In vitro activity of pertuzumab in combination with trastuzumab in uterine serous papillary adenocarcinoma

BACKGROUND: Uterine serous papillary adenocarcinoma (USPC) is a rare but highly aggressive variant of endometrial cancer. Pertuzumab is a new humanised monoclonal antibody (mAb) targeting the epidermal growth factor type II receptor (HER2/neu). We evaluated pertuzumab activity separately or in combination with trastuzumab against primary USPC cell lines expressing different levels of HER2/neu. METHODS: Six USPC cell lines were assessed by immunohistochemistry (IHC), flow cytometry, and real-time PCR for HER2/neu expression. c-erbB2 gene amplification was evaluated using fluorescent in situ hybridisation (FISH). Sensitivity to pertuzumab and trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) was evaluated in 5 h chromium release assays. Pertuzumab cytostatic activity was evaluated using proliferation-based assays. RESULTS: Three USPC cell lines stained heavily for HER2/neu by IHC and showed amplification of the c-erbB2 gene by FISH. The remaining FISH-negative USPCs expressed HER2/neu at 0/1\ufe levels. In cytotoxicity experiments against USPC with a high HER2/neu expression, pertuzumab and trastuzumab were similarly effective in inducing strong ADCC. The addition of complementcontaining plasma and interleukin-2 increased the cytotoxic effect induced by both mAbs. In low HER2/neu USPC expressors, trastuzumab was more potent than pertuzumab in inducing ADCC. Importantly, in this setting, the combination of pertuzumab with trastuzumab significantly increased the ADCC effect induced by trastuzumab alone (P\ubc0.02). Finally, pertuzumab induced a significant inhibition in the proliferation of all USPC cell lines tested, regardless of their HER-2/neu expression. CONCLUSION: Pertuzumab and trastuzumab induce equally strong ADCC and CDC in FISH-positive USPC cell lines. Pertuzumab significantly increases tratuzumab-induced ADCC against USPC with a low HER2/neu expression and may represent a new therapeutic agent in patients harbouring advanced/recurrent and/or refractory USPC

hI-con1, a factor VII-IgGFc chimeric protein targeting tissue factor for immunotherapy of uterine serous papillary carcinoma

BACKGROUND: Uterine serous papillary adenocarcinoma (USPC) is a highly aggressive variant of endometrial cancer. Human immunoconjugate molecule (hI-con1) is an antibody-like molecule targeted against tissue factor (TF), composed of two human Factor VII (fVII) as the targeting domain, fused to human immunoglobulin (Ig) G1 Fc as an effector domain. We evaluated hI-con1 potential activity against primary chemotherapy-resistant USPC cell lines expressing different levels of TF. METHODS: A total of 16 formalin-fixed, paraffin-embedded USPC samples were evaluated by immunohistochemistry (IHC) for TF expression. Six primary USPC cell lines, half of which overexpress the epidermal growth factor type II (HER2/neu) receptor at 3\ufe levels, were assessed by flow cytometry and real-time PCR for TF expression. Sensitivity to hI-con1-dependent cell-mediated cytotoxicity (IDCC) was evaluated in 5-hour-chromium release assays. Finally, to investigate the effect of interleukin-2 (IL-2) on IDCC, 5-h 51Cr assays were also conducted in the presence of low doses of IL-2 (i.e., 50\u2013100 IU ml 1). RESULTS: Cytoplasmic and/or membrane TF expression was observed in all 16 (100%) USPC samples tested by IHC, but not in normal endometrium. High expression of TF was found in 50% (three out of six) of the USPC cell lines tested by real-time PCR and flow cytometry when compared with normal endometrial cells (NECs; Po0.001). Uterine serous papillary adenocarcinoma cell lines overexpressing TF, regardless of their high or low HER2/neu expression, were highly sensitive to IDCC (mean killing\ub1s.d., 65.6\ub13.7%, range 57.5\u201377.0%, Po0.001), although negligible cytotoxicity against USPC was seen in the absence of hI-con1 or in the presence of Rituximab control antibody. The addition of low doses of IL-2 further increased the cytotoxic effect induced by hI-con1 against chemotherapy-resistant USPC. CONCLUSION: hI-con1 induces strong cytotoxicity against primary chemotherapy-resistant USPC cell lines overexpressing TF. The hI-con1 may represent a novel therapeutic agent for the treatment of patients harbouring advanced, recurrent and/or metastatic USPC refractory to standard treatment modalities

