Charlene Kay

Systems biology reveals new strategies for personalizing cancer medicine and confirms the role of PTEN in resistance to trastuzumab

Resistance to targeted cancer therapies such as trastuzumab is a frequent clinical problem not solely because of insufficient expression of HER2 receptor but also because of the overriding activation states of cell signaling pathways. Systems biology approaches lend themselves to rapid in silico testing of factors, which may confer resistance to targeted therapies. Inthis study, we aimed to develop a new kinetic model that could be interrogated to predict resistance to receptor tyrosine kinase (RTK) inhibitor therapies and directly test predictions in vitro and in clinical samples. The new mathematical model included RTK inhibitor antibody binding, HER2/HER3 dimerization and inhibition, AKT/mitogen-activated protein kinase cross-talk, and the regulatory properties of PTEN. The model was parameterized using quantitative phosphoprotein expression data from cancer cell lines using reverse-phase protein microarrays. Quantitative PTEN protein expression was found to be the key determinant of resistance to anti-HER2 therapy in silico, which was predictive of unseen experiments in vitro using the PTEN inhibitor bp(V). When measured in cancer cell lines, PTEN expression predicts sensitivity to anti-HER2 therapy; furthermore, this quantitative measurement is more predictive of response (relative risk, 3.0; 95% confidence interval, 1.6-5.5; P < 0.0001) than other pathway components taken in isolation and when tested by multivariate analysis in a cohort of 122 breast cancers treated with trastuzumab. For the first time, a systems biology approach has successfully been used to stratify patients for personalized therapy in cancer and is further compelling evidence that PTEN, appropriately measured in the clinical setting, refines clinical decision making in patients treated with anti-HER2 therapies.

Estrogen Receptor and Progesterone Receptor As Predictive Biomarkers of Response to Endocrine Therapy: A Prospectively Powered Pathology Study in the Tamoxifen and Exemestane Adjuvant Multinational Trial

PURPOSE The Tamoxifen and Exemestane Adjuvant Multinational (TEAM) trial included a prospectively planned pathology substudy testing the predictive value of progesterone receptor (PgR) expression for outcome of estrogen receptor-positive (ER-positive) early breast cancer treated with exemestane versus tamoxifen. PATIENTS AND METHODS Pathology blocks from 4,781 TEAM patients randomly assigned to exemestane versus tamoxifen followed by exemestane for 5 years of total therapy were collected centrally, and tissue microarrays were constructed from samples from 4,598 patients. Quantitative analysis of hormone receptors (ER and PgR) was performed by using image analysis and immunohistochemistry, and the results were linked to outcome data from the main TEAM trial and analyzed relative to disease-free survival and treatment. RESULTS Of 4,325 eligible ER-positive patients, 23% were PgR-poor (Allred < 4) and 77% were PgR- rich (Allred ≥ 5). No treatment-by-marker effect for PgR was observed for exemestane versus tamoxifen (PgR-rich hazard ratio [HR], 0.83; 95% CI, 0.65 to 1.05; PgR-poor HR, 0.85; 95% CI, 0.61 to 1.19; P = .88 for interaction). Both PgR and ER expression were associated with patient prognosis in univariate (PgR HR, 0.53; 95% CI, 0.43 to 0.65; P < .001; ER HR, 0.66; 95% CI, 0.51 to 0.86; P = .002), and multivariate analyses (P < .001 and P = .001, respectively). A trend toward a treatment-by-marker effect for ER-rich patients was observed. CONCLUSION Preferential exemestane versus tamoxifen treatment benefit was not predicted by PgR expression; conversely, patients with ER-rich tumors may derive additional benefit from exemestane. Quantitative analysis of ER and PgR expression provides highly significant information on risk of early relapse (within 1 to 3 years) during treatment.

Trastuzumab and Pertuzumab Produce Changes in Morphology and Estrogen Receptor Signaling in Ovarian Cancer Xenografts Revealing New Treatment Strategies

