Wendy C. Weinberg

FDA Approval: Ado-trastuzumab Emtansine for the Treatment of Patients with HER2-Positive Metastatic Breast Cancer

On February 22, 2013, the FDA licensed ado-trastuzumab emtansine (Kadcyla; Genentech, Inc.) for use as a single agent for the treatment of patients with human epidermal growth factor receptor 2 (HER2)–positive metastatic breast cancer (MBC) who previously received trastuzumab and a taxane, separately or in combination. The clinical basis for licensure was a phase III trial in 991 patients with HER2-positive MBC that randomly allocated patients to receive ado-trastuzumab emtansine (n = 495) or lapatinib in combination with capecitabine (n = 496). The coprimary endpoints were progression-free survival (PFS) based on tumor assessments by an independent review committee and overall survival (OS). Statistically significant improvements in PFS and OS were observed in patients receiving ado-trastuzumab emtansine compared with patients receiving lapatinib plus capecitabine [difference in PFS medians of 3.2 months, HR, 0.65 (95% confidence interval, CI, 0.55–0.77), P < 0.0001 and difference in OS medians of 5.8 months, HR, 0.68 (95% CI, 0.55–0.85), P = 0.0006]. The most common adverse reactions in patients receiving ado-trastuzumab emtansine were fatigue, nausea, musculoskeletal pain, thrombocytopenia, headache, increased aminotransferase levels, and constipation. Other significant adverse reactions included hepatobiliary disorders and left ventricular dysfunction. Given the PFS and OS results, the benefit–risk profile was considered favorable. Clin Cancer Res; 20(17); 4436–41. ©2014 AACR.

Development and regulation of monoclonal antibody products: Challenges and opportunities

SummaryAn increasing number of monoclonal antibodies for cancer diagnosis and treatment are in clinical use and in the development pipeline, with more expected as new molecular targets are identified. As with all drugs, product quality, an appropriate pre-clinical pharmacology-toxicology testing program, and well-designed clinical trials are essential for a successful drug development program. However, protein products such as monoclonal antibodies present unique regulatory concerns. The derivation from biological sources as well as the constantly evolving technologies utilized to develop these products demands continuous appraisal of safety concerns, even while the accumulated experience with these protein products has facilitated their safety evaluations. Because of the complex nature of these products and their inherent heterogeneity, a mechanistic understanding of the mode of action along with careful attention to product design and manufacture are critical to assuring a safe, effective and consistent product. Protein products may be highly species specific, thus pharmacologically relevant animal models are an important component in accurately assessing pre-clinical safety and establishing initial dosing. Furthermore, the immunogenicity of protein products can impact its safety profile, dose exposure, and efficacy. Mechanistic insight should form the basis of biological assays used for monitoring efficacy, safety, lot-to-lot consistency and manufacturing changes. The inherent uniqueness of each product necessitates a flexible case-by-case approach for biologics review that is based on a strong scientific understanding of relative risks. This review will provide an overview of approaches used in the development of antibody-based cancer therapeutics and the scientific basis of regulatory reviews.

First FDA Approval of Dual Anti-HER2 Regimen: Pertuzumab in Combination with Trastuzumab and Docetaxel for HER2-Positive Metastatic Breast Cancer

On June 8, 2012, the U.S. Food and Drug Administration (FDA) approved pertuzumab (Perjeta, Genentech) for use in combination with trastuzumab (Herceptin, Genentech) and docetaxel for the treatment of patients with HER2-positive metastatic breast cancer (MBC) who have not received prior anti-HER2 therapy or chemotherapy for metastatic disease. Approval was based on the results of a randomized, double-blind, placebo-controlled trial conducted in 808 patients with HER2-positive MBC. Patients were randomized (1:1) to receive pertuzumab (n = 402) or placebo (n = 406) in combination with trastuzumab and docetaxel. The primary endpoint was progression-free survival (PFS) and a key secondary endpoint was overall survival (OS). A statistically significant improvement in PFS (difference in medians of 6.1 months) was observed in patients receiving pertuzumab [HR, 0.62; 95% confidence interval (CI), 0.51–0.75; P < 0.0001]. A planned interim analysis suggested an improvement in OS (HR, 0.64; 95% CI, 0.47–0.88; P = 0.0053) but the HR and P value did not cross the stopping boundary. Common adverse reactions (>30%) observed in patients on the pertuzumab arm included diarrhea, alopecia, neutropenia, nausea, fatigue, rash, and peripheral neuropathy. No additive cardiac toxicity was observed. Significant manufacturing issues were identified during the review. On the basis of substantial evidence of efficacy for pertuzumab in MBC and the compelling public health need, FDA did not delay availability to patients pending final resolution of all manufacturing concerns. Therefore, FDA approved pertuzumab but limited its approval to lots not affected by manufacturing problems. The applicant agreed to multiple manufacturing and testing postmarketing commitments under third-party oversight to resolve manufacturing issues. Clin Cancer Res; 19(18); 4911–6. ©2013 AACR.

