Nuclear P27 expression in benign, borderline (LMP) and invasive tumors of the ovary and its association with prognosis: a gynecologic oncology group study.

OBJECTIVE: Nuclear p27 expression was examined in non-invasive and invasive ovarian tumors from a cross-sectional study, and clinical relevance of p27 was evaluated in the primary tumors from women participating in two randomized phase III treatment trials. METHODS: An immunohistochemistry assay was used to detect p27 in formalin-fixed paraffin-embedded ovarian tumors from 3 distinct sources. RESULTS: Among the initial 91 ovarian tumors tested, low p27 expression (<50% positive cells) was observed in 5.4% of non-invasive tumors versus 42.6% of invasive tumors (p<0.001). In 145 ovarian cancers with high-risk early stage disease, 16.5% exhibited low p27 expression, and categorized p27 was not associated with age, race, or performance status. Low expression of p27 was common in poorly differentiated tumors (35.7%) compared to moderately (15.0%) and well (9.5%) differentiated tumors (p=0.024) and rare in clear cell carcinomas (2.4%) compared to other histologies (p=0.014). In the 139 cancers with advanced disease, 60% displayed low p27 expression, and categorized p27 expression was not associated with age, race, performance status, tumor grade, histologic subtype, measurable disease status or survival. Exploratory analyses revealed an association of cyclin E to p27 ratio >1.0 with an increased risk of death (hazard ratio=1.53; p=0.017). CONCLUSIONS: Low p27 expression could be associated with malignant transformation of the ovarian epithelium and FIGO stage. A cyclin E to p27 ratio >1.0 may be associated with shorter survival in these patients. Further study is required to confirm the trend for increased recurrences with low p27 expression in early stage disease.

Preclinical characterization of SGN-70, a humanized antibody directed against CD70.

PURPOSE: CD70 (CD27L) is a member of the tumor necrosis factor family aberrantly expressed on a number of hematologic malignancies and some carcinomas. CD70 expression on malignant cells coupled with its highly restricted expression on normal cells makes CD70 an attractive target for monoclonal antibody (mAb)-based therapies. We developed a humanized anti-CD70 antibody, SGN-70, and herein describe the antitumor activities of this mAb. EXPERIMENTAL DESIGN: CD70 expression on primary tumors was evaluated by immunohistochemical staining of Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, and renal cell carcinoma tissue microarrays. The CD70-binding and cytotoxic activities of SGN-70 were tested in vitro using a number of cell-based assays. The in vivo antitumor properties of SGN-70 were tested in severe combined immunodeficient mice bearing disseminated lymphoma and multiple myeloma xenografts. Mechanism-of-action studies were conducted using SGN-70v, a variant mAb with equivalent target-binding activity but impaired Fcgamma receptor binding compared with SGN-70. RESULTS: Immunohistochemical analysis identified CD70 expression on approximately 40% of multiple myeloma isolates and confirmed CD70 expression on a high percentage of Hodgkin lymphoma Reed-Sternberg cells, non-Hodgkin lymphoma, and renal cell carcinoma tumors. SGN-70 lysed CD70+ tumor cells via Fc-dependent functions, including antibody-dependent cellular cytotoxicity and phagocytosis and complement fixation. In vivo, SGN-70 treatment significantly decreased tumor burden and prolonged survival of tumor-bearing mice. CONCLUSIONS: SGN-70 is a novel humanized IgG1 mAb undergoing clinical development for the treatment of CD70+ cancers. SGN-70 possesses Fc-dependent antibody effector functions and mediates antitumor activity in vivo.

Patterns of gene recombination shape var gene repertoires in Plasmodium falciparum: comparisons of geographically diverse isolates.

