Spatial predictors of immunotherapy response in triple-negative breast cancer.

Immune checkpoint blockade (ICB) benefits some patients with triple-negative breast cancer, but what distinguishes responders from non-responders is unclear1. Because ICB targets cell-cell interactions2, we investigated the impact of multicellular spatial organization on response, and explored how ICB remodels the tumour microenvironment. We show that cell phenotype, activation state and spatial location are intimately linked, influence ICB effect and differ in sensitive versus resistant tumours early on-treatment. We used imaging mass cytometry3 to profile the in situ expression of 43 proteins in tumours from patients in a randomized trial of neoadjuvant ICB, sampled at three timepoints (baseline, n = 243; early on-treatment, n = 207; post-treatment, n = 210). Multivariate modelling showed that the fractions of proliferating CD8+TCF1+T cells and MHCII+ cancer cells were dominant predictors of response, followed by cancer-immune interactions with B cells and granzyme B+ T cells. On-treatment, responsive tumours contained abundant granzyme B+ T cells, whereas resistant tumours were characterized by CD15+ cancer cells. Response was best predicted by combining tissue features before and on-treatment, pointing to a role for early biopsies in guiding adaptive therapy. Our findings show that multicellular spatial organization is a major determinant of ICB effect and suggest that its systematic enumeration in situ could help realize precision immuno-oncology.

The 27-gene IO score is associated with efficacy of PD-1/L1 inhibitors independent of FGFR expression in a real-world metastatic urothelial carcinoma cohort.

Multiple targeted therapeutics have been approved by the FDA for mUC, including immune checkpoint inhibitors (ICIs) and more recently targeted agents for both FGFR and Nectin-4. FGFR3-aberrant and Nectin-4 expressing cells have been associated with an immunosuppressed phenotype. Given that less than half of all patients respond to these agents as monotherapies and less than 20% are eligible to receive salvage therapy, effective personalized treatment plans are critical. Typical biomarkers for ICIs such as PD-L1 and TMB have not been definitive in mUC, yet a biomarker-driven optimization of first-line therapy and subsequent sequencing have the potential to achieve higher and more durable response rates. The IO score is a 27-gene tumor immune microenvironment (TIME) classifier that has been associated with the clinical benefits of ICIs in multiple cancer types, including mUC. This study demonstrates that the IO score was associated with both progression-free survival (PFS) and overall survival (OS) in a real-world cohort of mUC patients treated with ICIs. Furthermore, the IO score was independent of and provided information incremental to TMB. Interestingly, the IO score predicted benefit in patients with high FGFR expression, despite conflicting data regarding response rates among the FGFR aberrant population. Taken together, these results demonstrate that the IO score assessment of the TIME is associated with a clinical benefit from ICI therapy and that this novel biomarker may inform therapeutic sequencing decisions in mUC, potentially improving outcomes for this notoriously difficult-to-treat disease.

Translation of the 27-gene immuno-oncology test (IO score) to predict outcomes in immune checkpoint inhibitor treated metastatic urothelial cancer patients.

BACKGROUND: The IO Score is a 27-gene immuno-oncology (IO) classifier that has previously predicted benefit to immune checkpoint inhibitor (ICI) therapy in triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC). It generates both a continuous score and a binary result using a defined threshold that is conserved between breast and lung. Herein, we aimed to evaluate the IO Score's binary threshold in ICI-naïve TCGA bladder cancer patients (TCGA-BLCA) and assess its clinical utility in metastatic urothelial cancer (mUC) using the IMvigor210 clinical trial treated with the ICI, atezolizumab. METHODS: We identified a list of tumor immune microenvironment (TIME) related genes expressed across the TCGA breast, lung squamous and lung adenocarcinoma cohorts (TCGA-BRCA, TCGA-LUSQ, and TCGA-LUAD, 939 genes total) and then examined the expression of these 939 genes in TCGA-BLCA, to identify patients as having high inflammatory gene expression. Using this as a test of classification, we assessed the previously established threshold of IO Score. We then evaluated the IO Score with this threshold in the IMvigor210 cohort for its association with overall survival (OS). RESULTS: In TCGA-BLCA, IO Score positive patients had a strong concordance with high inflammatory gene expression (p < 0.0001). Given this concordance, we applied the IO Score to the ICI treated IMvigor210 patients. IO Score positive patients (40%) had a significant Cox proportional hazard ratio (HR) of 0.59 (95% CI 0.45-0.78 p < 0.001) for OS and improved median OS (15.6 versus 7.5 months) compared to IO Score negative patients. The IO Score remained significant in bivariate models combined with all other clinical factors and biomarkers, including PD-L1 protein expression and tumor mutational burden. CONCLUSION: The IMvigor210 results demonstrate the potential for the IO Score as a clinically useful biomarker in mUC. As this is the third tumor type assessed using the same algorithm and threshold, the IO Score may be a promising candidate as a tissue agnostic marker of ICI clinical benefit. The concordance between IO Score and inflammatory gene expression suggests that the classifier is capturing common features of the TIME across cancer types.