Induction of tumour-specific CD8+ cytotoxic T lymphocytes by tumour lysate-pulsed autologous dendritic cells in patients with uterine serous papillary cancer

Uterine serous papillary carcinoma is a highly aggressive variant of endometrial cancer histologically similar to high grade ovarian cancer. Unlike ovarian cancer, however, it is a chemoresistant disease from onset, with responses to combined cisplatinum-based chemotherapy in the order of 20% and an extremely poor prognosis. In this study, we demonstrate that tumour lysate-pulsed autologous dendritic cells can elicit a specific CD8+ cytotoxic T lymphocyte response against autologous tumour target cells in three patients with uterine serous papillary cancer. CTL from patients 1 and 2 expressed strong cytolytic activity against autologous tumour cells, did not lyse autologous lymphoblasts or autologous EBV-transformed cell lines, and were variably cytotoxic against the NK-sensitive cell line K-562. Patient 3 CD8+ T cells expressed a modest but reproducible cytotoxicity against autologous tumour cells only at the time of the first priming. Further priming attempts with PBL collected from patient 3 after tumour progression in the lumboaortic lymph nodes were unsuccesful. Cytotoxicity against autologous tumour cells could be significantly inhibited by anti-HLA class I (W6/32) and anti-LFA-1 MAbs. Highly cytotoxic CD8+ T cells from patients 1 and 2 showed a heterogeneous CD56 expression while CD56 was not expressed by non-cytotoxic CD8+ T cells from patient 3. Using two colour flow cytometric analysis of intracellular cytokine expression at the single cell level, a striking dominance of IFN-γ expressors was detectable in CTL populations of patients 1 and 2 while in patient 3 a dominant population of CD8+ T cells expressing IL-4 and IL-10 was consistently detected. Taken together, these data demonstrate that tumour lysate-pulsed DC can be an effective tool in inducing uterine serous papillary cancer-specific CD8+ CTL able to kill autologous tumour cells in vitro. However, high levels of tumour specific tolerance in some patients may impose a significant barrier to therapeutic vaccination. These results may have important implications for the treatment in the adjuvant setting of uterine serous papillary cancer patients with active or adoptive immunotherapy

Serum amyloid A (SAA): a novel biomarker for uterine serous papillary cancer

BACKGROUND: Uterine serous papillary carcinoma (USPC) is a biologically aggressive variant of endometrial cancer. We investigated the expression of Serum Amyloid A (SAA) and evaluated its potential as a serum biomarker in USPC patients. METHODS: SAA gene and protein expression levels were evaluated in USPC and normal endometrial tissues (NEC) by real-time PCR, immunohistochemistry (IHC), flow cytometry and by a sensitive bead-based immunoassay. SAA concentration in 123 serum samples from 51 healthy women, 42 women with benign diseases, and 30 USPC patients were also studied. RESULTS: SAA gene expression levels were significantly higher in USPC when compared with NEC (mean copy number by RT\u2013PCR\ubc162 vs 2.21; P\ubc0.0002). IHC revealed diffuse cytoplasmic SAA protein staining in USPC tissues. High intracellular levels of SAA were identified in primary USPC cell lines evaluated by flow cytometry and SAA was found to be actively secreted in vitro. SAA concentrations (mgml 1) had a median (95% CIs) of 6.0 (4.0\u20138.9) in normal healthy females and 6.0 (4.2\u20138.1) in patients with benign disease (P\ubc0.92). In contrast, SAA values in the serum of USPC patients had a median (95% CI) of 15.6 (9.2\u201356.2), significantly higher than those in the healthy group (P\ubc0.0005) and benign group (P\ubc0.0006). Receiver operating characteristics (ROC) analysis of serum SAA to classify advanced- and early-stage USPC yielded an area under the ROC curve of 0.837 (P\ubc0.0024). CONCLUSION: SAA is not only a liver-secreted protein but is also a USPC cell product. SAA may represent a novel biomarker for USPC to assist in staging patients preoperatively, and to monitor early-disease recurrence and response to therapy

Expression of Tissue factor in Adenocarcinoma and Squamous Cell Carcinoma of the Uterine Cervix: Implications for immunotherapy with hI-con1, a factor VII-IgGFc chimeric protein targeting tissue factor

