Integrated molecular pathway analysis informs a synergistic combination therapy targeting PTEN/PI3K and EGFR pathways for basal-like breast cancer

The basal-like breast cancer (BLBC) subtype is characterized by positive staining for basal mammary epithelial cytokeratin markers, lack of hormone receptor and HER2 expression, and poor prognosis with currently no approved molecularly-targeted therapies. The oncogenic signaling pathways driving basal-like tumorigenesis are not fully elucidated. Methods One hundred sixteen unselected breast tumors were subjected to integrated analysis of phosphoinositide 3-kinase (PI3K) pathway related molecular aberrations by immunohistochemistry, mutation analysis, and gene expression profiling. Incidence and relationships between molecular biomarkers were characterized. Findings for select biomarkers were validated in an independent series. Synergistic cell killing in vitro and in vivo tumor therapy was investigated in breast cancer cell lines and mouse xenograft models, respectively. Results Sixty-four % of cases had an oncogenic alteration to PIK3CA, PTEN, or INPP4B; when including upstream kinases HER2 and EGFR, 75 % of cases had one or more aberration including 97 % of estrogen receptor (ER)-negative tumors. PTEN-loss was significantly associated to stathmin and EGFR overexpression, positivity for the BLBC markers cytokeratin 5/14, and the BLBC molecular subtype by gene expression profiling, informing a potential therapeutic combination targeting these pathways in BLBC. Combination treatment of BLBC cell lines with the EGFR-inhibitor gefitinib plus the PI3K pathway inhibitor LY294002 was synergistic, and correspondingly, in an in vivo BLBC xenograft mouse model, gefitinib plus PI3K-inhibitor PWT-458 was more effective than either monotherapy and caused tumor regression. Conclusions Our study emphasizes the importance of PI3K/PTEN pathway activity in ER-negative and basal-like breast cancer and supports the future clinical evaluation of combining EGFR and PI3K pathway inhibitors for the treatment of BLBC. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2609-2) contains supplementary material, which is available to authorized users.BioMed Central open acces

Recurrent gross mutations of the PTEN tumor suppressor gene in breast cancers with deficient DSB repair

Basal-like breast cancer (BBC) is a subtype of breast cancer with poor prognosis. Inherited mutations of BRCA1, a cancer susceptibility gene involved in double-strand DNA break (DSB) repair, lead to breast cancers that are nearly always of the BBC subtype; however, the precise molecular lesions and oncogenic consequences of BRCA1 dysfunction are poorly understood. Here we show that heterozygous inactivation of the tumor suppressor gene Pten leads to the formation of basal-like mammary tumors in mice, and that loss of PTEN expression is significantly associated with the BBC subtype in human sporadic and BRCA1-associated hereditary breast cancers. In addition, we identify frequent gross PTEN mutations, involving intragenic chromosome breaks, inversions, deletions and micro copy number aberrations, specifically in BRCA1-deficient tumors. These data provide an example of a specific and recurrent oncogenic consequence of BRCA1-dependent dysfunction in DNA repair and provide insight into the pathogenesis of BBC with therapeutic implications. These findings also argue that obtaining an accurate census of genes mutated in cancer will require a systematic examination for gross gene rearrangements, particularly in tumors with deficient DSB repair

Basal-like phenotype is not associated with patient survival in estrogen-receptor-negative breast cancers