Association of a novel 27-gene immuno-oncology assay with efficacy of immune checkpoint inhibitors in advanced non-small cell lung cancer.

BACKGROUND: Immune checkpoint inhibitor (ICI) therapies represent a major advance in treating a variety of advanced-stage malignancies. Nevertheless, only a subset of patients benefit, even when selected based on approved biomarkers such as PD-L1 and tumor mutational burden. New biomarkers are needed to maximize the therapeutic ratio of these therapies. METHODS: In this retrospective cohort, we assessed a 27-gene RT-qPCR immuno-oncology (IO) gene expression assay of the tumor immune microenvironment and determined its association with the efficacy of ICI therapy in 67 advanced-stage NSCLC patients. The 27-gene IO test score (IO score), programmed cell death ligand 1 immunohistochemistry tumor proportion score (PD-L1 TPS), and tumor mutational burden (TMB) were analyzed as continuous variables for response and as binary variables for one-year progression free survival. The threshold for the IO score was prospectively set based upon a previously described training cohort. Prognostic implications of the IO score were evaluated in a separate cohort of 104 advanced-stage NSCLC patients from The Cancer Genome Atlas (TCGA) who received non-ICI therapy. RESULTS: The IO score was significantly different between responders or non-responders (p = 0.007) and associated with progression-free survival (p = 0.001). Bivariate analysis established that the IO score was independent of PD-L1 TPS and TMB in identifying patients benefiting from ICI therapy. In a separate cohort of late-stage NSCLC patients from TCGA, the IO score was not prognostic of outcome from non-ICI-treated patients. CONCLUSIONS: This study is the first application of this 27-gene IO RT-qPCR assay in a clinical cohort with outcome data. IO scores were significantly associated with response to ICI therapy and prolonged progression-free survival. Together, these data suggest the IO score should be further studied to define its role in informing clinical decision-making for ICI treatment in NSCLC.

Comparison of triple-negative breast cancer molecular subtyping using RNA from matched fresh-frozen versus formalin-fixed paraffin-embedded tissue.

BACKGROUND: Triple negative breast cancer (TNBC) is a heterogeneous disease that lacks unifying molecular alterations that can guide therapy decisions. We previously identified distinct molecular subtypes of TNBC (TNBCtype) using gene expression data generated on a microarray platform using frozen tumor specimens. Tumors and cell lines representing the identified subtypes have distinct enrichment in biologically relevant transcripts with differing sensitivity to standard chemotherapies and targeted agents. Since our initial discoveries, RNA-sequencing (RNA-seq) has evolved as a sensitive and quantitative tool to measure transcript abundance. METHODS: To demonstrate that TNBC subtypes were similar between platforms, we compared gene expression from matched specimens profiled by both microarray and RNA-seq from The Cancer Genome Atlas (TCGA). In the clinical care of patients with TNBC, tumor specimens collected for diagnostic purposes are processed by formalin fixation and paraffin-embedding (FFPE). Thus, for TNBCtype to eventually have broad and practical clinical utility we performed RNA-seq gene expression and molecular classification comparison between fresh-frozen (FF) and FFPE tumor specimens. RESULTS: Analysis of TCGA showed consistent subtype calls between 91% of evaluable samples demonstrating conservation of TNBC subtypes across microarray and RNA-seq platforms. We compared RNA-seq performed on 21-paired FF and FFPE TNBC specimens and evaluated genome alignment, transcript coverage, differential transcript enrichment and concordance of TNBC molecular subtype calls. We demonstrate that subtype accuracy between matched FF and FFPE samples increases with sequencing depth and correlation strength to an individual TNBC subtype. CONCLUSIONS: TNBC subtypes were reliably identified from FFPE samples, with highest accuracy if the samples were less than 4 years old and reproducible subtyping increased with sequencing depth. To reproducibly subtype tumors using gene expression, it is critical to select genes that do not vary due to platform type, tissue processing or RNA isolation method. The majority of differentially expressed transcripts between matched FF and FFPE samples could be attributed to transcripts selected for by RNA enrichment method. While differentially expressed transcripts did not impact TNBC subtyping, they will provide guidance on determining which transcripts to avoid when implementing a gene set size reduction strategy. TRIAL REGISTRATION: NCT00930930 07/01/2009.