<p>Abstract</p> <p>Background</p> <p>Cervical cancer continues to be an important worldwide health problem for women. Up to 35% of patients who are diagnosed with and appropriately treated for cervical cancer will recur and treatment results are poor for recurrent disease. Given these sobering statistics, development of novel therapies for cervical cancer remains a high priority. We evaluated the expression of Tissue Factor (TF) in cervical cancer and the potential of hI-con1, an antibody-like-molecule targeted against TF, as a novel form of immunotherapy against multiple primary cervical carcinoma cell lines with squamous- and adenocarcinoma histology.</p> <p>Methods</p> <p>Because TF is a transmembrane receptor for coagulation factor VII/VIIa (fVII), in this study we evaluated the <it>in vitro </it>expression of TF in cervical carcinoma cell lines by immunohistochemistry (IHC), real time-PCR (qRT-PCR) and flow cytometry. Sensitivity to hI-con1-dependent cell-mediated-cytotoxicity (IDCC) was evaluated in 5-hrs-⁵¹chromium-release-assays against cervical cancer cell lines <it>in vitro</it>.</p> <p>Results</p> <p>Cytoplasmic and/or membrane TF expression was observed in 8 out of 8 (100%) of the tumor tissues tested by IHC and in 100% (11 out of 11) of the cervical carcinoma cell lines tested by real-time-PCR and flow cytometry but not in normal cervical keratinocytes (<it>p </it>= 0.0023 qRT-PCR; <it>p </it>= 0.0042 flow cytometry). All primary cervical cancer cell lines tested overexpressing TF, regardless of their histology, were highly sensitive to IDCC (mean killing ± SD, 56.2% ± 15.9%, range, 32.4%-76.9%, <it>p </it>< 0.001), while negligible cytotoxicity was seen in the absence of hI-con1 or in the presence of rituximab-control-antibody. Low doses of interleukin-2 further increased the cytotoxic effect induced by hI-con1 (<it>p </it>= 0.025) while human serum did not significantly decrease IDCC against cervical cancer cell lines (<it>p </it>= 0.597).</p> <p>Conclusions</p> <p>TF is highly expressed in squamous and adenocarcinoma of the uterine cervix. hI-con1 induces strong cytotoxicity against primary cervical cancer cell lines overexpressing TF and may represent a novel therapeutic agent for the treatment of cervical cancer refractory to standard treatment modalities.</p

Gene expression fingerprint of uterine serous papillary carcinoma: identification of novel molecular markers for uterine serous cancer diagnosis and therapy

Uterine serous papillary cancer (USPC) represents a rare but highly aggressive variant of endometrial cancer, the most common gynecologic tumour in women. We used oligonucleotide microarrays that interrogate the expression of some 10 000 known genes to profile 10 highly purified primary USPC cultures and five normal endometrial cells (NEC). We report that unsupervised analysis of mRNA fingerprints readily distinguished USPC from normal endometrial epithelial cells and identified 139 and 390 genes that exhibited >5-fold upregulation and downregulation, respectively, in primary USPC when compared to NEC. Many of the genes upregulated in USPC were found to represent adhesion molecules, secreted proteins and oncogenes, such as L1 cell adhesion molecule, claudin-3 and claudin-4, kallikrein 6 (protease M) and kallikrein 10 (NES1), interleukin-6 and c-erbB2. Downregulated genes in USPC included SEMACAP3, ras homolog gene family, member I (ARHI), and differentially downregulated in ovarian carcinoma gene 1. Quantitative RT–PCR was used to validate differences in gene expression between USPC and NEC for several of these genes. Owing to its potential as a novel therapeutic marker, expression of the high-affinity epithelial receptor for Clostridium perfringens enterotoxin (CPE) claudin-4 was further validated through immunohistochemical analysis of formalin-fixed paraffin-embedded specimens from which the primary USPC cultures were obtained, as well as an independent set of archival USPC specimens. Finally, the sensitivity of primary USPC to the administration of scalar doses of CPE in vitro was also demonstrated. Our results highlight the novel molecular features of USPC and provide a foundation for the development of new type-specific therapies against this highly aggressive variant of endometrial cancer

Human kallikrein 6: a new potential serum biomarker for uterine serous papillary cancer.