INTRODUCTION: Basal-phenotype or basal-like breast cancers are characterized by basal epithelium cytokeratin (CK5/14/17) expression, negative estrogen receptor (ER) status and distinct gene expression signature. We studied the clinical and biological features of the basal-phenotype tumors determined by immunohistochemistry (IHC) and cDNA microarrays especially within the ER-negative subgroup. METHODS: IHC was used to evaluate the CK5/14 status of 445 stage II breast cancers. The gene expression signature of the CK5/14 immunopositive tumors was investigated within a subset (100) of the breast tumors (including 50 ER-negative tumors) with a cDNA microarray. Survival for basal-phenotype tumors as determined by CK5/14 IHC and gene expression signature was assessed. RESULTS: From the 375 analyzable tumor specimens, 48 (13%) were immunohistochemically positive for CK5/14. We found adverse distant disease-free survival for the CK5/14-positive tumors during the first years (3 years hazard ratio (HR) 2.23, 95% confidence interval (CI) 1.17 to 4.24, p = 0.01; 5 years HR 1.80, 95% CI 1.02 to 3.15, p = 0.04) but the significance was lost at the end of the follow-up period (10 years HR 1.43, 95% CI 0.84 to 2.43, p = 0.19). Gene expression profiles of immunohistochemically determined CK5/14-positive tumors within the ER-negative tumor group implicated 1,713 differently expressed genes (p < 0.05). Hierarchical clustering analysis with the top 500 of these genes formed one basal-like and a non-basal-like cluster also within the ER-negative tumor entity. A highly concordant classification could be constructed with a published gene set (Sorlie's intrinsic gene set, concordance 90%). Both gene sets identified a basal-like cluster that included most of the CK5/14-positive tumors, but also immunohistochemically CK5/14-negative tumors. Within the ER-negative tumor entity there was no survival difference between the non-basal and basal-like tumors as identified by immunohistochemical or gene-expression-based classification. CONCLUSION: Basal cytokeratin-positive tumors have a biologically distinct gene expression signature from other ER-negative tumors. Even if basal cytokeratin expression predicts early relapse among non-selected tumors, the clinical outcome of basal tumors is similar to non-basal ER-negative tumors. Immunohistochemically basal cytokeratin-positive tumors almost always belong to the basal-like gene expression profile, but this cluster also includes few basal cytokeratin-negative tumors