Generation of an algorithm based on minimal gene sets to clinically subtype triple negative breast cancer patients.

BACKGROUND: Recently, a gene expression algorithm, TNBCtype, was developed that can divide triple-negative breast cancer (TNBC) into molecularly-defined subtypes. The algorithm has potential to provide predictive value for TNBC subtype-specific response to various treatments. TNBCtype used in a retrospective analysis of neoadjuvant clinical trial data of TNBC patients demonstrated that TNBC subtype and pathological complete response to neoadjuvant chemotherapy were significantly associated. Herein we describe an expression algorithm reduced to 101 genes with the power to subtype TNBC tumors similar to the original 2188-gene expression algorithm and predict patient outcomes. METHODS: The new classification model was built using the same expression data sets used for the original TNBCtype algorithm. Gene set enrichment followed by shrunken centroid analysis were used for feature reduction, then elastic-net regularized linear modeling was used to identify genes for a centroid model classifying all subtypes, comprised of 101 genes. The predictive capability of both this new "lean" algorithm and the original 2188-gene model were applied to an independent clinical trial cohort of 139 TNBC patients treated initially with neoadjuvant doxorubicin/cyclophosphamide and then randomized to receive either paclitaxel or ixabepilone to determine association of pathologic complete response within the subtypes. RESULTS: The new 101-gene expression model reproduced the classification provided by the 2188-gene algorithm and was highly concordant in the same set of seven TNBC cohorts used to generate the TNBCtype algorithm (87%), as well as in the independent clinical trial cohort (88%), when cases with significant correlations to multiple subtypes were excluded. Clinical responses to both neoadjuvant treatment arms, found BL2 to be significantly associated with poor response (Odds Ratio (OR) =0.12, p=0.03 for the 2188-gene model; OR = 0.23, p < 0.03 for the 101-gene model). Additionally, while the BL1 subtype trended towards significance in the 2188-gene model (OR = 1.91, p = 0.14), the 101-gene model demonstrated significant association with improved response in patients with the BL1 subtype (OR = 3.59, p = 0.02). CONCLUSIONS: These results demonstrate that a model using small gene sets can recapitulate the TNBC subtypes identified by the original 2188-gene model and in the case of standard chemotherapy, the ability to predict therapeutic response.

Expression of a-Tocopherol-Associated protein (TAP) is associated with clinical outcome in breast cancer patients.

BACKGROUND: The role of vitamin E in breast cancer prevention and treatment has been widely investigated, and the different tocopherols that comprise this nutrient have been shown to have divergent associations with cancer outcome. Our previous studies have shown that α-Tocopherol-associated protein (TAP), a vitamin E binding protein, may function as a tumor suppressor-like factor in breast carcinogenesis. The current study addresses the association of TAP expression with breast cancer clinical outcomes. METHODS: Immunohistochemical stain for TAP was applied to a tissue microarray from a breast cancer cohort consisting of 271 patients with a median follow-up time of 5.2 years. The expression of TAP in tumor cells was compared with patient's clinical outcome at 5 years after diagnosis. The potential role of TAP in predicting outcome was also assessed in clinically relevant subsets of the cohort. In addition, we compared TAP expression and Oncotype DX scores in an independent breast cancer cohort consisting of 71 cases. RESULTS: We demonstrate that the expression of TAP was differentially expressed within the breast cancer cohort, and that ER+/PR ± tumors were more likely to exhibit TAP expression. TAP expression was associated with an overall lower recurrence rate and a better 5-year survival rate. This association was primarily in patients with ER+ tumors; exploratory analysis showed that this association was strongest in patients with node-positive tumors and was independent of stage and treatment with chemotherapy. TAP expression in ER/PR negative or triple negative tumors had no association with clinical outcome. In addition, we did not observe an association between TAP expression and Oncotype DX recurrence score. CONCLUSIONS: The significant positive association we found for α-Tocopherol-associated protein with outcome in breast cancer may help to better define and explain studies addressing α-tocopherol's association with cancer risk and outcome. Additionally, further studies to validate and extend these findings may allow TAP to serve as a breast-specific prognostic marker in breast cancer patients, especially in those patients with ER+ tumors.

