Abstract 1860: Bispecific claudin-6 x CD3 antibodies in a 2 + 1 format demonstrate selectivity and activity on human ovarian cancer cells

Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but is aberrantly expressed in various human cancers, such as ovarian cancer (OC) and testicular cancer (TC). A monoclonal antibody against CLDN6, IMAB027, has shown promising antitumor activity in preclinical human CLDN6-positive (CLDN6+) cancer models. In this series of nonclinical studies, we investigated CLDN6 expression in normal and cancer tissues, as well as the localization and possible function of CLDN6 in cancer cells. Methods Expression of CLDN6 was assessed in a wide range of human tissues (eg, lung, colon, skin, ovary) and cultured cells by quantitative RT-PCR, immunohistochemistry (IHC), flow cytometry, and western blotting. To investigate the effect of dedifferentiation on CLDN6 expression, human-induced pluripotent cells were generated by transfecting foreskin fibroblasts with a reprogramming cocktail, and then CLDN6 expression was evaluated. To characterize CLDN6 as a potential novel marker to identify cancer stem cells (CSCs) in OC, coexpression of CLDN6 with known CSC surface markers were analyzed by flow cytometry, and CLDN6+ and CLDN6-negative cells were tested in colony formation and sphere formation assay. Human OC cell lines were transplanted intraperitoneally into nude mice and assessed for metastasis to investigate tumorigenecity of CLDN6+ cells. Results Except for low mRNA levels measured in placenta, testis, umbilical cord, cerebellum, and lung samples, no CLDN6 (mRNA or protein) was detected in the vast majority of normal tissues. Additionally, there was also a lack of CLDN6 protein expression in tissue zones where stem cells for tissue homeostasis would normally be found as determined by IHC with an anti-CLDN6 antibody. CLDN6 was expressed on the cell surface of several solid tumors, including ovarian, testicular, uterine, and lung cancer tissues; OC and TC samples had high level expression. CLDN6 expression was strongly activated in human-induced pluripotent stem cells generated from fibroblasts. CLDN6 showed selective coexpression with known CSC markers such as CD44, CD24, and CD90 in OC and TC cell lines. In addition, some CLDN6+ OC cells exhibited CSC-like behavior in vitro: CLDN6+ populations were clonogenic and formed well-defined spheres in low attachment conditions; these spheres had the ability to self-renew into secondary spheres. Analysis of OC metastases in mouse xenografts showed when xenografts were generated by OC cells that had <10% of CLDN6+ cells, the metastases were enriched in CLDN6+ cells, suggesting CLDN6+ cells had selective growth advantage. Conclusions CLDN6 is a cancer cell-specific surface molecule aberrantly expressed in several cancers, and its expression may be an identifier for cells with CSC-like traits. These characteristics make CLDN6 an attractive target candidate for tumor-specific therapeutic antibodies. Citation Format: Özlem Türeci, Meike Wagner, Claudia Paret, Maria M. Kreuzberg, Stefan Wöll, Korden Walter, Sabine C. Häcker, Ikumi Nakajo, Tomohiro Yamada, Ugur Sahin. Claudin 6 is a carcinoembryonic antigen with cancer stem cell marker features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1907.

Long Noncoding RNA Ceruloplasmin Promotes Cancer Growth by Altering Glycolysis.

What’s next after bevaciumab resistance? Targeting metabolic vulnerabilities in ovarian cancer (022)

Impacts of transmembrane serine protease 4 expression on susceptibility to severe acute respiratory syndrome coronavirus 2.