BACKGROUND: Var genes encode a family of virulence factors known as PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) which are responsible for both antigenic variation and cytoadherence of infected erythrocytes. Although these molecules play a central role in malaria pathogenesis, the mechanisms generating variant antigen diversification are poorly understood. To investigate var gene evolution, we compared the variant antigen repertoires from three geographically diverse parasite isolates: the 3D7 genome reference isolate; the recently sequenced HB3 isolate; and the IT4/25/5 (IT4) parasite isolate which retains the capacity to cytoadhere in vitro and in vivo. RESULTS: These comparisons revealed that only two var genes (var1csa and var2csa) are conserved in all three isolates and one var gene (Type 3 var) has homologs in IT4 and 3D7. While the remaining 50 plus genes in each isolate are highly divergent most can be classified into the three previously defined major groups (A, B, and C) on the basis of 5' flanking sequence and chromosome location. Repertoire-wide sequence comparisons suggest that the conserved homologs are evolving separately from other var genes and that genes in group A have diverged from other groups. CONCLUSION: These findings support the existence of a var gene recombination hierarchy that restricts recombination possibilities and has a central role in the functional and immunological adaptation of var genes.

Potent cytotoxicity of an auristatin-containing antibody-drug conjugate targeting melanoma cells expressing melanotransferrin/p97.

Identifying factors that determine the sensitivity or resistance of cancer cells to cytotoxicity by antibody-drug conjugates is essential in the development of such conjugates for therapy. Here the monoclonal antibody L49 is used to target melanotransferrin, a glycosylphosphatidylinositol-anchored glycoprotein first identified as p97, a cell-surface marker in melanomas. L49 was conjugated via a proteolytically cleavable valine-citrulline linker to the antimitotic drug, monomethylauristatin F (vcMMAF). Effective drug release from L49-vcMMAF likely requires cellular proteases most commonly located in endosomes and lysosomes. Melanoma cell lines with the highest surface p97 expression (80,000-280,000 sites per cell) were sensitive to L49-vcMMAF whereas most other cancer cell lines with lower p97 expression were resistant, as were normal cells with low copy numbers (< or = 20,000 sites per cell). Cell line sensitivity to L49-vcMMAF was found by immunofluorescence microscopy to correlate with intracellular fate of the conjugate. Specifically, L49-vcMMAF colocalized with the lysosomal marker CD107a within sensitive cell lines such as SK-MEL-5 and A2058. In contrast, in resistant cells expressing lower p97 levels (H3677; 72,000 sites per cell), L49-vcMMAF colocalized with caveolin-1, a protein prominent in caveolae, but not with CD107a. Thus, for antibody-drug conjugates targeting p97, antigen level and trafficking to the lysosomes are important factors for achieving robust in vitro cytotoxicity against cancer cells. Immunohistochemical analysis with L49 revealed that 62% of metastatic melanoma tumors had strong staining for p97. Overexpression of p97 in melanoma as compared with normal tissue, in conjunction with the greater sensitivity of tumor cells to L49-vcMMAF, supports further evaluation of antibody-drug conjugates for targeting p97-overexpressing tumors.

Cyclin E expression is a significant predictor of survival in advanced, suboptimally debulked ovarian epithelial cancers: a Gynecologic Oncology Group study.

Cyclin E is a key regulator of the G(1)-S transition. Abnormalities in cyclin E expression have been related to survival in a variety of cancers. This study evaluated the prognostic relevance of cyclin E in human ovarian cancer. Immunohistochemical expression of cyclin E was evaluated in 139 advanced, suboptimally debulked epithelial ovarian cancer specimens from patients treated on Gynecologic Oncology Group protocol 111. High cyclin E protein expression (> or =40% cyclin E positive tumor cells) was seen in 62 (45%) of the advanced, suboptimally debulked ovarian cancer patients. Expression of cyclin E was not associated with age, race, stage, grade, cell type, or amount of residual disease. High verses low cyclin E expression was associated with a shorter median survival (29 +/- 2 versus 35 +/- 3 months) and worse overall survival (P < 0.05). Univariate and multivariate regression analyses revealed that high relative to low cyclin E was associated with a 40-50% increase in the risk of death (hazard rate, P < or = 0.05). Fluorescence in situ hybridization was used in a subset of 20 cases to examine cyclin E gene amplification. Eight of 10 cases with high cyclin E expression exhibited amplification of the cyclin E gene, whereas only 1 of 10 cases with low expression displayed gene amplification (P < 0.006). High cyclin E expression was an independent poor prognostic factor for patients with advanced ovarian cancer, and it was associated with amplification of the cyclin E gene.

Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia.

Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated viral (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in cynomolgus macaques. Three groups of animals (n = 2 males and 2 females per group) received a subretinal injection in one eye of 300 µl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two concentrations (4 × 10(11) or 4 × 10(12) vector genomes/ml) and were evaluated over a 3-month period before being euthanized. Administration of rAAV2tYF-PR1.7-hCNGB3 was associated with a dose-related anterior and posterior segment inflammatory response that was greater than that observed in eyes injected with the vehicle control. Most manifestations of inflammation improved over time except that vitreous cells persisted in vector-treated eyes until the end of the study. One animal in the lower vector dose group was euthanized on study day 5, based on a clinical diagnosis of endophthalmitis. There were no test article-related effects on intraocular pressure, visual evoked potential responses, hematology or clinical chemistry parameters, or gross necropsy observations. Histopathological examination demonstrated minimal mononuclear infiltrates in all vector-injected eyes. Serum anti-AAV antibodies developed in all vector-injected animals. No animals developed antibodies to CNGB3. Biodistribution studies demonstrated high levels of vector DNA in the injected eye but minimal or no vector DNA in any other tissue. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations.

Safety and Biodistribution Evaluation in CNGB3-Deficient Mice of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia.

Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated virus (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. We report here results of a study evaluating safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in CNGB3-deficient mice. Three groups of animals (n = 35 males and 35 females per group) received a subretinal injection in one eye of 1 µl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two dose concentrations (1 × 10(12) or 4.2 × 10(12) vg/ml) and were euthanized 4 or 13 weeks later. There were no test-article-related changes in clinical observations, body weights, food consumption, ocular examinations, clinical pathology parameters, organ weights, or macroscopic observations at necropsy. Cone-mediated electroretinography (ERG) responses were detected after vector administration in the treated eyes in 90% of animals in the higher dose group and 31% of animals in the lower dose group. Rod-mediated ERG responses were reduced in the treated eye for all groups, with the greatest reduction in males given the higher dose of vector, but returned to normal by the end of the study. Microscopic pathology results demonstrated minimal mononuclear cell infiltrates in the retina and vitreous of some animals at the interim euthanasia and in the vitreous of some animals at the terminal euthanasia. Serum anti-AAV antibodies developed in most vector-injected animals. No animals developed antibodies to hCNGB3. Biodistribution studies demonstrated high levels of vector DNA in vector-injected eyes but little or no vector DNA in nonocular tissue. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations.

Caveolin-1 down-regulation activates estrogen receptor alpha expression and leads to 17beta-estradiol-stimulated mammary tumorigenesis.

Constitutive activation of estrogen receptor alpha (ER-alpha) expression is an early event in breast cancer tumorigenesis. However, the mechanism whereby ER-alpha is constitutively activated during transformation of normal mammary cells has not been well established. Previously, we reported that haploinsufficiency of caveolin-1, a major structural protein that forms caveolae, resulted in anchorage-independent growth of a normal mammary epithelial cell line, MCF10A. Here, we further demonstrated that ER-alpha but not ER-beta expression was constitutively activated in these caveolin-1 haploinsufficient cells. Transient treatment of MCF10A cells with beta-methyl-cyclodextrin, a chemical that can displace caveolin-1 from the plasma membrane, also stimulated ER-alpha expression. We further found that the 17beta-estradiol (E2) accelerated anchorage-independent growth of these cells in vitro and promoted their tumorigenesis in nude mice. These results suggest that dysregulation of caveolin-1 is one of the mechanisms by which ER-alpha expression is activated during initiation of breast tumorigenesis.