27-gene Immuno-Oncology (IO) Score is Associated With Efficacy of Checkpoint Immunotherapy in Advanced NSCLC: A Retrospective BC Cancer Study.

BACKGROUND: Immune checkpoint inhibitors (ICI) are standard of care in advanced non-small cell lung cancer (NSCLC). However, not all patients benefit, even among PD-L1 tumor proportional score (TPS) ≥50%, indicating an unmet need for additional biomarkers such as those assessing the tumor immune microenvironment (TIME). DetermaIO is a 27-gene assay that classifies TIME and has previously demonstrated association with ICI response. METHODS: FFPE samples were selected from BC Cancer and West Clinic Cancer Center patients with performance status (PS) ≤2 who received at least 2 cycles of ICI monotherapy in the first (1L) or second line (2L). IO scores were generated and analyzed for association with PFS and OS. RESULTS: In the entire cohort (N=147), IO score was significantly associated with OS (HR=0.68, 95%CI 0.47-0.99, P = .042) and PFS (HR=0.62, 95%CI 0.43-0.88, P = .0069). In 1L treated patients (PD-L1≥50%, N=78), IO score was significantly associated with PFS (HR=0.55, 95%CI 0.32-0.94, P = .028). In exploratory analyses, IO score was associated with benefit in 1L PS2 patients for OS (HR = 0.26, 95%CI 0.091-0.74, P = .012) and PFS (HR = 0.27, 95%CI 0.098-0.72, P = .0095) which was confirmed in PFS subgroup analysis in the independent West Cancer Center study (N=13 HR=0.14, 95%CI 0.027-0.76, P = .023). CONCLUSION: These data confirm the association of DetermaIO with ICI clinical benefit in NSCLC, and expand on previous studies by demonstrating that first line treated PD-L1≥50% patients can further be stratified by IO score to identify efficacy. Exploratory analysis suggested that the IO score identifies benefit in patients with poor PS.

A phase Ib trial of pembrolizumab plus paclitaxel or flat-dose capecitabine in 1st/2nd line metastatic triple-negative breast cancer.

Chemoimmunotherapy with anti-programmed cell death 1/ligand 1 and cytotoxic chemotherapy is a promising therapeutic modality for women with triple-negative breast cancer, but questions remain regarding optimal chemotherapy backbone and biomarkers for patient selection. We report final outcomes from a phase Ib trial evaluating pembrolizumab (200 mg IV every 3 weeks) with either weekly paclitaxel (80 mg/m2 weekly) or flat-dose capecitabine (2000 mg orally twice daily for 7 days of every 14-day cycle) in the 1st/2nd line setting. The primary endpoint is safety (receipt of 2 cycles without grade III/IV toxicities requiring discontinuation or ≥21-day delays). The secondary endpoint is efficacy (week 12 objective response). Exploratory aims are to characterize immunologic effects of treatment over time, and to evaluate novel biomarkers. The trial demonstrates that both regimens meet the pre-specified safety endpoint (paclitaxel: 87%; capecitabine: 100%). Objective response rate is 29% for pembrolizumab/paclitaxel (n = 4/13, 95% CI: 10-61%) and 43% for pembrolizumab/capecitabine (n = 6/14, 95% CI: 18-71%). Partial responses are observed in two subjects with chemo-refractory metaplastic carcinoma (both in capecitabine arm). Both regimens are associated with significant peripheral leukocyte contraction over time. Response is associated with clinical PD-L1 score, non-receipt of prior chemotherapy, and the H&E stromal tumor-infiltrating lymphocyte score, but also by a novel 27 gene IO score and spatial biomarkers (lymphocyte spatial skewness). In conclusion, pembrolizumab with paclitaxel or capecitabine is safe and clinically active. Both regimens are lymphodepleting, highlighting the competing immunostimulatory versus lymphotoxic effects of cytotoxic chemotherapy. Further exploration of the IO score and spatial TIL biomarkers is warranted. The clinical trial registration is NCT02734290.

Changes in Triple-Negative Breast Cancer Molecular Subtypes in Patients Without Pathologic Complete Response After Neoadjuvant Systemic Chemotherapy.