Objective To detect the correlation between the expression of oneogene Bmi-1 gene and telomerase activity in ovarian cancer. Methods RT-PCR method and SP immunohistechemistry were adopted to detect the mRNA of Bmi-1 gene and the expression of Bmi-1 protein in tissues of 47 ovarian epithelial cancer cases. TRAP-silver staining technique was used to detect the telomerase activity. Western Blot was adopted to detect the expression of Telomerase hTERT. Results Compared with the normal ovary epithelium tissues, Bmi-1 pro-tein in tissues of 47 ovarian epithelial cancer cases processed more obvious expression and was related to patho-logical grade and clinical stage. Significant Bmi-1 expression levels were found among different clinopathological types of the cancer (P < 0.05). In ovarian epithelial cancer tissues, 87. 23% demonstrated positive telomerase activity in contrast to zero activity in normal tissues. Majority (90.24%) of specimens with positive telomerase activity possessed high Bmi-1 expression levels. Spearman correlation analysis indicated that expression of Bmi-1 protein was positively correlated with the elevated telomerase activity. Conclusion Bmi-1 protein is highly expressed in ovarian epithelial cancer tissues, and its expression level correlates with histological grade and clinical phase of the patients. Elevation of Bmi-1 expression is closely correlated to the increased telomerase ac-tivity, and plays a significant role in the pathogenesis of Ovarian Cancer, which deserves further researches. Key words: Bmi-1; Telomerase; Ovarian cancer

Purpose: Despite high initial response rates with cytoreductive surgery, conventional chemotherapy and the incorporation of biologic agents, ovarian cancer patients often relapse and die from their disease. New approaches are needed to improve patient outcomes. This study was designed to evaluate the antitumor activity of NEO-201 monoclonal antibody (mAb) in preclinical models of ovarian cancer where the NEO-201 target is highly expressed.Experimental Design: Functional analysis of NEO-201 against tumor cell lines was performed by antibody-dependent cellular cytotoxicity (ADCC) assays. Binding of NEO-201 to tumor tissues and cell lines were determined by immunohistochemistry (IHC) and flow cytometry, respectively. Further characterization of the antigen recognized by NEO-201 was performed by mass spectrometry. Ovarian cancer models were used to evaluate the anti-tumor activity of NEO-201 in vivo. NEO-201 at a concentration of 250 g/mouse was injected intraperitoneally (IP) on days 1, 4, and 8. Human PBMCs were injected IP simultaneously as effector cells.Results: Both IHC and flow cytometry revealed that NEO-201 binds prominently to the colon, pancreatic, and mucinous ovarian cancer tissues and cell lines. Immunoprecipitation of the antigen recognized by NEO-201 was performed in human ovarian, colon, and pancreatic cancer cell lines. From these screening, carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and CEACAM6 were identified as the most likely targets of NEO-201. Our results confirmed that NEO-201 binds different types of cancers; the binding is highly selective for the tumor cells without cross reactivity with the surrounding healthy tissue. Functional analysis revealed that NEO-201 mediates ADCC killing against human ovarian and colorectal carcinoma cell lines in vitro. In addition, NEO-201 inhibited tumor growth in the presence of activated human PBMCs in orthotopic mouse models of both primary and metastatic ovarian cancer. Importantly, NEO-201 prolonged survival of tumor-bearing mice.Conclusions: These data suggested that NEO-201 has an antitumor activity against tumor cells expressing its antigen. Targeting an antigen expressed in tumors, but not in normal tissues, allows patient selection for optimal treatment. These findings strongly indicate that NEO-201 warrants clinical testing as both a novel therapeutic and diagnostic agent for treatment of ovarian carcinomas. A first in human clinical trial evaluating NEO-201 in adults with chemo-resistant solid tumors is ongoing at the NIH clinical Center.

Objective : The inhibitor of apoptosis proteins (IAPs) constitutes a family of highly conserved apoptosis suppressor proteins that were originally identified in baculoviruses. Although IAP homologs have recently been demonstrated to suppress apoptosis in mammalian cells, their expression and role in human gynecologic cancers are unknown. In this study auther used ovarian and cervical cancer tissues to examine the role of IAP in the regulation of apoptosis in cervical and ovarian cancers compared with normal tissues. Methods : Fresh tissues were retrieved from 10 cases each with ovarian cancers, cervical cancers and 4 normal ovarian tissues, 6 benign ovarian tumors, and 10 normal cervical tissues. Expression of mRNA was determined by RT-PCR. Western blot analysis was also used for asseessment of protein expression. Results : The overexpression of c-IAP1,2 was identified in ovarian cancers compare with normal ovaries. All cases of cervical cancer tissues were negative and normal cervical tissues were positive for c-IAP1,2 by RT-PCR assay. Using Western blot analysis, the overexpression of c-IAP1,2 was observed in ovarian cancer tissues compared with normal ovarian tissues and overexpression of c-IAP1 in normal cervical tissues. However differences were not observed with expression of c-IAP2 in cervical cancer tissues. On immunohistochemical results, the expression of c-IAP1,2 was observed in ovarian cancer tissues, normal corpus luteum, and normal cervical tissues, whereas c-IAP1 was not shown in cervical cancer tissues. There was no correlation in c-IAP2 expression between cervical cancer and normal cervical tissues. Conclusion : These results suggest that c-IAP1,2 are important elements in growth of ovarian cancers, whereas c-IAP1 may play role of down regulation to cervical carcinogenesis.