Purpose: The aim of this study was to investigate the antitumor effects of HER2-directed combination therapy in ovarian cancer xenograft models to evaluate their potential. The combinations of trastuzumab and pertuzumab, and trastuzumab and aromatase inhibitor therapy were investigated. Experimental Design: The effects of trastuzumab, pertuzumab, and letrozole on growth response, apoptosis, morphology, and gene and protein expression were evaluated in the SKOV3 ovarian cancer cell line xenograft and a panel of five human ovarian xenografts derived directly from clinical specimens. Results: The combination of HER2-directed antibodies showed enhanced antitumor activity compared with single antibody therapy in the SKOV3 xenograft model. Apoptosis, morphology, and estrogen-regulated gene expression were modulated by these antibodies in both spatial and temporal manners. A panel of ovarian cancer xenografts showed differential growth responses to the combination of trastuzumab and pertuzumab. High HER2 expression and increasing HER3 protein expression on treatment were associated with growth response. In trastuzumab-treated SKOV3 tumors, there was a change in tumor morphology, with a reduction in frequency of estrogen receptor alpha (ERα)-negative clear cell areas. Trastuzumab, but not pertuzumab, increased expression of ERα in SKOV3 xenografts when analyzed by quantitative immunofluorescence. ERα and downstream signaling targets were modulated by trastuzumab alone and in combination. Trastuzumab enhanced the responsiveness of SKOV3 xenografts to letrozole when given in combination. Conclusions: These data suggest that trastuzumab in combination with pertuzumab could be an effective approach in high HER2-expressing ovarian cancers and could also enhance sensitivity to endocrine therapy in ERα-positive ovarian cancer. Clin Cancer Res; 17(13); 4451–61. ©2011 AACR.

Accurate Prediction and Validation of Response to Endocrine Therapy in Breast Cancer

PURPOSE Aromatase inhibitors (AIs) have an established role in the treatment of breast cancer. Response rates are only 50% to 70% in the neoadjuvant setting and lower in advanced disease. Accurate biomarkers are urgently needed to predict response in these settings and to determine which individuals will benefit from adjuvant AI therapy. PATIENTS AND METHODS Pretreatment and on-treatment (after 2 weeks and 3 months) biopsies were obtained from 89 postmenopausal women who had estrogen receptor-alpha positive breast cancer and were receiving neoadjuvant letrozole for transcript profiling. Dynamic clinical response was assessed with use of three-dimensional ultrasound measurements. RESULTS The molecular response to letrozole was characterized and a four-gene classifier of clinical response was established (accuracy of 96%) on the basis of the level of two genes before treatment (one gene [IL6ST] was associated with immune signaling, and the other [NGFRAP1] was associated with apoptosis) and the level of two proliferation genes (ASPM, MCM4) after 2 weeks of therapy. The four-gene signature was found to be 91% accurate in a blinded, completely independent validation data set of patients treated with anastrozole. Matched 2-week on-treatment biopsies were associated with improved predictive power as compared with pretreatment biopsies alone. This signature also significantly predicted recurrence-free survival (P = .029) and breast cancer -specific survival (P = .009). We demonstrate that the test can also be performed with use of quantitative polymerase chain reaction or immunohistochemistry. CONCLUSION A four-gene predictive model of clinical response to AIs by 2 weeks has been generated and validated. Deregulated immune and apoptotic responses before treatment and cell proliferation that is not reduced 2 weeks after initiation of treatment are functional characteristics of breast tumors that do not respond to AIs.

Defining the molecular response to trastuzumab, pertuzumab and combination therapy in ovarian cancer

Background:Trastuzumab and pertuzumab target the Human Epidermal growth factor Receptor 2 (HER2). Combination therapy has been shown to provide enhanced antitumour activity; however, the downstream signalling to explain how these drugs mediate their response is not clearly understood.Methods:Transcriptome profiling was performed after 4 days of trastuzumab, pertuzumab and combination treatment in human ovarian cancer in vivo. Signalling pathways identified were validated and investigated in primary ovarian xenografts at the protein level and across a timeseries.Results:A greater number and variety of genes were differentially expressed by the combination of antibody therapies compared with either treatment alone. Protein levels of cyclin-dependent kinase inhibitors p21 and p27 were increased in response to both agents and further by the combination; pERK signalling was inhibited by all treatments; but only pertuzumab inhibited pAkt signalling. The expression of proliferation, apoptosis, cell division and cell-cycle markers was distinct in a panel of primary ovarian cancer xenografts, suggesting the heterogeneity of response in ovarian cancer and a need to establish predictive biomarkers.Conclusion:This first comprehensive study of the molecular response to trastuzumab, pertuzumab and combined therapy in vivo highlights both common and distinct downstream effects to agents used alone or in combination, suggesting that complementary pathways may be involved.

Sprouty 2 is an independent prognostic factor in breast cancer and may be useful in stratifying patients for trastuzumab therapy.