PURPOSE: Lehmann et al have identified four molecular subtypes of triple-negative breast cancer (TNBC)-basal-like (BL) 1, BL2, mesenchymal (M), and luminal androgen receptor-and an immunomodulatory (IM) gene expression signature modifier. Our group previously showed that the response of TNBC to neoadjuvant systemic chemotherapy (NST) differs by molecular subtype, but whether NST affects the subtype was unknown. Here, we tested the hypothesis that in patients without pathologic complete response, TNBC subtypes can change after NST. Moreover, in cases with the changed subtype, we determined whether epithelial-to-mesenchymal transition (EMT) had occurred. MATERIALS AND METHODS: From the Pan-Pacific TNBC Consortium data set containing TNBC patient samples from four countries, we examined 64 formalin-fixed, paraffin-embedded pairs of matched pre- and post-NST tumor samples. The TNBC subtype was determined using the TNBCtype-IM assay. We analyzed a partial EMT gene expression scoring metric using mRNA data. RESULTS: Of the 64 matched pairs, 36 (56%) showed a change in the TNBC subtype after NST. The most frequent change was from BL1 to M subtypes (38%). No tumors changed from M to BL1. The IM signature was positive in 14 (22%) patients before NST and eight (12.5%) patients after NST. The EMT score increased after NST in 28 (78%) of the 36 patients with the changed subtype (v 39% of the 28 patients without change; P = .002254). CONCLUSION: We report, to our knowledge, for the first time that the TNBC molecular subtype and IM signature frequently change after NST. Our results also suggest that EMT is promoted by NST. Our findings may lead to innovative adjuvant therapy strategies in TNBC cases with residual tumor after NST.

Angiosarcoma: a study of 98 cases with immunohistochemical evaluation of TLE3, a recently described marker of potential taxane responsiveness.

Angiosarcomas may be primary in the skin, primary in soft tissue or viscera, or secondary to irradiation. All angiosarcomas have a poor prognosis. Taxanes may have efficacy in the treatment of angiosarcoma. Expression of TLE3 has been associated with improved outcome in taxane-treated breast cancers. We studied a series of angiosarcoma with TLE3 immunohistochemistry. Cases of angiosarcoma (98 total cases; 37 cutaneous, 48 soft tissue/visceral and 13 post-irradiation) were retrieved and follow up was obtained. Tumors were classified as 'vasoformative', 'spindled', 'epithelioid' and 'mixed'. TLE3 immunohistochemistry was performed. Statistical analyses were performed. Patients (50 males and 48 females) had a median age of 60.2 years. Tumors had a median size 7.5 cm and were vasoformative (N = 43, 44%), spindled (N = 21, 21%), epithelioid (N = 16, 16%) and mixed (N = 18, 18%). Follow up was available for 89/98 patients (91%): 32 (36%) were dead due to disease, 36 (41%) were dead due to other causes and 21 (24%) remained alive. The median time to death was 2.1 years. TLE3 reactivity was observed in 0/37 (0%) cutaneous angiosarcomas, in 28/48 (58%) cases from soft tissue/viscera and in 4/13 (31%) post-irradiation angiosarcomas. (p = <0.0001). Improved 5-year survival was seen in vasoformative angiosarcomas (p = 0.03). TLE3 expression was not associated with taxane response. However, only a subset of patients was treated with taxane. Our study confirms the poor prognosis of angiosarcoma. Vasoformative angiosarcoma may have a more favorable prognosis. A lack of TLE3 expression in cutaneous angiosarcoma may reflect differing pathogenesis.

A novel immuno-oncology algorithm measuring tumor microenvironment to predict response to immunotherapies.

Immune checkpoint inhibitor (ICI) therapies can improve clinical outcomes for patients with solid tumors, but relatively few patients respond. Because ICI therapies support an adaptive immune response, patients with an active tumor microenvironment (TME) may be more likely to respond, and thus biomarkers capable of discerning an active from a quiescent TME may be useful in patient selection. We developed an algorithm optimized for genes expressed in the mesenchymal and immunomodulatory subtypes of a 101-gene triple negative breast cancer model (Ring, BMC Cancer, 2016, 16:143) as a means to capture the immunological state of the TME. We compared the outcome of the algorithm (IO score) with the 101-gene model and found 88% concordance, indicating the models are correlated but not identical, and may be measuring different TME features. We found 92.5% correlation between IO scores of matched tumor epithelial and adjacent stromal tissues, indicating the IO score is not specific to these tissues, but reflects the TME as a whole. We observed a significant difference in IO score (p = 0.0092) between samples with high tumor-infiltrating lymphocytes and samples with increased neutrophil load, demonstrating agreement between IO score and these two prognostic markers. Finally, among non-small cell lung cancer patients receiving immunotherapy, we observed a significant difference in IO score (p = 0.0035) between responders and non-responders, and a significant odds ratio (OR = 5.76, 95% CI 1.30-25.51, p = 0.021), indicating the IO score can predict patient response. The immuno-oncology algorithm may offer independent and incremental predictive value over current biomarkers in the clinic.

