Cinzia Solinas

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.

Tumor-infiltrating lymphocyte composition, organization and PD-1/ PD-L1 expression are linked in breast cancer

ABSTRACT The clinical relevance of tumor-infiltrating lymphocytes (TIL) in breast cancer (BC) has been clearly established by their demonstrated correlation with long-term positive outcomes. Nevertheless, the relationship between protective immunity, observed in some patients, and critical features of the infiltrate remains unresolved. This study examined TIL density, composition and organization together with PD-1 and PD-L1 expression in freshly collected and paraffin-embedded tissues from 125 patients with invasive primary BC. Tumor and normal breast tissues were analyzed using both flow cytometry and immunohistochemistry. TIL density distribution is a continuum with 25% of tumors identified as TIL-negative at a TIL density equivalent to normal breast tissues. TIL-positive tumors (75%) were equally divided into TIL-intermediate and TIL-high. Tumors had higher mean frequencies of CD4+ T cells and CD19+ B cells and a lower mean frequency of CD8+ T cells compare with normal tissues, increasing the CD4+/CD8+ T-cell ratio. Tertiary lymphoid structures (TLS), principally located in the peri-tumoral stroma, were detected in 60% of tumors and correlated with higher TIL infiltration. PD-1 and PD-L1 expression were also associated with higher TIL densities and TLS. TIL density, TLS and PD-L1 expression were correlated with more aggressive tumor characteristics, including higher proliferation and hormone receptor negativity. Our findings reveal an important relationship between PD-1/PD-L1 expression, increased CD4+ T and B-cell infiltration, TIL density and TLS, suggesting that evaluating not only the extent but also the nature and location of the immune infiltrate should be considered when evaluating antitumor immunity and the potential for benefit from immunotherapies.

Adjuvant trastuzumab: a 10-year overview of its benefit

ABSTRACT Introduction: Anti-HER2 targeted therapy is one of the key advances in the treatment of breast cancer that have occurred in the last 20 years. In the adjuvant setting, the use of trastuzumab has led to prolonged and sustained survival benefit with very little toxicity as also confirmed by the 10-year follow-up results from the pivotal trials. Despite the survival improvement, several key issues are not entirely resolved in this field. These issues have led to multiple research efforts in de-escalating or escalating the standard treatment with chemotherapy and 1 year of adjuvant trastuzumab. Areas covered: In this paper, we present an in depth overview on the state of the art on these key issues of refining decision-making in adjuvant anti-HER2 therapy. Expert commentary: Despite many important research efforts in the field, chemotherapy plus trastuzumab for a total duration of 1 year remains the standard of care. However, recent data showed that besides standard anthracycline- and taxane-based cytotoxic therapy, alternative chemotherapy regimens can now be proposed to patients with small tumors without nodal involvement and to women at high-risk of developing cardiotoxicity. Of note, besides HER2 itself, biomarkers predicting patients who may truly benefit from anti-HER2 agents are still lacking.

Critical features and challenges associated with imaging in patients undergoing cancer immunotherapy

Manipulating an individuals immune system through immune checkpoint blockade is revolutionizing the paradigms of cancer treatment. Peculiar patterns and kinetics of response have been observed with these new drugs, rendering the assessment of tumor burden particularly challenging in cancer immunotherapy. The mechanisms of action for immune checkpoint blockade, based upon engagement of the adaptive immune system, can generate unusual response patterns, including pseudoprogression, hyperprogression, atypical and delayed responses. In patients treated with immune checkpoint blockade and radiotherapy, a reduction in tumor burden at metastatic sites distant from the irradiation field (abscopal effect) has been observed, with synergistic systemic immune effects provoked by this combination. New toxicities have also been observed, due to excessive immune activity in several organs, including lung, colon, liver and endocrine glands. Efforts to standardize assessment of cancer immunotherapy responses include novel consensus guidelines derived by modifying World Health Organization (WHO) and Response Evaluation Criteria In Solid Tumors (RECIST) criteria. The aim of this review is to evaluate imaging techniques currently used routinely in the clinic and those being used as investigational tools in immunotherapy clinical trials.