PURPOSE: The discovery of novel biomarkers might greatly contribute to improve clinical management and outcomes in uterine serous papillary carcinoma (USPC), a highly aggressive variant of endometrial cancer. EXPERIMENTAL DESIGN: Human kallikrein 6 (hK6) gene expression levels were evaluated in 29 snap-frozen endometrial biopsies, including 13 USPC, 13 endometrioid carcinomas, and 3 normal endometrial cells by real-time PCR. Secretion of hK6 protein by 14 tumor cultures, including 3 USPC, 3 endometrioid carcinoma, 5 ovarian serous papillary carcinoma, and 3 cervical cancers, was measured using a sensitive ELISA. Finally, hK6 concentration in 79 serum and plasma samples from 22 healthy women, 20 women with benign diseases, 20 women with endometrioid carcinoma, and 17 USPC patients was studied. RESULTS: hK6 gene expression levels were significantly higher in USPC when compared with endometrioid carcinoma (mean copy number by real-time PCR, 1,927 versus 239, USPC versus endometrioid carcinoma; P < 0.01). In vitro hK6 secretion was detected in all primary USPC cell lines tested (mean, 11.5 microg/L) and the secretion levels were similar to those found in primary ovarian serous papillary carcinoma cultures (mean, 9.6 microg/L). In contrast, no hK6 secretion was detectable in primary endometrioid carcinoma and cervical cancer cultures. hK6 serum and plasma concentrations (mean +/- SE) among normal healthy females (2.7 +/- 0.2 microg/L), patients with benign diseases (2.4 +/- 0.2 microg/L), and patients with endometrioid carcinoma (2.6 +/- 0.2 microg/L) were not significantly different. In contrast, serum and plasma hK6 values in USPC patients (6.1 +/- 1.1) were significantly higher than those in the noncancer group (P = 0.006), benign group (P = 0.003), and endometrioid carcinoma patients (P = 0.005). CONCLUSIONS: hK6 is highly expressed in USPC and is released in the plasma and serum of USPC patients. hK6 may represent a novel biomarker for USPC for monitoring early disease recurrence and response to therapy

Induction of tumor-specific cytotoxicity in tumor infiltrating lymphocytes by HPV16 and HPV18 E7-pulsed autologous dendritic cells in patients with cancer of the uterine cervix.

OBJECTIVE: To evaluate the potential of autologous dendritic cells (DC) pulsed with HPV16 and HPV18 E7 oncoprotein in restoring tumor-specific cytotoxicity in populations of tumor infiltrating lymphocytes (TIL) for adoptive immunotherapy of cervical cancer patients. METHODS: Full-length E7-pulsed DC-stimulated CD8(+) T cells derived from peripheral blood (PBL) and from tumor tissues (TIL) were tested and compared for their ability to induce a HLA class-I-restricted cytotoxic T lymphocyte (CTL) response against autologous tumor cells. In addition, in order to correlate cytotoxic activity by CTL with a particular lymphoid subset, analysis of surface antigens and intracellular CD3 zeta chain and two-color flow cytometric analysis of intracellular cytokine expression (IFN-gamma vs IL-4) at the single cell level were performed. RESULTS: DC stimulation induced powerful cytotoxicity against autologous tumor target cells by TIL-derived CD8(+) T cells from all three cervical cancer patients, while autologous Epstein-Barr virus-transformed lymphoblastoid cell lines were not lysed. Killing of autologous tumor cells was higher by CD8(+) T cells from TIL compared to PBL (P &gt; 0.01) and was more strongly inhibited by anti-HLA class I MAb (P &gt; 0.05). Phenotypically, all CTL populations were CD3(+)/CD8(+), with higher levels of CD56 expression by TIL-derived CTL. Finally, although a marked Type 1 cytokine bias (i.e., IFN-gamma(high)/IL-4(low)) was observable in both PBL- and TIL-derived DC-stimulated CD8(+) T cell populations, TIL-derived CD8(+) T cells showed a higher percentage of IFN-gamma-positive cells compared to PBL. CONCLUSIONS: Full-length E7-pulsed DC can consistently restore strong CD8(+) CTL responses against autologous HPV16- and HPV18-infected cervical cancer cells. DC-stimulated TIL may represent a superior source of tumor-specific CTL compared to PBL for adoptive T cell immunotherapy of patients harboring metastatic or recurrent cervical cancer refractory to standard treatment modalities

High serum levels of interleukin-6 in endometrial carcinoma are associated with uterine serous papillary histology, a highly aggressive and chemotherapy-resistant variant of endometrial cancer.