Characterization of the tumor immune-microenvironment of adenocarcinoma of lung with a metastatic lesion in the pancreas treated successfully with first-line, single-agent pembrolizumab.

Single-agent immune checkpoint inhibitor therapy in advanced non-small cell lung cancer can significantly prolong progression-free and overall survival when compared with cytotoxic chemotherapy. Here, we report a case of newly diagnosed adenocarcinoma of the lung with a solitary brain metastasis and a biopsy confirmed adenocarcinoma in the tail of the pancreas. Cytomorphology and immunohistochemistry suggested the lung and pancreas tumors were distinct primaries. However, molecular analysis of the lung primary and tumor in the pancreas revealed the same mutations of functional significance in PIK3CA, NF1 and TP53, suggesting the tumors were clonal. A total of three cycles of single-agent pembrolizumab, and radiation to the lung and brain administered between cycles 1 and 2, resulted in marked responses in lung, brain and pancreatic tumors. Despite the discontinuation of the pembrolizumab after three cycles due to severe immune-mediated toxicities, the patient has had no progression 11 months after stopping all active treatment. Results of a novel 27-gene immuno-oncology (IO) expression assay revealed strong IO scores for the lung and pancreatic tumors, indicating a favorable tumor immune-microenvironment and possibly explaining the significant response.

Mammostrat as a tool to stratify breast cancer patients at risk of recurrence during endocrine therapy.

INTRODUCTION: Patients with early-stage breast cancer, treated with endocrine therapy, have approximately 90% 5-year disease-free survival. However, for patients at higher risk of relapse despite endocrine therapy, additional adjuvant therapy, such as chemotherapy, may be indicated. The challenge is to prospectively identify such patients. The Mammostrat® test uses five immunohistochemical markers to stratify patients on tamoxifen therapy into risk groups to inform treatment decisions. We tested the efficacy of this panel in a mixed population of cases treated in a single center with breast-conserving surgery and long-term follow-up. METHODS: Tissue microarrays from a consecutive series (1981 to 1998) of 1,812 women managed by wide local excision and postoperative radiotherapy were collected following appropriate ethical review. Of 1,390 cases stained, 197 received no adjuvant hormonal or chemotherapy, 1,044 received tamoxifen only, and 149 received a combination of hormonal therapy and chemotherapy. Median age at diagnosis was 57, 71% were postmenopausal, 23.9% were node-positive and median tumor size was 1.5 cm. Samples were stained using triplicate 0.6 mm2 tissue microarray cores, and positivity for p53, HTF9C, CEACAM5, NDRG1 and SLC7A5 was assessed. Each case was assigned a Mammostrat risk score, and distant recurrence-free survival (DRFS), relapse-free survival (RFS) and overall survival (OS) were analyzed by marker positivity and risk score. RESULTS: Increased Mammostrat scores were significantly associated with reduced DRFS, RFS and OS in estrogen receptor (ER)-positive breast cancer (P < 0.00001). In multivariate analyses the risk score was independent of conventional risk factors for DRFS, RFS and OS (P < 0.05). In node-negative, tamoxifen-treated patients, 10-year recurrence rates were 7.6 ± 1.5% in the low-risk group versus 20.0 ± 4.4% in the high-risk group. Further, exploratory analyses revealed associations with outcome in both ER-negative and untreated patients. CONCLUSIONS: This is the fifth independent study providing evidence that Mammostrat can act as an independent prognostic tool for ER-positive, tamoxifen-treated breast cancer. In addition, this study revealed for the first time a possible association with outcome regardless of node status and ER-negative tumors. When viewed in the context of previous results, these data provide further support for this antibody panel as an aid to patient management in early-stage breast cancer.

The expression of TRMT2A, a novel cell cycle regulated protein, identifies a subset of breast cancer patients with HER2 over-expression that are at an increased risk of recurrence.