p63: Defining roles in morphogenesis, homeostasis, and neoplasia of the epidermis

p63 is a member of a gene family also including the p53 tumor suppressor and p73. In contrast to p53, p63 is rarely mutated in human cancers. Rather, gene amplification and dysregulated expression of p63 protein have been observed, particularly in squamous cell carcinomas. p63 is essential for development of stratified squamous epithelium, including the epidermis. The p63 gene is expressed as multiple protein isoforms with different functional capacities, and the balance of these isoforms, along with the presence or absence of the other family members, p53 and p73, can impact biological outcome. Both gene silencing and overexpression approaches have been utilized to elucidate the contributions of specific p63 isoforms to normal epidermal morphogenesis and tissue maintenance. While numerous studies have established the essential nature of p63 in the epidermis, the basis of this requirement, and the unique, as well as, overlapping functions of the individual isoforms, remain controversial. In this review, we summarize the current understanding of roles played by specific p63 isoforms within the context of epidermal morphogenesis and homeostasis of the established epidermis, and the potential impact of p63 dysregulation on cancer development.

Dysregulated ΔNp63α Inhibits Expression of Ink4a/arf, Blocks Senescence, and Promotes Malignant Conversion of Keratinocytes

p63 is critical for squamous epithelial development, and elevated levels of the ΔNp63α isoform are seen in squamous cell cancers of various organ sites. However, significant controversy exists regarding the role of p63 isoforms as oncoproteins or tumor suppressors. Here, lentiviruses were developed to drive long-term overexpression of ΔNp63α in primary keratinocytes. Elevated levels of ΔNp63α in vitro promote long-term survival and block both replicative and oncogene-induced senescence in primary keratinocytes, as evidenced by the expression of SA-β-gal and the presence of nuclear foci of heterochromatin protein 1γ. The contribution of ΔNp63α to cancer development was assessed using an in vivo grafting model of experimental skin tumorigenesis that allows distinction between benign and malignant tumors. Grafted lenti-ΔNp63α keratinocytes do not form tumors, whereas lenti-GFP/v-rasHa keratinocytes develop well-differentiated papillomas. Lenti-ΔNp63α/v-rasHa keratinocytes form undifferentiated carcinomas. The average volume of lenti-ΔNp63α/v-rasHa tumors was significantly higher than those in the lenti-GFP/v-rasHa group, consistent with increased BrdU incorporation detected by immunohistochemistry. The block in oncogene-induced senescence corresponds to sustained levels of E2F1 and phosphorylated AKT, and is associated with loss of induction of p16ink4a/p19arf. The relevance of p16ink4a/p19arf loss was demonstrated in grafting studies of p19arf-null keratinocytes, which develop malignant carcinomas in the presence of v-rasHa similar to those arising in wildtype keratinocytes that express lenti-ΔNp63α and v-rasHa. Our findings establish that ΔNp63α has oncogenic activity and its overexpression in human squamous cell carcinomas contributes to the malignant phenotype, and implicate its ability to regulate p16ink4a/p19arf in the process.

Dysregulated ΔNp63α negatively regulates the maspin promoter in keratinocytes via blocking endogenous p73 binding

