrent EBV infection also demonstrated large-vessel arteriopathy characterized by thickening of the intimae with collagen and smooth muscle. Venular endothelium from the lung of children with LIP, but not uninflamed lung from other children with AIDS or lung from children with nonspecific pneumonitis, expressed high levels of vascular cell adhesion molecule-1 (VCAM-1) protein. In turn, inflammatory cells expressing very late activation antigen-4 (VLA-4), the leukocyte ligand for VCAM-1, were the predominant perivascular infiltrate associated with vessels expressing VCAM-1. Expression of other endothelial adhesion molecules, including intracellular adhesion molecule-1 and E-selectin, was not uniformly associated with LIP. Using a tissue adhesion assay combined with immunohistochemistry for VCAM-1, we show that CD8(+) T cell clones that express VLA-4 bind preferentially to pulmonary vessels in sites of LIP: vessels that expressed high levels of VCAM-1. When tissues and cells were pretreated with antibodies to VCAM-1 or VLA-4, respectively, adhesion was inhibited by >/=80%. Thus, infiltration of alveolar septae with CD8(+) T cells was highly correlative with VCAM-1/VLA-4 adhesive interactions, and focal expansion of B cells was coincidental to co-infection with EBV.

Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses

BackgroundStandardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation) and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt), and cytomegalovirus (CMV) antigens. These antigens were determined to be low (HER-2/neu), moderate (tt), and robustly (CMV) immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response.ResultsLinear regression analysis indicates that both stimulation index (SI) (p = 0.011) and IFN-gamma secreting precursor frequency (p < 0.001) are significant indicators of antigen specific immunity. ROC curves plotted to assess the performance of tritiated thymidine incorporation and the ELISPOT assay indicate that SI is a significant indicator of low T cell response to the HER-2/neu vaccine (p = 0.05), and of moderate and robust responses to tt (p = 0.01) and CMV (p = 0.016), respectively. IFN-gamma precursor frequency is a significant indicator of a robust T cell response to CMV (p = 0.03), but not of moderate tt (p = 0.09), or low HER-2/neu (p = 0.09) T cell responses.ConclusionThese data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients.

Phenotype and in vitro function of mature MDDC generated from cryopreserved PBMC of cancer patients are equivalent to those from healthy donors

BackgroundMonocyte-derived-dendritic-cells (MDDC) are the major DC type used in vaccine-based clinical studies for a variety of cancers. In order to assess whether in vitro differentiated MDDC from cryopreserved PBMC of cancer patients are functionally distinct from those of healthy donors, we compared these cells for their expression of co-stimulatory and functional markers. In addition, the effect of cryopreservation of PBMC precursors on the quality of MDDC was also evaluated using samples from healthy donors.MethodsUsing flow cytometry, we compared normal donors and cancer patients MDDC grown in the presence of GM-CSF+IL-4 (immature MDDC), and GM-CSF+IL-4+TNFα+IL-1β+IL-6+PGE-2 (mature MDDC) for (a) surface phenotype such as CD209, CD83 and CD86, (b) intracellular functional markers such as IL-12 and cyclooxygenase-2 (COX-2), (c) ability to secrete IL-8 and IL-12, and (d) ability to stimulate allogeneic and antigen-specific autologous T cells.ResultsCryopreservation of precursors did affect MDDC marker expression, however, only two markers, CD86 and COX-2, were significantly affected. Mature MDDC from healthy donors and cancer patients up-regulated the expression of CD83, CD86, frequencies of IL-12+ and COX-2+ cells, and secretion of IL-8; and down-regulated CD209 expression relative to their immature counterparts. Compared to healthy donors, mature MDDC generated from cancer patients were equivalent in the expression of nearly all the markers studied and importantly, were equivalent in their ability to stimulate allogeneic and antigen-specific T cells in vitro.ConclusionOur data show that cryopreservation of DC precursors does not significantly affect the majority of the MDDC markers, although the trends are towards reduced expression of co-stimulatory makers and cytokines. In addition, monocytes from cryopreserved PBMC of cancer patients can be fully differentiated into mature DC with phenotype and function equivalent to those derived from healthy donors.