BACKGROUND: Over-expression of HER2 in a subset of breast cancers (HER2+) is associated with high histological grade and aggressive clinical course. Despite these distinctive features, the differences in response of HER2+ patients to both adjuvant cytotoxic chemotherapy and targeted therapy (e.g. trastuzumab) suggests that unrecognized biologic and clinical diversity is confounding treatment strategies. Furthermore, the small but established risk of cardiac morbidity with trastuzumab therapy compels efforts towards the identification of biomarkers that might help stratify patients. METHODS: A single institution tissue array cohort assembled at the Clearview Cancer Institute of Huntsville (CCIH) was screened by immunohistochemistry staining using a large number of novel and commercially available antibodies to identify those with a univariate association with clinical outcome in HER2+ patients. Staining with antibody directed at TRMT2A was found to be strongly associated with outcome in HER2+ patients. This association with outcome was tested in two independent validation cohorts; an existing staining dataset derived from tissue assembled at the Cleveland Clinic Foundation (CCF), and in a new retrospective study performed by staining archived paraffin blocks available at the Roswell Park Cancer Institute (RPCI). RESULTS: TRMT2A staining showed a strong correlation with likelihood of recurrence at five years in 67 HER2+ patients from the CCIH discovery cohort (HR 7.0; 95% CI 2.4 to 20.1, p < 0.0004). This association with outcome was confirmed using 75 HER2+ patients from the CCF cohort (HR 3.6; 95% CI 1.3 to 10.2, p < 0.02) and 64 patients from the RPCI cohort (HR 3.4; 95% CI 1.3-8.9, p < 0.02). In bivariable analysis the association with outcome was independent of grade, tumor size, nodal status and the administration of conventional adjuvant chemotherapy in the CCIH and RPCI cohorts. CONCLUSIONS: Studies from three independent single institution cohorts support TRMT2A protein expression as a biomarker of increased risk of recurrence in HER2+ breast cancer patients. These results suggest that TRMT2A expression should be further studied in the clinical trial setting to explore its predictive power for response to adjuvant cytotoxic chemotherapy in combination with HER2 targeted therapy.

TLE3 as a candidate biomarker of response to taxane therapy.

INTRODUCTION: The addition of taxanes (Ts) to chemotherapeutic regimens has not demonstrated a consistent benefit in early-stage breast cancer. To date, no clinically relevant biomarkers that predict T response have been identified. METHODS: A dataset of immunohistochemistry stains in 411 patients was mined to identify potential markers of response. TLE3 emerged as a candidate marker for T response. To test the association with T sensitivity, an independent 'triple-negative' (TN) validation cohort was stained with anti-TLE3 antibody. RESULTS: TLE3 staining was associated with improved 5-year disease-free interval (DFI) in the overall cohort (n = 441, P < 0.004), in patients treated with cyclophosphamide (C), methotrexate, and 5-fluorouracil (n = 72, P < 0.02), and in those treated with regimens containing doxorubicin (A) and a T (n = 65, P < 0.04). However, no association was shown with outcome in untreated patients (n = 203, P = 0.49) or those treated with a regimen containing A only (n = 66, P = 0.97). In the TN cohort, TLE3 staining was significantly associated with improved 5-year DFI in all patients (n = 81, P < 0.015), in patients treated with AC + T (n = 45, P < 0.02), but not in patients treated with AC (n = 17, P = 0.81). TLE3 was independent of tumor size, nodal status, and grade by bivariable analysis in both cohorts. CONCLUSION: TLE3 staining is associated with improved DFI in T-treated patients in two independent cohorts. Since the validation study was performed in a TN cohort, TLE3 is not serving as a surrogate for estrogen receptor or HER2 expression. TLE3 should be studied in large clinical trial cohorts to establish its role in T chemotherapy selection.

A novel five-antibody immunohistochemical test for subclassification of lung carcinoma.