While overexpression of the p63 isoform, ΔNp63α, has been reported in squamous cell cancers, the contribution of p63 to cancer pathogenesis remains unclear. We previously demonstrated that overexpressed ΔNp63α aberrantly maintains proliferation of primary mouse keratinocytes under conditions that normally induce growth arrest and differentiation. To identify genes downstream of dysregulated ΔNp63α that may contribute to squamous cancer development and progression, we performed microarray analyses using primary mouse keratinocytes. Herein we report that elevated ΔNp63α differentially regulates genes involved in a variety of cellular functions. Of note, multiple protease inhibitor mRNAs were downregulated including: maspin (serpinB5); plasminogen activator inhibitor‐2 (PAI‐2; serpinB2); and tissue inhibitor of metalloproteinase‐3 (TIMP‐3). Correspondingly, secreted TIMP‐3 and PAI‐2 protein declined in the presence of dysregulated ΔNp63α, however secreted maspin remained stable. Intracellular maspin protein expression decreased in response to overexpressed ΔNp63α, as did PAI‐2. In contrast, TIMP‐3 protein was not detected intracellularly, supporting a solely extracellular function. Electrophoretic mobility shift assays (EMSAs) using a maspin promoter p53/p63 consensus sequence revealed endogenous transcription factor(s) binding to this sequence in keratinocytes that was disrupted by overexpressed ΔNp63α. This was confirmed by ChIP assays. This binding was interrupted by the addition of antibodies recognizing p73, but not p53 or p63, and significantly diminished in EMSA reactions from p73(−/−) keratinocytes, confirming p73 as a constituent. Physical association between p73/ΔNp63α was observed in control β‐gal overexpressing keratinocytes and was enhanced in the presence of overexpressed ΔNp63α These findings underscore the importance of properly balanced p53 homologs for tissue homeostasis.

Abstract 768: A role for c-Rel in ΔNp63α/v-RASHA-driven carcinogenesis?.

The p63 isoform ΔNp63α is expressed in basal keratinocytes and overexpressed in human squamous cell carcinomas, but the mechanisms whereby this p53 homologue contributes to cancer pathogenesis have yet to be elucidated fully. In mimicking the overexpression of ΔNp63α observed in squamous cell carcinomas using transient adenoviral transduction of primary murine kerationcytes, we showed that overexpression of ΔNp63α inhibits Ca 2+ -mediated growth arrest and biochemical differentiation. We previously reported that this block in growth arrest is mediated via the NF-κB subunit, c-Rel, which accumulates in a phosphorylated form in the nucleus of ΔNp63α-overexpressing cells and physically associates in a phosphorylation-dependent manner with ΔNp63α. Consistent with the observed effects on growth regulation, ΔNp63α:c-Rel complexes bind to a p63 binding site on the cdk inhibitor p21 WAF1 . To explore the biological impact of long-term ΔNp63α overexpression in primary murine keratinocytes, lentiviruses were developed. Consistent with our transient adenoviral vector studies, keratinocytes expressing lenti-ΔNp63α have enhanced proliferation rates over 15 days in culture relative to lenti-GFP controls. Using a nude mouse grafting model that allows distinction between normal, benign, and malignant growths, we previously reported that lenti-GFP keratinocytes expressing oncogenic v-ras Ha form well differentiated papillomas at the graft site, while keratinocytes expressing lenti-ΔNp63α in combination with v-ras Ha form undifferentiated carcinomas. To expand this model system to study c-Rel we first confirmed that lentivirus-driven ΔNp63α overexpression results in sustained nuclear accumulation of the NF-kB subunit c-Rel, consistent with our observations using the adenovirally transduced cultures. In the lenti-ΔNp63α expressing cultures, enhancement of c-Rel is observed beginning at day 5 post-infection and is maintained through the latest time point tested, 14 days. Lentiviral c-Rel shRNAs have been developed to assess the contribution of altered nuclear c-Rel expression to ΔNp63α-mediated growth regulation in vitro and in vivo and have been successfully used to knockdown c-Rel to the latest time point tested, 28 days. Taken together, these data support a role for ΔNp63α in facilitating keratinocyte transformation and provide a model system for elucidating the contribution of NFkB/c-rel to ΔNp63α /v-ras Ha -driven carcinogenesis. Citation Format: Kathryn E. King, Roshini M. Ponnamperuma, Sa Ra Park, Steven Jay, Linan Ha, Wendy C. Weinberg. A role for c-Rel in ΔNp63α/v-RAS HA -driven carcinogenesis?. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 768. doi:10.1158/1538-7445.AM2013-768

Abstract 1105: Dysregulated ΔNp63α displaces p73 from the promoter of the tumor suppressor gene maspin in keratinocytes