Background Resistance to trastuzumab is a clinical problem, partly due to overriding activation of MAPK/PI3K signalling. Sprouty-family proteins are negative regulators of MAPK/PI3K signalling, but their role in HER2-therapy resistance is unknown. Patients and Methods Associations between Sprouty gene expression and clinicopathological features were investigated in a breast cancer microarray meta-analysis. Changes in expression of Spry2 and feedback inhibition on trastuzumab resistance were studied in SKBr3 and BT474 breast carcinoma cell lines using cell viability assays. Spry2 protein expression was measured by quantitative immunofluorescence in a cohort of 122 patients treated with trastuzumab. Results Low gene expression of Spry2 was associated with increased pathological grade, high HER2 expression, and was a significant independent prognostic factor. Overexpression of Spry2 in SKBr3s resulted in enhanced inhibition of cell viability after trastuzumab treatment, and the PI3K-inhibitor LY294002 had a similar effect. Low Spry2 expression was associated with increased risk of death (HR = 2.28, 95% CI 1.22–4.26; p = 0.008) in trastuzumab-treated patients, including in multivariate analysis. Stratification of trastuzumab-treated patients using PTEN and Spry2 was superior to either marker in isolation. Conclusion In breast cancers with deficient feedback inhibition, combinatorial therapy with negative regulators of growth factor signalling may be an effective therapeutic strategy.

Evaluation of carbonic anhydrase IX as a therapeutic target for inhibition of breast cancer invasion and metastasis using a series of in vitro breast cancer models

Triple negative, resistant or metastatic disease are major factors in breast cancer mortality, warranting novel approaches. Carbonic anhydrase IX (CAIX) is implicated in survival, migration and invasion of breast cancer cells and inhibition provides an innovative therapeutic strategy. The efficacy of 5 novel ureido-substituted sulfamate CAIX inhibitors were assessed in increasingly complex breast cancer models, including cell lines in normoxia and hypoxia, 3D spheroids and an ex-vivo explant model utilizing fresh biopsy tissue from different breast cancer subtypes. CAIX expression was evaluated in a tissue microarray (TMA) of 92 paired lymph node and primary breast cancers and 2 inhibitors were appraised in vivo using MDA-MB-231 xenografts. FC11409B, FC9398A, FC9403, FC9396A and S4 decreased cell proliferation and migration and inhibited 3D spheroid invasion. S4, FC9398A and FC9403A inhibited or prevented invasion into collagen. FC9403A significantly reversed established invasion whilst FC9398A and DTP348 reduced xenograft growth. TMA analysis showed increased CAIX expression in triple negative cancers. These data establish CAIX inhibition as a relevant therapeutic goal in breast cancer, targeting the migratory, invasive, and metastatic potential of this disease. The use of biopsy tissue suggests efficacy against breast cancer subtypes, and should provide a useful tool in drug testing against invasive cancers.

Predicting response to the anti-estrogen fulvestrant in recurrent ovarian cancer

BACKGROUND Anti-estrogen therapy appears to have efficacy in a subset of ovarian cancers, as demonstrated in multiple phase II studies. Identifying sensitive patients early in treatment may allow for targeted, low-toxicity primary therapy or prevention of recurrence. We have previously demonstrated that the likelihood of response to letrozole could be improved by patient selection based on estrogen-pathway marker expression. We sought to identify ovarian cancer biomarkers that might indicate sensitivity to fulvestrant, an estrogen receptor antagonist. METHODS Tissue samples from the primary tumors of patients enrolled in a phase II study of fulvestrant for the treatment of multiply-recurrent ovarian cancer were embedded randomly in a tissue microarray (TMA). Estrogen receptor alpha (ERα) expression was assessed by both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (IF) (AQUA) while expression of 14 other estrogen-regulated markers was assessed by quantitative IF and correlated with clinical outcomes. RESULTS Almost half of patients experienced clinical benefit (CR+PR+SD) at 90 days despite a median of 5 previous treatment regimens. 24 of 26 patient samples were available and included in the TMA. ERα expression, measured either by conventional IHC or by AQUA analysis, was associated with clinical benefit, while TFF1 and vimentin expression (measured by IF AQUA score) was predictive of progression-free survival. CONCLUSIONS These results confirm our previous observation that clinical ovarian cancer includes a subset of tumors with sensitivity to estrogen pathway blockade. Expression profile of sensitive tumors appears to be detectably different from insensitive tumors, suggesting that further improvements in treatment efficacy can be obtained through appropriate patient selection.