Malignant epithelial lung carcinoma can be subclassified by histology into several tumor types, including adenocarcinoma and squamous cell carcinoma. The need for a uniform method of classifying lung carcinomas is growing as clinical trials reveal treatment and side effect differences associated with histological subtypes. Diagnosis is primarily performed by morphological assessment. However, the increased use of needle biopsy has diminished the amount of tissue available for interpretation. These changes in how lung carcinomas are diagnosed and treated suggest that the development of improved molecular-based classification tools could improve patient management. We used a 551-patient surgical specimen lung carcinoma retrospective cohort from a regional hospital to assess the association of a large number of proteins with histological type by immunohistochemistry. Five of these antibodies, targeting the proteins TRIM29, CEACAM5, SLC7A5, MUC1, and CK5/6, were combined into one test using a weighted algorithm trained to discriminate adenocarcinoma from squamous cell carcinoma. Antibody-based classification on 600 muM tissue array cores with the five-antibody test was compared to standard histological evaluation on surgical specimens in three independent lung carcinoma cohorts (combined population of 1111 patients). In addition, the five-antibody test was tested against the two-marker panel thyroid transcription factor-1 (TTF-1) and TP63. Both the five-antibody test and TTF-1/TP63 panel had similarly low misclassification rates on the validation cohorts compared to morphological-based diagnosis (4.1 vs 3.5%). However the percentage of patients remaining unclassifiable by TTF-1/TP63 (22%, 95% CI: 20-25%) was twice that of the five-antibody test (11%, 95% CI: 8-13%). The results of this study suggest the five-antibody test may have an immediate function in the clinic for helping pathologists distinguish lung carcinoma histological types. The results also suggest that if validated in prospectively defined clinical trials this classifier might identify candidates for targeted therapy that are overlooked with current diagnostic approaches.

Chemosensitivity and stratification by a five monoclonal antibody immunohistochemistry test in the NSABP B14 and B20 trials.

PURPOSE: To test the association between risk stratification and outcome in a prospectively designed, blinded retrospective study using tissue arrays of available paraffin blocks from the estrogen receptor-expressing, node-negative samples from the National Surgical Adjuvant Breast and Bowel Project B14 and B20 tamoxifen and chemotherapy trials. EXPERIMENTAL DESIGN: Tissue arrays were stained by immunohistochemistry targeting p53, NDRG1, SLC7A5, CEACAM5, and HTF9C. Risk stratification was done using predefined scoring rules, algorithm for combining scores, and cutoff points for low-risk, moderate-risk, and high-risk patient strata. RESULTS: In a univariate Cox model, this test was significantly associated with recurrence-free interval [HR, 1.3 (95% confidence interval, 1.1-1.6); P = 0.006]. In a multivariate model it contributed information independent of age, tumor size, and menopausal status (P = 0.007). The Kaplan-Meier estimates of the proportion of recurrence-free after 10 years were 73%, 86%, and 85% for the high-risk, moderate-risk, and low-risk groups (P = 0.001). The Kaplan-Meier estimates of the breast-cancer-specific-death rate were 23%, 10%, and 9% (P < 0.0001). Exploratory analysis in patients >/=60 years old showed Kaplan-Meier estimates of the proportion of recurrence-free of 78%, 89%, and 92%. Both high-risk and low-risk groups showed significant improvement on treatment with cytotoxic chemotherapy. CONCLUSIONS: Immunohistochemistry using five monoclonal antibodies assigns breast cancer patients to a risk index that was significantly associated with clinical outcome among the estrogen receptor-expressing, node-negative tamoxifen-treated patients. It seems that the test may be able to identify patients who have greater absolute benefit from adjuvant chemotherapy compared with unstratified patient populations. Exploratory analysis suggests that this test will be most useful in clinical decision making for postmenopausal patients.

Gene expression patterns within cell lines are predictive of chemosensitivity.

BACKGROUND: The NCI has undertaken a twenty-year project to characterize compound sensitivity patterns in a selected set of sixty tumor derived cell lines. Previous studies have explored the relationship between compound sensitivity patterns to gene expression, protein expression, and DNA copy number for these same cell lines. A strong correlation between the pattern of expression of a biomarker and sensitivity to a compound could suggest a clinically interesting biological relationship between the two. RESULTS: We isolated RNA's and measured expression of 40000 genes using cDNA microarrays from the fifty-nine publicly available cell lines. Analysis of this data set in comparison with published gene expression data sets demonstrates a high degree of reproducibility in expression level measurements even using completely independent RNA preparations and array technologies. Using the fifty-nine cell lines for discovery and an additional seven cell lines for which extensive compound sensitivity data were available as a test set, we determined that gene-compound pairs with a correlation coefficient above 0.6 had a false discovery rate of approximately 5%. Large scale features of the gene expression and chemosensitivity data, such as tissue of origin and other physiological factors, did not seem to explain the majority of correlations between gene and compound patterns. CONCLUSION: A comparison of gene expression and compound sensitivity in panels of cell lines was demonstrated to have a relatively high validation and low false discovery rate supporting the use of this approach and datasets for identifying candidate biomarkers and targeted biologically active compounds.