Stephen J. Luen

Tumour-infiltrating lymphocytes in advanced HER2-positive breast cancer treated with pertuzumab or placebo in addition to trastuzumab and docetaxel: a retrospective analysis of the CLEOPATRA study

BACKGROUND High quantities of tumour-infiltrating lymphocytes (TILs) in primary HER2-positive breast cancer are associated with improved prognosis and response to therapy. We aimed to investigate the prognostic role of host antitumour immunity as represented by baseline quantities of TILs in patients with advanced HER2-positive breast cancer treated with either pertuzumab or placebo in addition to trastuzumab and docetaxel. METHODS CLEOPATRA was a randomised phase 3 study comparing the addition of either pertuzumab or placebo to first-line therapy with trastuzumab and docetaxel for patients with locally recurrent, unresectable, or metastatic HER2-positive breast cancer. We assessed the quantity of stromal TILs in prospectively collected tumour samples and investigated their association with progression-free survival, overall survival, clinicopathological characteristics, and pertuzumab treatment. We estimated hazard ratios (HR) and 95% CIs with multivariate Cox regression models fitting stromal TILs as a continuous variable (per 10% increment). The CLEOPATRA trial is registered with ClinicalTrials.gov, number NCT00567190. FINDINGS Tumour samples from 678 (84%) of 808 participants were evaluable for TILs, including 519 (77%) archival samples, 155 (23%) freshly obtained samples (collected 45 days or fewer before randomisation), and four samples of unknown archival status. Median follow-up was 50 months (IQR 41-54) for progression-free survival and 51 months (IQR 46-57) for overall survival. 519 progression-free survival events occurred and 358 patients died. The median TIL value was 10% (IQR 5-30). Freshly obtained tumour samples had significantly lower TIL values than did archival samples (10·00% [95% CI 5·00-20·00] vs 15·00% [5·00-35·00]; p=0·00036). We detected no significant association between TIL values and progression-free survival (adjusted HR 0·95, 95% CI 0·90-1·00, p=0·063). However, for overall survival, each 10% increase in stromal TILs was significantly associated with longer overall survival (adjusted HR 0·89, 95% CI 0·83-0·96, p=0·0014). The treatment effect of pertuzumab did not differ significantly by stromal TIL value for either progression-free survival (pinteraction=0·23) or overall survival (pinteraction=0·21). INTERPRETATION In patients with advanced HER2-positive breast cancer treated with docetaxel, trastuzumab, and pertuzumab or placebo, higher TIL values are significantly associated with improved overall survival, suggesting that the effect of antitumour immunity extends to the advanced setting. Future clinical studies in this cancer subtype should consider TILs as a stratification factor and investigate whether therapies that can augment immunity could potentially further improve survival. FUNDING F Hoffmann-La Roche-Genentech and the Breast Cancer Research Foundation.

Assessing tumor-infiltrating lymphocytes in solid tumors: a practical review for pathologists and proposal for a standardized method from the International Immuno-Oncology Biomarkers Working Group: part 2: TILs in melanoma, gastrointestinal tract carcinomas, non-small cell lung carcinoma and mesothelioma, endometrial and ovarian carcinomas, squamous cell carcinoma of the head and neck, genitourinary carcinomas, and primary brain Tumors

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

Combined CDK4/6 and PI3Kα inhibition is synergistic and immunogenic in triple negative breast cancer

New treatments for triple-negative breast cancer (TNBC) are urgently needed. Despite there being little evidence of clinical activity as single-agent therapies, we show that dual blockade of PI3Kα and CDK4/6 is synergistically effective against multiple RB1-wild-type TNBC models. Combined PI3Kα and CDK4/6 inhibition significantly increased apoptosis, cell-cycle arrest, and tumor immunogenicity and generated immunogenic cell death in human TNBC cell lines. Combination treatment also significantly improved disease control in human xenograft models compared with either monotherapy. Combined PI3Kα and CDK4/6 inhibition significantly increased tumor-infiltrating T-cell activation and cytotoxicity and decreased the frequency of immunosuppressive myeloid-derived suppressor cells in a syngeneic TNBC mouse model. Notably, combined PI3Kα and CDK4/6 inhibition, along with inhibition of immune checkpoints PD-1 and CTLA-4, induced complete and durable regressions (>1 year) of established TNBC tumors in vivo Overall, our results illustrate convergent mechanisms of PI3Kα and CDK4/6 blockade on cell-cycle progression, DNA damage response, and immune-modulation and may provide a novel therapeutic approach for TNBC. Cancer Res; 77(22); 6340-52. ©2017 AACR.

Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis

The quantity of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is a robust prognostic factor for improved patient survival, particularly in triple-negative and HER2-overexpressing BC subtypes1. Although T cells are the predominant TIL population2, the relationship between quantitative and qualitative differences in T cell subpopulations and patient prognosis remains unknown. We performed single-cell RNA sequencing (scRNA-seq) of 6,311 T cells isolated from human BCs and show that significant heterogeneity exists in the infiltrating T cell population. We demonstrate that BCs with a high number of TILs contained CD8+ T cells with features of tissue-resident memory T (TRM) cell differentiation and that these CD8+ TRM cells expressed high levels of immune checkpoint molecules and effector proteins. A CD8+ TRM gene signature developed from the scRNA-seq data was significantly associated with improved patient survival in early-stage triple-negative breast cancer (TNBC) and provided better prognostication than CD8 expression alone. Our data suggest that CD8+ TRM cells contribute to BC immunosurveillance and are the key targets of modulation by immune checkpoint inhibition. Further understanding of the development, maintenance and regulation of TRM cells will be crucial for successful immunotherapeutic development in BC.Extensive, high-dimensional characterization of T cells in breast cancer reveals activated TRM population and a gene signature associated with improved prognosis.

Agonist immunotherapy restores T cell function following MEK inhibition improving efficacy in breast cancer

The presence of tumor-infiltrating lymphocytes in triple-negative breast cancers is correlated with improved outcomes. Ras/MAPK pathway activation is associated with significantly lower levels of tumor-infiltrating lymphocytes in triple-negative breast cancers and while MEK inhibition can promote recruitment of tumor-infiltrating lymphocytes to the tumor, here we show that MEK inhibition adversely affects early onset T-cell effector function. We show that α-4-1BB and α-OX-40 T-cell agonist antibodies can rescue the adverse effects of MEK inhibition on T cells in both mouse and human T cells, which results in augmented anti-tumor effects in vivo. This effect is dependent upon increased downstream p38/JNK pathway activation. Taken together, our data suggest that although Ras/MAPK pathway inhibition can increase tumor immunogenicity, the negative impact on T-cell activity is functionally important. This undesirable impact is effectively prevented by combination with T-cell immune agonist immunotherapies resulting in superior therapeutic efficacy.MEK inhibition in breast cancer is associated with increased tumour infiltrating lymphocytes (TILs), however, MAPK activity is required for T cells function. Here the authors show that TILs activity following MEK inhibition can be enhanced by agonist immunotherapy resulting in synergic therapeutic effects.

Clinical Validity and Utility of Tumor-Infiltrating Lymphocytes in Routine Clinical Practice for Breast Cancer Patients: Current and Future Directions

The interest in tumor-infiltrating lymphocytes (TILs) as a prognostic biomarker in breast cancer has grown in recent years. Biomarkers must undergo comprehensive evaluation in terms of analytical validity, clinical validity and clinical utility before they can be accepted as part of clinical practice. The International Immuno-Oncology Biomarker Working Group has developed a practice guideline on scoring TILs in breast cancer in order to standardize TIL assessment. The prognostic value of TILs as a biomarker in early-stage breast cancer has been established by assessing tumor samples in thousands of patients from large prospective clinical trials of adjuvant therapy. There is a strong linear relationship between increase in TILs and improved disease-free survival for triple-negative and HER2-positive disease. Higher levels of TILs have also been associated with increased rates of pathological complete response to neoadjuvant therapy. TILs have potential clinical utility in breast cancer in a number of areas. These include prediction of responders to immune checkpoint blockade, identification of primary HER2-positive and triple-negative patients who have excellent prognoses and may thus be appropriate for treatment de-escalation, and potentially incorporation into a neoadjuvant endpoint which may be a better surrogate maker for drug development.

Advancing Immunotherapy in Metastatic Breast Cancer

Opinion statementDespite many advances in the treatment of breast cancer, the development of metastatic disease remains an incurable and frequent cause of cancer death for women worldwide. An improved understanding of the role of host immunosurveillance in modulating breast cancer disease biology, as well as impressive survival benefits seen to checkpoint blockade in other malignancies have provided great hope for an expanding role of immunotherapies in breast cancer management. While these novel therapies are currently being investigated in clinical trials, signals of efficacy, and tolerability in early phase studies suggest these will eventually make their way into standard practice algorithms. Ongoing research has highlighted a high degree of intertumoural heterogeneity in tumour lymphocytic infiltrates, suggesting some tumours or subtypes are more immunogenic than others. Furthermore, tumour intrinsic mechanisms of immune evasion are beginning to be uncovered, potentially representing key therapeutic targets to use in combination with checkpoint blockade, exemplifying the emerging concept of personalised medicine approaches to immune therapies. Subsequently, different immunotherapeutic strategies may be required based on stratification by these factors—for the minority of tumours with a high level of pre-existing immunity, immune checkpoint blockade monotherapy may be sufficient. However, for the majority of tumours with lower levels of pre-existing immunity, combination approaches will likely be required to achieve maximal therapeutic effect. Results of ongoing clinical trials including combinations with chemotherapy, radiation therapy, and targeted therapies are eagerly awaited.