Molecular Changes in Lobular Breast Cancers in Response to Endocrine Therapy

Invasive lobular carcinoma (ILC) accounts for approximately 10% to 15% of breast carcinomas, and although it responds poorly to neoadjuvant chemotherapy, it appears to respond well to endocrine therapy. Pre- and on-treatment (after 2 weeks and 3 months) biopsies and surgical samples were obtained from 14 postmenopausal women with estrogen receptor-positive (ER(+)) histologically confirmed ILC who responded to 3 months of neoadjuvant letrozole and were compared with a cohort of 14 responding invasive ductal carcinomas (IDC) matched on clinicopathologic features. RNA was extracted and processed for whole human genome expression microarray. Dynamic clinical response was assessed using periodic three-dimensional ultrasound measurements performed during treatment and defined as a reduction of >70% in tumor volume by 3 months. Pretreatment profiles of ILC and IDC tumors showed distinctive expression of genes associated with E-cadherin signaling, epithelial adhesion, and stromal rearrangement. The changes in gene expression in response to letrozole were highly similar between responding ILC and IDC tumors; genes involved in proliferation were downregulated and those involved with immune function and extracellular matrix remodeling were upregulated. However, molecular differences between the histologic subtypes were maintained upon treatment. This is the first study of molecular changes in ILC in response to endocrine therapy to date. The genes that change on letrozole are highly consistent between ILC and IDC. Differences in gene expression between ILC and IDC at diagnosis are maintained at each time point on treatment.

A prospectively planned pathology study within the TEAM trial confirms that progesterone receptor expression is prognostic but is not predictive for differential response to exemestane versus tamoxifen.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #81 Background: The multinational TEAM trial evaluated the steroidal aromatase inactivator Exemestane (E) compared to Tamoxifen (T) as initial adjuvant endocrine therapy. The trial was initiated in 2001 with a primary endpoint of DFS between T and E. In 2004, based on results of the Intergroup Exemestane Study (IES) TEAM was modified so all patients on T were switched to E after 2.5-3 years. The modified design includes 2 co-primary endpoints: DFS of T vs E at 2.75 years, and DFS of E vs T followed by E at 5 years. A prospective pathology study was initiated in 2001 involving patients from 5/9 countries participating in the main trial. Methods: Pathology blocks were collected and centralized in the Edinburgh laboratory. The prospective hypotheses defined in 2001 were 1) PgR poor and 2) HER1-3+ve tumors derive additional benefit from EvsT. Sample size was increased to 5000 cases in 2004. TMAs were constructed in sextuplet. Results: 4805 tumor samples collected centrally and TMAs constructed from 4556 cases (UK/Ireland, Netherlands/Belgium, Germany and Greece contributed tumour samples). Quantititive ER & PgR data for 4300 cases were included in this analysis of ER/PgR status and treatment benefit, for ER 106 cases were excluded (<100 cells) and 40 cases were ER-ve (of which 30 were PgR positive) by central pathology review (1.0%), 85% of cases had ER Allred scores of 7-8, 3.6% were ER poor (Allred 2-4). Of 4052 ER+ve cases with quantitative PgR data, (excluding 95 cases with <100 cells) 912 were PgR poor (Allred 4 or below, 22.5%) and 3140 were PgR rich (Allred 5 or above, 77.5%) in line with our original power calculations for this interaction. In these 4052 cases there were 352 DFS events (April 2008, locoregional or distant recurrence, second breast cancers, or death without recurrence) were recorded in the TEAM trial within 2.75 years of randomization. There was a statistically significant benefit of exemestane versus tamoxifen in this sub-group (HR = 0.81, 95% CI 0.654-0.996, p =0.046) Using Cox regression analysis to test the interaction between PgR status and EvsT no evidence for a treatment by marker effect for PgR with Exemestane was observed (PgR Rich EvT HR 0.77, 95% CI 0.59-1.00, PgR poor HR 0.87, 95% CI 0.62-1.18, p value for interaction 0.58). However PgR was a significant prognostic factor in univariate (PgR rich vs poor HR = 0.49, 95% CI 0.39-0.61, p<0.0001) and multivariate (p<0.0001) regression analysis in this predominantly ER rich population. Conclusion: The TEAM pathology study is the only prospective analysis of the interaction between PgR and efficacy of an AI versus tam as initial endocrine therapy. This study supports the prognostic value of PgR in ER rich early breast cancer but does not provide evidence that PgR poor tumors respond preferentially to AIs vs Tam. This prospectively planned and powered study has 90% power to detect such an interaction with a HR of 1.9. We therefore conclude that PgR status is not a predictive marker for AIvT in line with previous retrospective pathology analyses in BIG-I-98 and Trans-ATAC but in contrast with the original report from ATAC. However the use of PgR as a marker of increased risk of early relapse has been further validated by this study. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 81.