Background: Claudin 6 (CLDN6) is a clinically validated target for many solid tumors notably ovarian, endometrial, and testicular cancer. Its expression is restricted to tumor cells and fetal tissues with little or no detectable in normal tissues. As such, CLDN6 represents an attractive therapeutic target for the development of an antibody drug conjugate (ADC). Here, we describe the development and preclinical characterization of a novel ADC called PLB-002, which consists of a highly selective CLDN6 targeting antibody, PLB-002-ab7, conjugated to a FDA-approved microtubule inhibitor, eribulin, via a Primelink Biotherapeutics proprietary enzyme-cleavable linker with an optimized average drug-to-antibody ratio (DAR) of 4.0. PLB-002-ab7 is a novel humanized anti-CLDN6 single-domain antibody (Mw. 78.8 kDa) with highly binding affinity and selectivity to CLDN6 with barely binding to related CLDN family members (CLDN9, CLDN3, and CLDN4) in protein and cell based binding assays. Methods: The in vitro binding affinity of the PLB-002 was determined by flow cytometry on OVCAR3, PA-1, OVCA429, OV90, and NEC-8 cells. Cell internalization effect was evaluated by an in-direct flow cytometry assay on OVCAR3, PA-1, and OV90 cells. In vitro cytotoxicity was measured using Cell-Titer Glo assay on OVCAR3, OV90, and NEC-8 cells. In vivo anti-tumor efficacy was investigated using several cancer cell derived xenograft (CDX) models of OVCAR3, OV90, and NEC-8 cells, as well as patient derived xenograft (PDX) models of ovarian cancer with CLDN6 high, moderate, low and negative expression. A dose-range finding (DRF) safety study of PLB-002 was performed in cynomolgus monkeys. Pharmacokinetics (PK) and pharmacodynamics (PD) data of PLB-002 in CDX mice were determined by LC-MS/MS method. Results: PLB-002 exhibited strong binding, rapid internalization effect, and potent antitumor activity in vitro on OV90 (CLDN6 low expression) and OVCAR3 (CLDN6 high expression) cells with nano-molar range of IC50. When evaluated in OVCAR3 and OV90 CDXs of ovarian cancer, NEC-8 CDX of testicular germ cell tumor, and OV PDXs, PLB-002 showed strong tumor regression in a dose-dependent manner, which is consistent with the PK analyses showing high exposure, slow clearance and long half-life for both total antibody and conjugated drug. In a non-human primate study using cynomolgus monkey, PLB-002 showed favorable safety profile with no clinical symptoms of toxicities up to the highest tested dose. Conclusions: These results show that PLB-002 has remarkable antitumor activity and support the clinical development as a therapeutic ADC for the treatment of ovarian cancer and other CLDN6 expressing solid cancer. Citation Format: Haitao Pan, Qing Zhang, Ganyuan Xiao, Guangchao Zhang, Jiaxin Li, Ling Xin, Kia J. Puan, Ling Xu, Yingdong Lu, Mao Yin. PLB-002 is a novel Claudin 6 antibody-drug conjugate for ovarian cancer and testicular germ cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2945.