To evaluate and compare autocrine expression and production of interleukin-6 (IL-6), a pleiotropic cytokine involved in the resistance to cytotoxic agents and inhibition of anti-tumor immune function in endometrial carcinoma in vitro as well as in vivo. PATIENTS AND METHODS: IL-6 gene expression levels were evaluated in twenty-four primary endometrial tumors including 14 endometrioid carcinomas (EC) and 10 uterine serous papillary carcinoma (USPC) as well as in normal control endometrial cells (NEC) by real-time PCR. Secretion of IL-6 protein by 6 primary endometrial tumor cultures including USPC and EC was measured using a sensitive enzyme-linked immunosorbent assay (ELISA) in vitro. Finally, IL-6 concentration in 71 serum samples including 20 apparently healthy women, 19 women with benign abdominal diseases, 19 women with primary EC, and 13 USPC patients was studied. RESULTS: IL-6 gene expression levels were significantly higher in USPC when compared to EC (mean copy number by RT-PCR = 313 +/- 55 vs. 53 +/- 11, USPC vs. EC, respectively: P < 0.01). IL-6 serum concentrations between normal healthy females (range 0.01-21.23 pg/ml; mean 3.1 pg/ml) and benign disease patients (range 0.01-95.77 pg/ml; mean 13.07 pg/ml) were not statistically different. In contrast, significantly higher levels of IL-6 were detected in both patients with EC (range 2.86-82.13 pg/ml; mean 20.43 pg/ml) and patients with UPSC (range 16.3-500.1 pg/ml; mean 125.7 pg/ml) when compared to the healthy females (P < 0.01), with a mean serum IL-6 level in USPC patients 6.1-fold higher when compared to EC patients (P < 0.03). Accordingly, higher levels of IL-6 secretion were noted in primary USPC cell lines (mean 3121 pg/ml, range between 1099 and 5017 pg/ml/10(5) cells/48 h) when compared to primary EC (mean 88, range between 19 and 112 pg/ml/10(5) cells/48 h) (P < 0.01) in vitro. CONCLUSIONS: IL-6 is highly expressed in USPC, and it is released in high concentration in the serum of USPC patients. IL-6 may be a novel biomarker for USPC. Drugs used to inhibit the expression of IL-6 or the IL-6 signal transduction pathway may potentially be highly beneficial in USP

Restoration of tumor specific human leukocyte antigens class I-restricted cytotoxicity by dendritic cell stimulation of tumor infiltrating lymphocytes in patients with advanced ovarian cancer

Despite the large number of potentially cytotoxic tumor-infiltrating (TIL) and tumor-associated (TAL) lymphocytes accumulated in the peritoneal cavity ascitic fluid and tumor tissue, advanced ovarian cancer is a progressive disease, suggesting that TIL and TAL populations eventually become functionally suppressed in vivo. Dendritic cells (DC) are the most powerful professional antigen presenting cells known in humans and recently, ovarian tumor antigen pulsed DC have been shown to elicit tumor specific human leukocyte antigens (HLA)-class I-restricted cytotoxicity from the peripheral blood of advanced ovarian cancer patients. In this study, we have evaluated the potential of tumor antigen-pulsed fully mature DC stimulation in restoring tumor-specific cytotoxicity in anergic TIL populations from advanced ovarian cancer patients. In addition, we have compared tumor-specific T-cell responses induced by tumor antigen-loaded DC in TIL to those induced in TAL and peripheral blood lymphocytes (PBL). DC stimulation induced powerful cytotoxicity against autologous tumor target cells in TIL-derived CD8+ T-cells from all patients tested, while autologous Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) were not lysed. Killing of autologous tumor cells was higher by CD8+ T-cells from TIL compared to PBL and TAL (P < 0.01) and was more strongly inhibited by anti-HLA class I MAb (P < 0.05 compared to PBL and TAL). Phenotypically, all cytotoxic T lymphocyte (CTL) populations were CD3+/CD8+, with variable levels of CD56 expression. Finally, although a marked Type 1 cytokine bias [ie, interferon-gamma/interleukin-4 (IFN-gammahigh/IL-4low)] was observable in all DC-stimulated CD8+ T-cell populations, TIL derived CD8+ T-cells showed a higher percentage of IFN-gamma positive cells compared to TAL and PBL. Taken together, these data show that tumor lysate-pulsed DC can consistently restore strong CD8+ CTL responses from TIL against autologous ovarian cancer cells. DC-stimulated TIL may represent a superior source of tumor-specific CTL for adoptive T-cell immunotherapy for advanced ovarian cancer