Tumor-infiltrating lymphocytes in patients with HER2-positive breast cancer treated with neoadjuvant chemotherapy plus trastuzumab, lapatinib or their combination: A meta-analysis of randomized controlled trials

BACKGROUND A relationship between baseline tumor-infiltrating lymphocytes (TIL) and outcomes has been described in HER2-positive breast cancer. Nevertheless, the magnitude of this association and whether this effect differs based on the type of anti-HER2 agent remain controversial. This meta-analysis investigated the association between baseline TIL and pathologic complete response (pCR) rates in HER2-positive breast cancer patients treated with neoadjuvant chemotherapy plus trastuzumab and lapatinib either alone or in combination. METHODS A literature search covering PubMed, Embase and the Cochrane library up to October 31, 2016 identified randomized, controlled trials investigating neoadjuvant chemotherapy plus trastuzumab and lapatinib either alone or in combination where published data for pCR based on pre-treatment TIL scores were available. Two subgroups were considered: high baseline TIL vs. non-high TIL, according to each study definition. Summary risk estimates (odds ratio) and 95% confidence intervals (CI) were calculated for pCR using pre-treatment TIL levels for each trial. Pooled analyses were conducted using random and fixed effects models. Interaction P-values were computed using a Monte Carlo permutation test. RESULTS A total of 5 studies (N=1256 patients) were included. Overall, high TIL subgroup was associated with a significantly increased pCR rate (OR 2.46; 95% CI 1.36-4.43; P=0.003). No interaction was observed between TIL subgroup (high vs. non-high TIL) and response to anti-HER2 agent(s) (trastuzumab vs. lapatinib vs. their combination; P=0.747) and chemotherapy (anthracycline and taxanes vs. taxanes only; P=0.201). A stronger association between high TIL subgroup and pCR rates was observed when examining only the 4 studies using anthracycline- and taxane- based neoadjuvant chemotherapy and the 60% cut-off for high TIL (N=869, NeoALTTO excluded) with an OR of 2.88 (95% CI 2.03-4.08; P<0.001). CONCLUSIONS In HER2-positive breast cancer, high baseline TIL are associated with increased pCR probability irrespective of neoadjuvant anti-HER2 agent(s) and chemotherapy regimens used.

Immune Checkpoint Molecules on Tumor-Infiltrating Lymphocytes and Their Association with Tertiary Lymphoid Structures in Human Breast Cancer.

There is an exponentially growing interest in targeting immune checkpoint molecules in breast cancer (BC), particularly in the triple-negative subtype where unmet treatment needs remain. This study was designed to analyze the expression, localization, and prognostic role of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, and TIM3 in primary BC. Gene expression analysis using the METABRIC microarray dataset found that all six immune checkpoint molecules are highly expressed in basal-like and HER2-enriched compared to the other BC molecular subtypes. Flow cytometric analysis of fresh tissue homogenates from untreated primary tumors show that PD-1 is principally expressed on CD4+ or CD8+ T cells and CTLA-4 is expressed on CD4+ T cells. The global proportion of PD-L1+, PD-L2+, LAG3+, and TIM3+ tumor-infiltrating lymphocytes (TIL) was low and detectable in only a small number of tumors. Immunohistochemically staining fixed tissues from the same tumors was employed to score TIL and tertiary lymphoid structures (TLS). PD-L1+, PD-L2+, LAG3+, and TIM3+ cells were detected in some TLS in a pattern that resembles secondary lymphoid organs. This observation suggests that TLS are important sites of immune activation and regulation, particularly in tumors with extensive baseline immune infiltration. Significantly improved overall survival was correlated with PD-1 expression in the HER2-enriched and PD-L1 or CTLA-4 expression in basal-like BC. PD-1 and CTLA-4 proteins were most frequently detected on TIL, which supports the correlations observed between their gene expression and improved long-term outcome in basal-like and HER2-enriched BC. PD-L1 expression by tumor or immune cells is uncommon in BC. Overall, the data presented here distinguish PD-1 as a marker of T cell activity in both the T and B cell areas of BC associated TLS. We found that immune checkpoint molecule expression parallels the extent of TIL and TLS, although there is a noteworthy amount of heterogeneity between tumors even within the same molecular subtype. These data indicate that assessing the levels of immune checkpoint molecule expression in an individual patient has important implications for the success of therapeutically targeting them in BC.