Stanniocalcin 1 (STC1) is a secreted glycoprotein hormone. High expression of STC1 has been associated with several cancers including ovarian cancer, but its role in the development of ovarian cancer is not clear. We used five human ovarian epithelial cancer cell lines (OVCA420, OVCA432, OVCA433, SKOV3, and HEY), immortalized human ovarian surface epithelial cells (T29 and T80), ovarian cancer tissues from 342 patients, serum from 73 ovarian cancer patients and from58 control subjects, and 116 mice, with six or eight per group. Protein expression was assessed. Cells overexpressing STC1 protein were generated by ectopic expression of human STC1 cDNA. STC1 expression was silenced by using small interfering RNA against STC1. Cell proliferation, migration, colony formation, and apoptosis were assessed. Xenograft tumor growth in mice was studied. Neutralizing anti-STC1 antibody was used to inhibit STC1 function. All statistical tests were two-sided. STC1 protein expression was higher in all human ovarian cancer cell lines examined than in immortalized human ovarian epithelial cell lines, higher in ovarian cancer tissue than in normal ovarian tissue (P < .001), and higher in serum from ovarian cancer patients than from control subjects (P = .021). Ovarian cancer cells with STC1 overexpression, compared with corresponding control cells, had increased cell proliferation, migration, and colony formation in cell culture and increased growth of xenograft tumors in mice. These activities in normal or malignant ovarian cells with STC1 overexpression, compared with control cells, were also accompanied by increased expression of cell cycle regulatory proteins and antiapoptotic proteins but decreased cleavage of several caspases. Within 24 hours of treatment, apoptosis in cultures of HEY ovarian cancer cells treated with neutralizing anti-STC1 monoclonal antibody was higher (17.3% apoptotic cells) than that in cultures treated with mouse IgG control cells (4.4%) (12.9% difference, 95% confidence interval = 11.6% to 14.2%). STC1 protein may be involved in ovarian tumorigenesis.

Highlights from Recent Cancer Literature

This study investigates the correlation between the oncoprotein Bmi-1 and telomerase activity in ovarian cancer. A real-time polymerase chain reaction (PCR) method is used to detect the messenger RNA (mRNA) expression of Bmi-1 protein in 47 ovarian epithelial cancer cases, and immunohistochemistry is used to detect Bmi-1 protein expression in the tissues. A modified telomeric repeat amplification protocol (TRAP) is used to detect telomerase activity. Western blotting is used to detect the expression of telomerase hTERT in the tissues studied. Compared to normal ovarian epithelial tissue, Bmi-1 protein in the 47 ovarian epithelial cancer cases showed higher expression and was related to pathological grade and clinical stage. Significantly higher Bmi-1 levels were found among different clinocopathological types of the cancer (P<0.05). Grade G3 cases expressed Bmi-1 at a higher rate (93.10%) than did grade G2 cases (61.11%). Expression in phase II and phase III cases was lower (66.67%) than in phase IV (92.31%). In ovarian epithelial cancer tissues, 87.23% (41/47) cases demonstrated positive telomerase activity, whereas no activity was observed in normal tissues. The majority (90.24%) of specimens with positive telomerase activity showed high Bmi-1 expression levels. Spearman correlation analysis indicated that expression of Bmi-1 protein correlated positively with elevated telomerase activity. Bmi-1 protein is highly expressed in ovarian epithelial cancer tissues, and expression correlates with histological grade and clinical phase. Elevated Bmi-1 expression correlates closely with increased telomerase activity and plays a significant role in the pathogenesis of ovarian cancer.

