Ignatiadis, Michail

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

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.

Abstract Background: CTCs are associated with clinical outcomes in metastatic breast cancer irrespective of ER/PR/HER2 status. Some data support the prognostic relevance of serial CTC enumeration relative to adjuvant chemotherapy in early stage breast cancer. However, data from a large scale study focused on HER2 directed therapy for HER2+ disease have been lacking. We therefore sought to prospectively evaluate the effect of trastuzumab +/- lapatinib on CTCs and assess the prognostic/predictive value of CTC monitoring in HER2+ early stage breast cancer patients (pts). Methods: The Adjuvant Lapatinib and/or Trastuzumab Treatment Optimisation (ALTTO; NCT00490139) Trial is an international, randomized, open-label phase III study of two targeted agents for HER2+ breast cancer. From June 2007 to July 2011, 8381 pts were randomised from 946 sites in 44 countries to 1 of 4 arms with sequential or concurrent chemotherapy: (i) 52 wks of trastuzumab (T); (ii) 52 wks of oral lapatinib (L); (iii) 12 or 18 wks of T followed by a washout and then 34 or 38 wks of L; or (iv) 52 wks of L+T. 540 (6%) pts provided optional informed consent and up to 30 mL peripheral blood suitable for CTC analyses at baseline with additional collections at 13 or 19 wks, 52 wks, 18 mos, 24 mos, and recurrence. CTC analyses are being conducted in three laboratories (Mayo Clinic Rochester, n=431; Institut Jules Bordet and University of Munich, n=109). 2-3 x 10 mL CellSave™ samples are pooled and processed at each time point for CTC enumeration and HER2 expression using the immunomagnetic/immunofluorescence assay (CellSearch™). A round-robin concordance project was done between Mayo Clinic Rochester and Institut Jules Bordet before embarking on the primary correlative work. Results: At baseline, 20% pts had detectable (i.e., ≥1) EpCAM+/CK+/DAPI+/CD45- CTCs, and 16% pts had detectable EpCAM+/CK+/DAPI+/CD45-/HER2+ CTCs. Correlative analyses with clinical outcome are ongoing with plans for completion by Fall 2014. Conclusions: CTCs were detected in 20% of pts with HER2+ early stage breast cancer. This is similar to the frequency of detection in mixed early stage breast cancer populations relative to ER/PR and HER2 status. Concordance of enumeration and HER2 assessments between the two experienced laboratories, and correlation between disease free survival and CTC findings (from serial samples collected at baseline, during the course of HER2 directed therapy, and at set intervals of follow-up) will be reported. Citation Format: Minetta C Liu, Brigitte Rack, Amylou C Dueck, David W Hillman, Michael B Campion, Monica M Reinholz, Kevin C Halling, Christos Sotiriou, Françoise Rothé, Marion Maetens, Ghizlane Rouas, Wolfgang Janni, Antonio C Wolff, Lyndsay N Harris, Julie R Gralow, Kathleen I Pritchard, Susan Ellard, Nguyet A Le-Lindqwister, Frances Boyle, Evandro De Azambuja, Martine J Piccart-Gebhart, Michail Ignatiadis, Edith A Perez. Circulating tumor cell (CTC) enumeration and HER2 assessment as predictors of breast cancer outcomes in the ALTTO (BIG 2-06, Alliance N063D) Trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-01.

Abstract Background: Preliminary results from the Success and Remagus trials showed that CTC detection by CellSearch® has adverse prognostic value in non-metastatic breast cancer. Moreover, several investigators have characterized HER2 expression on CTCs in early breast cancer. Since the majority of CTC-positive women with non-metastatic breast cancer have only 1 CTC detected / blood volume analyzed, we wanted to evaluate the inter-reader variability in this setting. This is a crucial step before moving forward with multi-lab/multi-center prospective testing of the clinical utility of the CellSearch® technology in non-metastatic breast cancer. Methods: Five galleries of CellSearch® images from 3 European and one US institution (a total of 307 images) were mailed to 22 independent readers from 14 European and US academic laboratories and 8 readers from two CellSearch® Veridex laboratories in a blinded fashion. These images came mainly from studies on CTC and/or HER2−positive CTC detection in breast cancer and included healthy women (negative controls), women with metastatic (positive controls) and non-metastatic disease (Pierga et al SABCS 2010, Pierga et al ASCO 2011, Riedthorf et al CCR 2010, Ignatiadis et al PLoS ONE 2011). Each reader reported the images as either CTC-negative or CTC-positive/HER2−negative or CTC-positive/HER2−positive. Kappa statistics were used to assess inter-reader agreement. The 8 Veridex readers were summarized by a majority voting system to derive a gold standard in order to compare each independent reader using discordance rates. Results: Kappa statistics showed moderate to good agreement between independent readers depending on the gallery evaluated (gallery 1 K: 0.55, gallery 2 K:0.57 gallery 3 K:0.64, gallery 4 K:0.72, gallery 5 K:0.85). These differences were attributed to differences in scoring difficulty of each gallery. Discordances in CTC-positive vs CTC-negative events between each reader and the gold standard ranged from 2.3%-31.3% with 7 readers showing discordance rates <5%, 11 readers between 5–10%, 10 readers between 10–14% and only 2 readers showing discordance rates >14% as compared to the gold standard (median discordance rate: 9.3%). Image analysis showed that investigators with high discordance rates compared to the Veridex gold standard were not always taking into account “morphological characteristics” for defining an event as CTC-positive. Discordant results between readers were mainly due to discordance in CTC definition and to a lesser extent in the definition of HER2−positivity. Indeed, discordances in assigning a CTC-positive event as either HER2−negative or HER2−positive ranged from 0.3−13.7% with 19 readers showing discordance rates <5%, 7 readers between 5–10% and 4 readers with discordance rates >10% (median discordance rate: 3.9%). Conclusion: In non-metastatic breast cancer, we observed low discordance between most independent readers and the gold standard for defining a CellSearch® event as CTC with very few readers showing high discordance rates. Concordance can be improved through appropriate training and the use of all the tools provided by the CellSearch® system for image interpretation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-14.