Targeting immune checkpoints in breast cancer: an update of early results

The immune tumour microenvironment has been shown to play a crucial role in the development and progression of cancer. Expression of gene signatures, reflecting immune activation, and the presence of tumour-infiltrating lymphocytes were associated with favourable outcomes in HER2-positive and triple-negative breast cancer. Recently, immunotherapy with immune checkpoint blockade induced long-lasting responses and improved survival in hard-to-treat malignancies (ie, melanoma and non-small cell lung cancer) and are changing treatment paradigms in a variety of neoplastic diseases. Immune checkpoint blockade has been evaluated in breast cancer, particularly in the triple-negative subtype, with promising results observed in monotherapy or in combination with chemotherapy in the metastatic and neoadjuvant settings. However, identification of patients who are most likely to benefit from immune checkpoint blockade remains challenging, with many patients not responding to treatments and a significant financial cost. The combination of immune checkpoint blockade with conventional cancer treatments such as chemotherapy, radiotherapy, targeted therapies or with other immunotherapies is a promising strategy to potentiate its efficacy in breast cancer although further research is required to effectively identify who will respond to these immunotherapies. In this review we report the most recent results that emerged from trials testing immune checkpoint blockade and potential predictive biomarkers and emphasise the new strategies that are under clinical development in breast cancer.

The Abscopal Effect in the Era of Cancer Immunotherapy: a Spontaneous Synergism Boosting Anti-tumor Immunity?

Radiotherapy is one of the main treatment strategies used in cancer. Aside from the local control of the disease, which is mediated by a direct cytotoxic effect on tumor cells, radiotherapy has also been shown to exert immune-mediated local and systemic effects. Radiotherapy can elicit anti-tumor responses in distant sites from the radiation field; this phenomenon is known as the abscopal effect and has been described in patients previously treated with immune checkpoint blockade (ICB). Considering that the efficacy of immunotherapy has been demonstrated only in a subset of patients—who often benefit with lasting responses—efforts are ongoing to potentiate its activity with the development of new combination strategies. Radiotherapy might represent a potential candidate for a synergistic combination with immunotherapy, by improving the immunogenicity of tumors and by enhancing local and systemic immune effects. This review aims to summarize the current pre-clinical and clinical data on the immune effects of radiotherapy and their potential implications for cancer immunotherapy.

Cancer immunotherapy-associated hypophysitis

Side effects of immune checkpoint blockade are often said to be infrequent and usually mild. The uniqueness of endocrine immune-related adverse events is their non-reversibility, with incidence and prevalence destined to increase in the coming years, particularly if immunotherapy is used at earlier stages of neoplastic disease. Immune-related hypophysitis is one of these observed endocrine adverse events. It is often difficult to diagnose, sometimes occurring without specific symptoms. It can lead to irreversibly altered functioning of diverse endocrine glands. Radiographically, the differential diagnosis of hypophysitis includes pituitary apoplexy and primary and secondary neoplastic lesions. Immune-related hypophysitis is most common with single-agent anti-CTLA-4, followed by the combination of anti-CTLA-4 and anti-PD-1, while occurs infrequently when anti-PD-1 or anti-PD-L1 agents are administered alone. Hypophysitis with immune checkpoint blockade requires early recognition, diagnosis, and treatment. Patients can present with headache, visual disturbances or other endocrine-related syndromes or they can be asymptomatic. The manifestation of symptoms should prompt blood analysis and magnetic resonance imaging of the brain. Imaging is important to exclude secondary meningeal or parenchymal lesions. Management should include discontinuation of the immune checkpoint blockade, initiation of corticosteroid therapy and eventually hormone replacement therapy. Hypophysitis impacts treatment of the disease and usually requires long-term management of this irreversible side effect. A multidisciplinary team approach is merited to insure the correct diagnosis and management of immune-related hypophysitis.

Quantifying Tertiary Lymphoid Structure-Associated Genes in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissues

Tertiary lymphoid structures (TLS) have been detected in several types of human solid tumors. These structures are thought to regulate local adaptive immune responses that can promote or antagonize tumor progression. Despite positive prognostic values associated with a TLS presence in several studies, discrepancies still exist. TLS are structurally organized entities composed of varying numbers of multiple cell types making their assessment in tumor tissues, particularly biopsies, challenging. Immunohistochemical staining of TLS-related cell populations is the most frequently used method for identifying and scoring them; however, TLS-related gene expression has also been explored. The protocols described are detailed to allow the user to quantify TLS-related gene expression on formalin-fixed paraffin-embedded human breast tumor tissues.