Basal Cytokeratins and HER-2 Oncogene in Breast Cancer

Rintasyöpään sairastuu melkein joka kymmenes suomalainen nainen elinaikanaan. Rintasyöpä on siis kansanterveydellisesti merkittävä tautiryhmä, jota tulee nykytietämyksen mukaan käsitellä monimuotoisena sairautena, joka jakautuu useisiin biologisesti ja kliinisesti toisistaan poikkeaviin alaryhmiin. Jotta yksittäisen rintasyöpäpotilaan hoitoa ja ennustetta voitaisiin tarkentaa, nämä alaryhmät tulisi olla tunnistettavissa ja niiden hoidollinen ja ennusteellinen merkitys tulisi tietää. Väitöskirjassa tutkittiin epiteelin tyvisolukon tukirankaproteiineja eli sytokeratiineja ilmentäviä ns. basaalityypin rintasyöpiä, jotka muodostavat oman rintasyöpäalaryhmänsä. Tutkimuksessa kehitettiin immunohistokemiallinen tunnistusmenetelmä basaalityypin rintasyövälle. Basaalityypin rintasyöviksi on luokiteltavissa noin 10 % ei-perinnöllisistä rintasyövistä ja noin 80 % perinnöllisistä kasvaimista, joissa on syövälle altistava BRCA1-rintasyöpägeenin mutaatio. Tutkimus osoitti, että basaalityypin rintasyövät ovat tyypillisesti aggressiivisia, hormonaaliselle lääkehoidolle reagoimattomia ja nopeasti jakautuvia kasvaimia, jotka ilmentävät niille tunnusomaisia proteiineja. Vaikka basaalityypin kasvaimissa saattaa olla HER-2-syöpägeenin monistuma, joka mahdollistaa vasta-aineperusteisen täsmähoidon, basaalityypin kasvaimet ja HER-2 monistuneet rintasyövät ovat pääasiassa erillisiä tautiryhmiä. Tutkimus tuo tämän lisäksi ensimmäistä kertaa selvästi ilmi, että basaalityypin rintasyövät jakautuvat kahdeksi erilliseksi alaryhmäksi, joilla on erilaiset biologiset ja ennusteelliset ominaisuudet. Tulosten perusteella basaalityypin kasvaimilla on taipumus uusiutua aikaisessa vaiheessa. Kuitenkaan basaalityypin rintasyövän ennuste ei eroa muiden hormonaaliselle lääkehoidolle reagoimattomien kasvainten ennusteesta. Vaikka ennusteellista eroa ei havaittukaan näiden ryhmien välillä, komplementaarisella DNA-sirutekniikalla (cDNA microarray) saatujen tulosten perusteella basaalityypin rintasyövät eroavat muista hormonaaliselle lääkehoidolle reagoimattomista rintasyövistä geenien ilmentymisen suhteen. Väitöskirjatutkimuksessa kehitettiin kaksivärinen kromogeeninen in situ hybridisaatio -menetelmä (dc-CISH), jolla voidaan tunnistaa kasvaimista yksinkertaisesti ja luotettavasti erilaisten geenien kopiolukuja. Geenikopiolukujen määrä saattaa muuttua kasvaimissa, ja niillä on useissa tapauksissa vaikutusta potilaan hoitoon ja ennusteeseen. Tutkimus toi uutta tietoa basaalityypin rintasyövän luokittelusta, biologisista ominaisuuksista ja ennusteesta. Tuloksia voidaan hyödyntää basaalityypin rintasyövän kliinisissä tutkimuksissa ja jatkossa mahdollisesti diagnostiikassa, kun kyseinen rintasyöpäalaryhmä otetaan mitä todennäköisimmin huomioon kasvainten luokittelussa hoidollisten ja ennusteellisten seikkojen tarkennuttua.Breast cancer is no longer regarded as a single disease group but it represents a variety of subgroups with unique biological and clinical characteristics. Basal phenotype breast cancers form one of the subgroups with a unique gene expression profile and cytokeratin expression pattern. Another important breast cancer subgroup determinant is HER-2 oncogene amplification, which is both a prognostic and predictive factor in breast cancer. In this study, basal phenotype breast cancer was studied by both immunohistochemistry and cDNA microarrays. Immunohistochemical identification of basal phenotype breast cancer was conducted using a triple antibody cocktail of p63 and basal cytokeratins 5 and 14. Since there was no association between p63 and basal cytokeratin expression, breast cancer was regarded as basal phenotype if basal cytokeratin expression was present. The basal phenotype was seen in ~10% of sporadic invasive ductal, sporadic HER-2 amplified, and hereditary BRCA2 germ-line mutated tumors, while BRCA1 germ-line mutated tumors showed 78% prevalence of the basal phenotype. Sporadic basal phenotype tumors always expressed luminal cytokeratins, which supports the progenitor cell origin of basal phenotype tumors. In contrast, a small number of CK5/14-positive BRCA1 germ-line mutated hereditary tumors lacked luminal cytokeratin expression. The most prominent features associated with basal phenotype breast cancer were histologic grade III and steroid hormone receptor negative status. Other features characteristic of basal phenotype breast cancers were high proliferation activity, immunopositivity for vimentin, c-kit, p53, EGFR immunopositivity, and negativity for Bcl-2. Additionally, this study showed that even if HER-2 amplification and the basal phenotype can co-exist, they are inversely associated within the ER-negative tumor subgroup. Interestingly, this study showed for the first time that basal phenotype tumors are divided into two microscopically distinguishable tumor subtypes, which differ in the biological features typical of basal phenotype tumors. "Basoluminal" tumors show heterogeneous basal cytokeratin expression in lower proportion of the tumor cells while "basal" subtype tumors stain uniformly with basal cytokeratins. High proliferation activity and c-kit and vimentin immunopositivity were seen more frequently in the basal than in the basoluminal tumor subtype. Conversely, basoluminal tumors were larger in size than basal tumors and included almost all of the HER-2 amplified CK5/14 expressing tumors. When basal phenotype tumors were studied as one group for prognosis, it was shown that they have significantly worse survival estimates during the first years of follow-up than the basal cytokeratin negative tumors. Although, when survival was studied within the ER-negative subgroup, no adverse survival was seen at any stage for basal phenotype tumors identified either by immunohistochemistry or cDNA microarrays. Still, the basal phenotype tumors differ biologically from other ER-negative tumors since they display a unique gene expression profile within the ER-negative tumor subgroup. Further, the newly defined basal phenotype breast cancer subtypes differed in prognosis. The basoluminal subtype tumors had worse survival estimates than the basal subtype breast cancers. This difference was not explained by more frequent HER-2 amplification in the basoluminal subtype. In this study a dual-color chromogenic in situ hybridization (dc-CISH) method was developed for the simultaneous detection of probes against the HER-2 oncogene and the chromosome 17 centromere. Distinct probe detection was achieved by using two enzymes and a combination of green and red chromogens. The HER-2/chomosome 17 centromere ratios obtained by dc-CISH were highly concordant with the results gained by the FDA-approved fluorescence in situ hybridization (FISH) method. dc-CISH combines the good qualities of both FISH and CISH and represents a simple and reliable method for gene copy number assessment in conjunction with good morphological examination and brightfield microscopy