Claudins (CLDNs), key components of tight junctions, are dysregulated in various cancers. However, the roles and therapeutic potential of specific CLDN family members-particularly CLDN6, CLDN9, and CLDN10-in ovarian cancer (OC) remain incompletely defined. To address this gap, we conducted a comprehensive analysis of the CLDN family to identify novel diagnostic and prognostic biomarkers as well as potential therapeutic targets for OC. Gene expression profiles and corresponding clinical data from The Cancer Genome Atlas ovarian cancer cohort (TCGA-OV) and two Gene Expression Omnibus (GEO) datasets (GSE18520, GSE26712) were analyzed. Differential expression of CLDN genes between OC and normal tissues was evaluated using R with appropriate bioinformatics packages (e.g., limma). Logistic regression models were employed to calculate odds ratios (ORs), and receiver operating characteristic (ROC) curves were generated across all datasets to identify consistently dysregulated CLDNs associated with OC. Prognostic hazard ratios (HRs) for these CLDNs were extracted from the Kaplan-Meier Plotter (KM Plotter) database and synthesized using a random-effects model to assess their associations with overall survival. Intersection analysis was performed to identify CLDNs exhibiting both significant differential expression and prognostic significance. Candidate targets underwent comprehensive validation, including single-cell RNA sequencing (scRNA-seq) to characterize cell-type-specific expression patterns. Notably, Key findings regarding CLDN6 were further validated by immunohistochemistry (IHC) on an independent tissue microarray (TMA), as well as functional assays in OC cell lines following siRNA-mediated knockdown. These included transwell invasion, wound healing (scratch) test, and measurements of mitochondrial depolarization, reactive oxygen species (ROS) accumulation, cell cycle arrest, and apoptosis. CLDN6, CLDN9, and CLDN10 were consistently and significantly upregulated in OC compared to normal tissues across all datasets. Single-cell RNA sequencing revealed that CLDN6 and CLDN10 were predominantly expressed in malignant epithelial cell subsets, a pattern associated with aggressive tumor phenotypes. Meta-analysis of HRs showed that HR >1 in CLDN6 and HR <1 in CLDN10. Although CLDN10 is highly expressed in tumor cells, its hazard ratio (HR) is less than 1, and the underlying mechanism of this gene remains unclear. Experiments have confirmed that CLDN6 is closely associated with tumor invasion. Computational analysis, meta-analysis, and single-cell data collectively confirm that only CLDN6 is a clearly defined gene closely associated with tumor progression, a finding subsequently validated by experimental results. Notably, the combined signature comprising CLDN6, CLDN9, and CLDN10 exhibited superior diagnostic performance, with higher area under the curve (AUC) values in ROC analysis, compared to individual CLDNs or established OC biomarkers such as carbohydrate antigen 125 (CA125), human epididymis 4 (HE4), carcinoembryonic antigen (CEA), and alpha-fetoprotein (AFP). The signature also showed enhanced prognostic discrimination, as indicated by time-dependent ROC analysis. Protein overexpression of these targets was validated by IHC and Western blot. Functional assays further demonstrated that siRNA-mediated knockdown of CLDN6 significantly inhibited the proliferation of OC cells, promoted cell apoptosis, increased production of ROS, induced G1 phase arrest, inhibited cell invasion and migration in vitro. Furthermore, western blot analysis identified that knockdown of CLDN6 repressed the Wnt/β-catenin pathway. Nude mice experiments indicated that CLDN6 knockdown in OC cells dramatically suppresses the tumor growth and lung metastasis in vivo. CLDN6, CLDN9, and CLDN10 are critically involved in the pathogenesis and progression of OC. A biomarker panel combining these three claudins demonstrates superior diagnostic and prognostic performance compared to individual markers and established clinical biomarkers such as CA125 and HE4. Notably, functional evidence indicates that CLDN6 plays a pivotal role in regulating malignant phenotypes, highlighting its potential as a novel therapeutic target. These findings collectively support the clinical utility of the CLDN6/9/10 axis as both a non-invasive biomarker signature and a promising avenue for targeted intervention in ovarian cancer.

BackgroundCTIM-76 is a bispecific Claudin 6 (CLDN6) T-cell engager antibody in preclinical development for treatment of CLDN6-positive cancers, including ovarian, testicular, and non-small cell lung cancers. CLDN6 is a tight...

Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but is aberrantly expressed in various human cancers. The anti-CLDN6 monoclonal antibody (mAb), IMAB027, has shown promising antitumor activity in preclinical human CLDN6-positive (CLDN6+) cancer models. Conjugation of IMAB027 with monomethyl auristatin E (MMAE) may utilize the precision tumor-targeting of the mAb to deliver a highly effective cytotoxic drug to the tumor. In this report we present the preclinical characterization of this antibody–drug conjugate, IMAB027–vcMMAE. Methods Internalization of IMAB027 in various CLDN6+ human ovarian (OC) and testicular cancer (TC) cell lines was assessed by immunofluorescence, flow cytometry, and Fab-ZAP internalization assay. Binding characteristics of IMAB027–vcMMAE were examined by flow cytometry. Cell viability and IMAB027–vcMMAE-mediated cytotoxic effects (direct and indirect [bystander]) were assessed in cell cultures by the XTT metabolic assay. Apoptosis was evaluated by caspase 3/7, annexin V, and TUNEL assays. Xenograft mouse tumors were generated by injecting human OC cells into nude mice to assess the safety and antitumor activity of IMAB027–vcMMAE. Results IMAB027–vcMMAE binds robustly to, and is internalized by, cell lines expressing CLDN6. IMAB027–vcMMAE reduced the viability of CLDN6+ OC and TC cells by up to 100% with EC50 values in the ng/mL order. IMAB027–vcMMAE induced apoptosis in CLDN6+ cells in a dose-dependent manner. Additionally, after conjugation, IMAB027–vcMMAE retained IMAB027's ability to induce CLDN6+ cell death via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Cell lines that did not express CLDN6 were unaffected by IMAB027–vcMMAE in monocultures; however, in cocultures of CLDN6+ and CLDN6-negative cells, IMAB027–vcMMAE exerted bystander effect, resulting in the death of cocultured CLDN6-negative cells in addition to the target-bearing CLDN6+ cells. In vivo, significant antitumor effects were observed after a single intravenous administration of 16 mg/kg IMAB027–vcMMAE in mouse OC xenografts. Further, xenograft tumors with low and/or heterogeneous CLDN6 expression treated with IMAB027–vcMMAE showed efficient tumor size reduction. Repeated dosing of IMAB027–vcMMAE was well tolerated in mice, with no physical abnormalities, changes in behavior, or alterations in appearance observed. Conclusions IMAB027–vcMMAE is a specific antibody–drug conjugate against CLDN6 that induces potent antitumor activity in CLND6+ tumor cells in vitro and in vivo. Furthermore, IMAB027–vcMMAE was able to induce antitumor effects in tumors with low and/or heterogeneous target expression, which may be driven by bystander activity. Citation Format: Özlem Türeci, Maria Kreuzberg, Korden Walter, Stefan Wöll, Ramona Schmitt, Tomohiro Yamada, Ikumi Nakajo, Ugur Sahin. Preclinical characterization of the safety and antitumor activity of IMAB027-vcMMAE, an anticlaudin 6 antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1778.

Breast cancer heterogeneity in histology and molecular subtype influences metabolic and proliferative activity and hence the acid load on cancer cells. We hypothesized that acid-base transporters and intracellular pH (pHi) dynamics contribute inter-individual variability in breast cancer aggressiveness and prognosis. We show that Na+,HCO3– cotransport and Na+/H+ exchange dominate cellular net acid extrusion in human breast carcinomas. Na+/H+ exchange elevates pHi preferentially in estrogen receptor-negative breast carcinomas, whereas Na+,HCO3– cotransport raises pHi more in invasive lobular than ductal breast carcinomas and in higher malignancy grade breast cancer. HER2-positive breast carcinomas have elevated protein expression of Na+/H+ exchanger NHE1/SLC9A1 and Na+,HCO3– cotransporter NBCn1/SLC4A7. Increased dependency on Na+,HCO3– cotransport associates with severe breast cancer: enlarged CO2/HCO3–-dependent rises in pHi predict accelerated cell proliferation, whereas enhanced CO2/HCO3–-dependent net acid extrusion, elevated NBCn1 protein expression, and reduced NHE1 protein expression predict lymph node metastasis. Accordingly, we observe reduced survival for patients suffering from luminal A or basal-like/triple-negative breast cancer with high SLC4A7 and/or low SLC9A1 mRNA expression. We conclude that the molecular mechanisms of acid-base regulation depend on clinicopathological characteristics of breast cancer patients. NBCn1 expression and dependency on Na+,HCO3– cotransport for pHi regulation, measured in biopsies of human primary breast carcinomas, independently predict proliferative activity, lymph node metastasis, and patient survival.