Functional interdependence of the DBLbeta domain and c2 region for binding of the Plasmodium falciparum variant antigen to ICAM-1.

Cytoadherence of Plasmodium falciparum-infected erythrocytes is associated with severe malaria and is primarily mediated through binding of the variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1) to specific host ligands. Infected erythrocyte binding to Intercellular Adhesion Molecule 1 (ICAM-1) has been implicated as having a role in cerebral malaria, a major cause of death from P. falciparum infection. We have examined ICAM-1-binding PfEMP1 proteins in the cytoadhesive P. falciparum strain IT4/25/5 in order to extend our understanding of binding. For A4tres, the ICAM-1 binding region was previously shown to reside within contiguous DBL2beta and c2 domains. We determined the gene sequence encoding IT-ICAM var, and showed that ICAM-1 binding in this protein also maps to DBL2betac2 domains that have 48% amino acid identity to A4tres. By truncation and chimera analysis, most of the DBL2beta and the first half of the c2 region were required for A4tres binding to ICAM-1, suggesting this tandem should be considered a structural-functional combination for ICAM-1 binding. Of interest, a chimera formed between two different ICAM-1 binding domains did not bind ICAM-1, suggesting a functional interdependence between DBL2beta and c2 from the same protein. As gene recombination and gene conversion are important mechanisms for generating diversity in the PfEMP1 protein family, this finding implies an extra level of constraint on the functional evolution of binding traits. Knowledge about the PfEMP1::ICAM-1 interaction may allow the development of interventions to prevent binding and disease.

Caveolin-1 haploinsufficiency leads to partial transformation of human breast epithelial cells.

BACKGROUND: Caveolin-1 (Cav-1), a principal component of caveolae membranes, may function as a tumor suppressor in several types of human cancer. MATERIALS AND METHODS: In this report, we employed a retrovirus-mediated poly-A gene trapping approach to inactivate expression of genes that may be involved in transformation of mammary cells and screened for cell growth in soft agar. RESULTS: We found two anchorage-independent clones (ST1 and ST3) that expressed reduced levels (approximately 50%) of Cav-1 mRNA and protein, suggesting the inactivation of one allele of Cav gene. However, haploinsufficiency of Cav-1 expression did not induce significant tumor formation when tested in nude mice. CONCLUSION: Cav-1 haploinsufficiency in human breast epithelial cells can lead to partial transformation.

SGN-LIV1A: a novel antibody-drug conjugate targeting LIV-1 for the treatment of metastatic breast cancer.

In this article, we describe a novel antibody-drug conjugate (ADC; SGN-LIV1A), targeting the zinc transporter LIV-1 (SLC39A6) for the treatment of metastatic breast cancer. LIV-1 was previously known to be expressed by estrogen receptor-positive breast cancers. In this study, we show that LIV-1 expression is maintained after hormonal therapy in primary and metastatic sites and is also upregulated in triple-negative breast cancers. In addition to breast cancer, other indications showing LIV-1 expression include melanoma, prostate, ovarian, and uterine cancer. SGN-LIV1A consists of a humanized antibody conjugated through a proteolytically cleavable linker to monomethyl auristatin E, a potent microtubule-disrupting agent. When bound to surface-expressed LIV-1 on immortalized cell lines, this ADC is internalized and traffics to the lysozome. SGN-LIV1A displays specific in vitro cytotoxic activity against LIV-1-expressing cancer cells. In vitro results are recapitulated in vivo where antitumor activity is demonstrated in tumor models of breast and cervical cancer lineages. These results support the clinical evaluation of SGN-LIV1A as a novel therapeutic agent for patients with LIV-1-expressing cancer.