ImmunoRatio: a publicly available web application for quantitative image analysis of estrogen receptor (ER), progesterone receptor (PR), and Ki-67

Introduction Accurate assessment of estrogen receptor (ER), progesterone receptor (PR), and Ki-67 is essential in the histopathologic diagnostics of breast cancer. Commercially available image analysis systems are usually bundled with dedicated analysis hardware and, to our knowledge, no easily installable, free software for immunostained slide scoring has been described. In this study, we describe a free, Internet-based web application for quantitative image analysis of ER, PR, and Ki-67 immunohistochemistry in breast cancer tissue sections. Methods The application, named ImmunoRatio, calculates the percentage of positively stained nuclear area (labeling index) by using a color deconvolution algorithm for separating the staining components (diaminobenzidine and hematoxylin) and adaptive thresholding for nuclear area segmentation. ImmunoRatio was calibrated using cell counts defined visually as the gold standard (training set, n = 50). Validation was done using a separate set of 50 ER, PR, and Ki-67 stained slides (test set, n = 50). In addition, Ki-67 labeling indexes determined by ImmunoRatio were studied for their prognostic value in a retrospective cohort of 123 breast cancer patients. Results The labeling indexes by calibrated ImmunoRatio analyses correlated well with those defined visually in the test set (correlation coefficient r = 0.98). Using the median Ki-67 labeling index (20%) as a cutoff, a hazard ratio of 2.2 was obtained in the survival analysis (n = 123, P = 0.01). ImmunoRatio was shown to adapt to various staining protocols, microscope setups, digital camera models, and image acquisition settings. The application can be used directly with web browsers running on modern operating systems (e.g., Microsoft Windows, Linux distributions, and Mac OS). No software downloads or installations are required. ImmunoRatio is open source software, and the web application is publicly accessible on our website. Conclusions We anticipate that free web applications, such as ImmunoRatio, will make the quantitative image analysis of ER, PR, and Ki-67 easy and straightforward in the diagnostic assessment of breast cancer specimens.BioMed Central Open acces

Additional file 2: Figure S2. of Integrated molecular pathway analysis informs a synergistic combination therapy targeting PTEN/PI3K and EGFR pathways for basal-like breast cancer

Chronic treatment with gefitinib and PWT-458, alone or in combination, does not cause eight loss in mice. Mice bearing MDA-MB-468 xenograft tumors were treated with PWT-458 (100 mg/kg five imes/week), gefitinib (150 mg/kg five times/week), combination of both drugs, or vehicle control. The mouse body weight was measured in control and treated groups using a weighing scale. The results represent the mean body weight Âą S.E.M. (n = 5 mice per group). (PDF 880 kb