Supplementary Text from Genetic Alterations in the Tyrosine Kinase Transcriptome of Human Cancer Cell Lines

Background: During early clinical development, prospective identification of a predictive biomarker and validation of an assay method may not always be feasible. Dichotomizing a continuous biomarker measure to classify responders also leads to challenges. We present a case study of a prospective-retrospective approach for a continuous biomarker identified after patient enrollment but defined prospectively before the unblinding of data. An analysis of the strengths and weaknesses of this approach and the challenges encountered in its practical application are also provided.Methods: HERALD (NCT02134015) was a double-blind, phase 2 study in patients with non-small cell lung cancer (NSCLC) randomized to erlotinib with placebo or with high or low doses of patritumab, a monoclonal antibody targeted against human epidermal growth factor receptor 3 (HER3). While the primary objective was to assess safety and progression-free survival (PFS), a secondary objective was to determine a single predictive biomarker hypothesis to identify subjects most likely to benefit from the addition of patritumab. Although not identified as the primary biomarker in the study protocol, on the basis of preclinical results from 2 independent laboratories, expression levels of the HER3 ligand heregulin (HRG) were prospectively declared the predictive biomarker before data unblinding but after subject enrollment. An assay to measure HRG mRNA was developed and validated. Other biomarkers, such as epidermal growth factor receptor (EGFR) mutation status, were also evaluated in an exploratory fashion. The cutoff value for high vs. low HRG mRNA levels was set at the median delta threshold cycle. A maximum likelihood analysis was performed to evaluate the provisional cutoff. The relationship of HRG values to PFS hazard ratios (HRs) was assessed as a measure of internal validation. Additional NSCLC samples were analyzed to characterize HRG mRNA distribution.Results: The subgroup of patients with high HRG mRNA levels ("HRG-high") demonstrated clinical benefit from patritumab treatment with HRs of 0.37 (P = 0.0283) and 0.29 (P = 0.0027) in the high-and low-dose patritumab arms, respectively. However, only 102 of the 215 randomized patients (47.4%) had sufficient tumor samples for HRG mRNA measurement. Maximum likelihood analysis showed that the provisional cutoff was within the optimal range. In the placebo arm, the HRG-high subgroup demonstrated worse prognosis compared with HRG-low. A continuous relationship was observed between increased HRG mRNA levels and lower HR. Additional NSCLC samples (N = 300) demonstrated a similar unimodal distribution to that observed in this study, suggesting that the defined cutoff may be applicable to future NSCLC studies.Conclusions: The prospective-retrospective approach was successful in clinically validating a probable predictive biomarker. Post hoc in vitro studies and statistical analyses permitted further testing of the underlying assumptions. However, limitations of this analysis include the incomplete collection of adequate tumor tissue and a lack of stratification. In a phase 3 study, findings are being confirmed, and the HRG cutoff value is being further refined. (C) 2015 The Authors. Published by Elsevier B.V.

e19016 Background: HER3, an ErbB receptor with 6 docking sites for PI3K, is implicated in resistance to cancer treatment. P is a fully human mAb specific to HER3. Heregulin (HRG) is the ligand for HER3. In preclinical studies, HRG expression predicted sensitivity to P (Schneider et al., Yonesaka et al. ASCO 2014). P, in combination with erlotinib vs. erlotinib alone, was assessed in a randomized Ph2 study in advanced NSCLC (HERALD, von Pawel et al., ASCO 2014). Methods: Based on preclinical data after HERALD study initiation, HRG was identified as the primary predictive biomarker hypothesis for P clinical benefit prior to unblinding (prospective), albeit after subject enrollment (retrospective). Various methods were explored to measure HRG protein or mRNA in FFPE tissue, and a validated quantitative PCR assay for HRG mRNA measurment was developed. The median delta threshold cycle ΔCt value was specified as the cut-off for HRG high/low, prior to study unblinding. Other biomarkers were also measured, but not specified as the primary biomarker hypothesis. Results: Of the 215 FFPE samples collected in HERALD, 102 provided sufficient tumor sample for HRG mRNA measurement. Subjects in HERALD showed PFS improvement for P combined with erlotinib vs. erlotinib alone in the HRG high group (< ΔCt 3.9), but no PFS improvement in HRG low group, suggesting HRG is predictive for P benefit (von Pawel, ASCO 2014). A continuous relationship (Table) was observed between increased HRG mRNA expression (lower ΔCt) and greater clinical benefit in terms of PFS. Conclusions: Avalidated assay method was established to measure HRG mRNA in FFPE. Utilizing a prospective-retrospective approach in a Ph2 study, HRG mRNA appears to be a predictive biomarker for P, confirming preclinical data. The distribution of HRG mRNA in NSCLC will be further characterized and the cut-off refined to optimally define the recommended patient population that will benefit from P. Clinical trial information: NCT01211483. Cutoff for HRG high (ΔCt) N (#events) HR (pooled dose vs. Pbo) for PFS in HRG high group Log-rank p value 2.7 (1st quartile) 24 (18) 0.180 0.0039 3.0 33 (24) 0.177 0.0009 3.5 46 (36) 0.283 0.0009 3.9 (median) 51 (41) 0.324 0.0013 4.5 65 (50) 0.490 0.0190 5.0 (3rd quartile) 76 (58) 0.561 0.0429