Association of Somatic Driver Alterations With Prognosis in Postmenopausal, Hormone Receptor–Positive, HER2-Negative Early Breast Cancer: A Secondary Analysis of the BIG 1-98 Randomized Clinical Trial

Importance A range of somatic driver alterations has been described in estrogen receptor–positive, HER2-negative (ER+/HER2−) early breast cancer (BC); however, the clinical relevance is unknown. Objective To investigate associations of driver alterations with prognosis and the role of PIK3CA mutations in prediction of benefit associated with endocrine therapy in postmenopausal patients with ER+/HER2− early BC treated with tamoxifen or letrozole. Design, Setting, and Participants The Breast International Group (BIG) 1-98 trial randomized 8010 postmenopausal patients with hormone receptor–positive, operable, invasive BC to monotherapy with letrozole, tamoxifen, or a sequential strategy for 5 years. Driver alterations were characterized using next-generation sequencing in primary tumors from a subset of 764 patients from 7329 eligible patients with ER+/HER2− BC, with 841 distant recurrences after a median of 8.1 years of follow-up. To correct for the oversampling of distant recurrences, weighted analysis methods were used. This analysis was conducted from April 4, 2016, to November 30, 2016. Main Outcomes and Measures The prevalence of driver alterations, associations with clinicopathologic factors, distant recurrence-free interval, and treatment interactions were analyzed. Multivariable analyses were performed to adjust for clinicopathologic factors. Results Of 764 samples, 538 (70.4%), including 140 distant recurrence events, were successfully sequenced. Nineteen driver alterations were observed with 5% or greater frequency, with a mean of 4 alterations (range, 0-15) per tumor. PIK3CA mutations were the most common (49%) and were significantly associated with reduction in the risk for distant recurrence (hazard ratio [HR], 0.57; 95% CI, 0.38-0.85; P = .006). TP53 mutations (HR, 1.92; 95% CI, 1.21-3.04; P = .006), amplifications on 11q13 (HR, 2.14; 95% CI, 1.36-3.37; P = .001) and 8p11 (HR, 3.02; 95% CI, 1.88-4.84; P < .001), and increasing number of driver alterations (HR per additional alteration, 1.18; 95% CI, 1.11-1.25; P < .001) were associated with significantly greater risk. Amplifications on 11q13 and 8p11 remained significant predictors in multivariable analysis, but not PIK3CA and TP53 mutations. Patients with tumors harboring kinase or helical domain PIK3CA mutations derived significantly greater benefit from letrozole over tamoxifen than patients whose tumors did not (P interaction = .002). Conclusions and Relevance In ER+/HER2− postmenopausal, early-stage BC, amplifications on 11q13 and 8p11 were significantly associated with increased risk for distant recurrence and PIK3CA mutations were predictive of greater magnitude of benefit from letrozole. With these findings, DNA-based classification may aid adjuvant treatment decision making in this setting. Trial Registration ClinicalTrials.gov Identifier: NCT00004205

Primary Tumor Thickness is a Prognostic Factor in Stage IV Melanoma: A Retrospective Study of Primary Tumor Characteristics.

Introduction: Stage IV melanoma exhibits a diverse range of tumor biology from indolent to aggressive disease. Many important prognostic factors have already been identified. Despite this, the behavior of metastatic melanoma remains difficult to predict. We sought to determine if any primary tumor characteristics affect survival following the diagnosis of stage IV melanoma. Methods: All patients diagnosed with stage IV melanoma between January 2003 and December 2012 were identified from the Victorian Melanoma Service database. Retrospective chart review was performed to collect data on primary tumor characteristics (thickness, ulceration, mitotic rate, melanoma subtype, or occult primary). Known and suspected prognostic factors were additionally collected (time to diagnosis of stage IV disease, age, sex, stage, receipt of chemotherapy, and era of recurrence). The effect of primary tumor characteristics on overall survival from the date of diagnosis of stage IV disease was assessed. Results: A total of 227 patients with a median follow-up of 5 years from diagnosis of stage IV disease were identified. Median overall survival of the cohort was 250 days. Of the primary tumor characteristics assessed, only tumor thickness affected survival from diagnosis of stage IV disease, hazard ratio=1.09 (1.02 to 1.16), P=0.008. This remained significant in multivariate analysis, P=0.007. Other primary tumor characteristics did not significantly influence survival. Conclusions: Primary tumor thickness is a significant prognostic factor in stage IV melanoma. Our data suggest that the biology of the primary melanoma may persist to influence the behavior of metastatic disease.

Publisher Correction: Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis.

In the version of this article originally published, the institution in affiliation 10 was missing. Affiliation 10 was originally listed as Department of Surgery, Royal Melbourne Hospital and Royal Womens’ Hospital, Melbourne, Victoria, Australia. It should have been Department of Surgery, Royal Melbourne Hospital and Royal Womens’ Hospital, University of Melbourne, Melbourne, Victoria, Australia. The error has been corrected in the HTML and PDF versions of this article.