The p53 homologue p63 is critical for normal epidermal development. While overexpression of p63, in particular ΔNp63α, has been reported in many squamous cell cancers including those of the skin, the role of p63 in cancer pathogenesis remains unclear. We have previously shown that elevated levels of ΔNp63α aberrantly maintain keratinocyte proliferation under differentiating conditions. We performed microarray analyses of primary murine epidermal keratinocytes overexpressing adenoviral (Ad)-driven ΔNp63α vs. β-gal to identify target genes impacted by dysregulated ΔNp63α. Our studies revealed a coordinated downregulation of RNA transcripts for multiple protease inhibitors, including two members of the serine protease inhibitor (serpin) family, maspin (serpinB5) and plasminogen activator inhibitor-2, PAI-2 (serpinB2), in addition to the metalloproteinase inhibitor, tissue inhibitor of metalloproteinase-3 (TIMP-3). Downregulation of all three protease inhibitors was confirmed by RT-PCR and examined further by western analysis. TIMP-3 protein was detected in keratinocyte conditioned medium but not in whole cell lysates, supporting a primarily extracellular function for TIMP-3. PAI-2 and maspin were detected in both whole cell lysates and conditioned medium, consistent with both intracellular and extracellular roles for these proteins. Secreted levels of TIMP-3 and PAI-2 declined in Ad-ΔNp63α-cultures compared to Ad-β-gal-cultures, whereas maspin levels remained stable. Intracellular expression of both PAI-2 and maspin was negatively regulated by ΔNp63α. To investigate the mechanism by which ΔNp63α down-regulates maspin, we performed electrophoretic mobility shift assays (EMSAs) using a known p53 consensus DNA binding sequence in the maspin promoter. An endogenous complex was seen by EMSA in normal keratinocytes that was disrupted in the presence of increasing levels of ΔNp63α. Supershift analysis revealed that this endogenous complex was interrupted by the addition of a p73 antibody, but no supershift or block was seen with antibodies to p63 or p53. EMSA analysis of nuclear extracts prepared from p73(-/-) keratinocytes confirmed that p73 is required for this complex formation. The current data suggest additional roles for ΔNp63α in cancer pathogenesis by disrupting p73 transcriptional activity to down-modulate protease inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1105.

Abstract 2464: Dysregulated DeltaNp63alpha modulates p16 expression, blocks senescence, and promotes malignant conversion of keratinocytes

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC P63 is critical for squamous epithelial development, and elevated levels of the p63 isoform ΔNp63 were reported in squamous cell cancers of the head, neck, skin, lung and cervix. Our lab has previously shown that transient overexpression of ΔNp63α via adenoviral transduction in primary mouse epidermal keratinocytes is associated with enhanced cell proliferation and inhibition of differentiation. However, long-term biological effects of sustained ΔNp63α dysregulation and its contribution to squamous cancer pathogenesis remain unknown. Here, lentiviruses were developed to drive long-term overexpression of ΔNp63α in an in vivo grafting model of experimental skin cancer. Lenti-GFP- and lenti-ΔNp63α-expressing keratinocytes consistently formed normal skin following grafting to nude mice (10 grafted mice/group). Lenti-GFP /v-rasHa keratinocytes developed benign tumors (well-differentiated papillomas) in 4/15 grafted mice. In contrast, lenti- ΔNp63α/v-ras-Ha keratinocytes formed undifferentiated carcinomas in 20/20 mice. The average volume of lenti-ΔNp63α/v-rasHa tumors was 831.9mm3, compared to 25mm3 in the lenti-GFP/v-rasHa group, consistent with increased BrdU incorporation detected by immunohistochemistry. To understand the mechanism of ΔNp63α-induced malignant transformation, we further studied in vitro cell proliferation and senescence in the presence of ΔNp63α dysregulation. Proliferation status was assessed by BrdU uptake. The S-phase population was consistently higher in lenti-ΔNp63α cultures compared to lenti-GFP control cultures (18.18%, 11.12% and 5.12% at days 5, 10, and 14, respectively, versus 17.58%, 4.12% and 1.59%). Moreover, keratinocytes expressing lenti-GFP, but not lenti-ΔNp63α, underwent spontaneous replicative senescence by day 14, as evidenced by the expression of SA-β-gal or the presence of nuclear foci of heterochromatin protein 1γ (HP1γ). Lenti-ΔNp63α also blocked oncogene-induced senescence typically seen within 14 days following introduction of v-rasHa. Both replicative and oncogene-induced senescence were accompanied by an upregulation of p16, which corresponded with a decreased nuclear level of E2F1, starting at day 7 post-infection in lenti-GFP keratinocytes. The upregulation of p16 is much delayed and attenuated in lenti-ΔNp63α cells. The relationship between ΔNp63α and p16 was further confirmed following transient adenoviral-mediated expression of ΔNp63α, which delayed p16 induction until ΔNp63α decreased to endogenous levels. The modulation of p16 by ΔNp63α was shown by RT-PCR to be regulated at the transcriptional level. Taken together, our findings suggest that long-term overexpression of ΔNp63α, as seen in human squamous cell cancers, promotes keratinocyte proliferation and inhibits senescence, thereby facilitating keratinocyte transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2464.