2618 Background: Activation of the receptor tyrosine kinase HER3 and its oncogenic downstream signaling pathways are thought to be critically involved in the development of various cancer types, including non-small cell lung cancer (NSCLC). HER3 serves as a scaffold for PI3K/AKT signaling via heterodimeric interaction with other HER family members, which can be mediated by binding of heregulin (HRG), the natural high affinity ligand for HER3. Patritumab (P) is an internalizing, fully human anti-HER3 monoclonal antibody that competes with HRG for receptor binding. We examined whether HRG expression correlates with P efficacy in vitro and in vivo. Methods: Protein and phosphoprotein levels in tumor cell lines were determined by western blot. Endogenous HRG mRNA expression was analyzed by RT-PCR. To examine signaling in vitro, tumor cells were treated with 10 µg/ml of P. Cells then remained untreated or were stimulated with recombinant HRG for 1 hour prior to lysis. Cell lysates were analyzed for HER3/pHER3- and AKT/pAKT-levels. To determine the efficacy of P in vivo, mice bearing ~200 mm3 tumor xenografts were treated with P twice weekly, and tumor volumes were assessed. Results: Cell lines endogenously expressing HRG showed decreased HER3- and AKT-phosphorylation following treatment with 10 µg/ml of P, whereas no inhibitory effects of P were observed in HRG-negative cell lines. Remarkably, while HCC1569 cells are normally insensitive to P, the addition of exogenous HRG conveyed sensitivity to these cells. In line with these in vitro observations, HRG expression was found to correlate with P single-agent efficacy in 13/15 in vivo tumor models, from a range of tissue types. In contrast, neither levels of HER3 expression nor levels of HER3 phosphorylation were found to consistently correlate with P single-agent efficacy. Conclusions: Unlike HER3 expression or activation, HRG expression reliably correlated with P-mediated inhibition of signaling in vitro and tumor growth inhibition in vivo. Based on these data, HRG expression levels were nominated as the primary predictive biomarker for P efficacy and were clinically evaluated in the HERALD phase 1b/2 trial in patients with NSCLC (Mendell-Harary et al., von Pawel et al. ASCO 2014).

Background/Aims: FGFR4, a member of the fibroblast growth factor receptor family, has been recently associated with progression of melanoma, breast and head and neck carcinoma. Given its uniquely high expression in the liver, we investigated its contributory role to hepatocellular carcinoma (HCC).Methods: We performed a comprehensive sequencing of full-length FGFR4 transcript in 57 tumor/normal HCC tissue pairs, and quantified their mRNA expressions. Notable mutations and expression patterns were correlated with patient data. Clinically significant trends were examined in in vitro models.Results: We found eight genetic alterations including two highly frequent polymorphisms (V101 and G338r). Secretion of alpha-fetoprotein (AFP), a HCC biomarker, was increased among patients bearing homozygous Arg388 alleles. One-third of these patients exhibited increased FGFR4 mRNA expression in the matched tumor/normal tissue. Subsequent in vitro perturbation of FGFR4 signaling through both FGF19-stimulation and FGFR4 silencing confirmed a mechanistic link between FGFR4 activities and tumor aggressiveness. More importantly, inhibition of FGFR activity with PD173074 exquisitely blocked HuH7 (high FGFR4 expression) proliferation as compared to control cell lines.Conclusions: FGFR4 contributes significantly to HCC progression by modulating AFP secretion, proliferation and antiapoptosis. Its frequent overexpression in patients renders its inhibition a novel and much needed pharmacological approach against HCC. (C) 2008 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

AbstractProtein tyrosine kinases (PTKs) play a critical role in the manifestation of cancer cell properties, and respective signaling mechanisms have been studied extensively on immortalized tumor cells. To characterize and analyze commonly used cancer cell lines with regard to variations in the primary structure of all expressed PTKs, we conducted a cDNA-based sequence analysis of the entire tyrosine kinase transcriptome of 254 established tumor cell lines. The profiles of cell line intrinsic PTK transcript alterations and the evaluation of 155 identified polymorphisms and 234 somatic mutations are made available in a database designated “Tykiva” (tyrosine kinome variant). Tissue distribution analysis and/or the localization within defined protein domains indicate functional relevance of several genetic alterations. The cysteine replacement of the highly conserved Y367 residue in fibroblast growth factor receptor 4 or the Q26X nonsense mutation in the tumor-suppressor kinase CSK are examples, and may contribute to cell line–specific signaling characteristics and tumor progression. Moreover, known variants, such as epidermal growth factor receptor G719S, that were shown to mediate anticancer drug sensitivity could be detected in other than the previously reported tumor types. Our data therefore provide extensive system information for the design and interpretation of cell line–based cancer research, and may stimulate further investigations into broader clinical applications of current cancer therapeutics. [Cancer Res 2007;67(23):11368–76]

C73 Hepatocellular carcinoma (HCC) is a major malignancy inflicting most ethnicities with close to half a million fatalities annually. While the etiology of the disease is relatively well-established (mostly due to viral hepatitis and chronic liver injuries), the progression and the treatment of the disease is still poorly understood and under-explored. Hence, we investigated the contributory role of FGFR4 to HCC, a member of the fibroblast growth factor receptor family that has been recently associated with progression of melanoma, breast and head and neck carcinoma. FGFR4 also presents a unique interest to HCC because liver is the tissue type with the highest mRNA expression of FGFR4. We performed a comprehensive resequencing of the entire coding region of FGFR4 in 62 tumor-normal HCC tissue pairs, and also quantified their respective transcript expression by real-time PCR. We found 4 novel genetic alterations (D126N, T179A, G426D, D709G) as well as a dominant representation of two known polymorphisms among the patients (V10I and G338R). Increased FGFR4 mRNA expression was observed in the tumor tissues as compared to the adjacent normal tissue in 31.6% of the patients. Correlation studies with available clinical follow-ups revealed an increased α-fetoprotein (AFP) production in patients bearing the homozygous Arg388 allele, an observation suggestive of the role of FGFR4 in disease progression. Hence, in vitro studies were subsequently performed to confirm the role of FGFR4 in HCC progression. FGFR4 siRNA administration significantly reduced the production of AFP in HuH7, a high AFP-producing cell line. Stimulation of HepG2 cells (low AFP-producing) with FGF19, a specific FGFR4 ligand, also increased the production of AFP. These effects were independent from the impact of FGFR4 activities on cell proliferation. In summary, our data (both in vivo and in vitro) demonstrated that FGFR4 may represent a drug target against the progression of HCC. The specific role of FGFR4 in HCC and the signal transduction pathways implicated will require further investigation.

Signal transduction via tyrosine phosphorylation, normally fine-tuned by the concerted action of both protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is a key mechanism in tumorigenesis. PTP-PEST, a ubiquitously expressed cytoplasmic tyrosine phosphatase, is thought to play an important role in cell adhesion and motility, and may be involved in metastasis. A search for sequence variations within the gene PTPN12 (alias PTP-PEST) was performed in breast cancer cell lines, leading to the identification of three amino acid substitutions at positions 322, 573, and 709. These alterations were also found in squamous cell carcinoma cell lines and could be verified in primary human breast and kidney tumor samples. Analysis of peripheral blood samples confirmed the germline origin of these alterations. Furthermore, functional characterization of the Ile322 and Ala573 PTP-PEST mutants revealed an enhancement of in vitro phosphatase activity, whereas the Lys709 variant showed reduced catalytic activity. These data demonstrate the existence of PTP-PEST variants that might be meaningful for human cancer and underscore the need for further characterizing PTP-PEST and its signaling pathways in context of this disease.

Proteolytic processing and ectodomain shedding have been described for a broad spectrum of transmembrane proteins under both normal and pathophysiological conditions and has been suggested as one mechanism to regulate a protein's function. It has also been documented for the receptor-like protein tyrosine phosphatase PTP-LAR, induced by treating cells with the tumor promoter TPA or the calcium ionophor A23187. Here we identified the epidermal growth factor receptor (EGFR) as both an association partner of PTP-LAR, that mediates phosphorylation of the latter, as well as an inducer of LAR-cleavage. Both overexpression of this kinase and stimulation of endogenous EGFR in various tumor cell lines were shown to induce proteolytic processing of the catalytic LAR-P-subunit. In contrast to TPA-induced shedding of PTP-LAR, EGFR-mediated cleavage did not require PKC-activity. For both stimuli, however, processing of the P-subunit turned out to be dependent on the activation of the MAP kinases ERK1 and ERK2, and was completely abrogated upon pre-treating cells with Batimastat, indicating the involvement of a metalloproteinase in this pathway. Being strongly impaired in fibroblasts derived from ADAM-17/TACE-knockout-mice or tumor cells that express a dominant negative mutant of ADAM-17/TACE, cleavage of PTP-LAR is suggested to be mediated by this metalloproteinase. Paralleled by rapid reduction of cell surface-localized LAR-E-subunit, EGFR-induced cleavage could be shown to lead to degradation of the catalytic LAR-P-subunit, thereby resulting in a significantly reduced overall cellular phosphatase activity of PTP-LAR. These results for the first time identify a protein tyrosine phosphatase as a potential substrate of TACE and describe proteolytic processing of PTP-LAR as a means of regulating phosphatase activity downstream and thus under the control of EGFR-mediated signaling pathways.