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.

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.

Fusions involving NTRK1, NTRK2, and NTRK3 are oncogenic drivers occurring in a spectrum of mesenchymal neoplasms ranging from benign to highly malignant tumors. To gain further insights into the staining profile with the pan-TRK assay, we analyzed a large number of soft tissue sarcomas and correlated our findings with molecular testing. Additionally, we expand the spectrum of NTRK-fusion tumors by reporting a mesenchymal lesion in the lung as well as a mesenchymal skin lesion in the spectrum of benign fibrous histiocytoma with NTRK—fusion. We retrospectively reviewed soft tissue sarcomas diagnosed at the Diagnostic and Research Institute of Pathology, Medical University of Graz, between 1999 and 2019, and cases from the consultation files of one of the authors (BLA). In total, 494 cases were analyzed immunohistochemically with pan-TRK antibody (clone EPR17341, RTU, Roche/Ventana) and positive cases (defined as any cytoplasmic/nuclear staining in more than 1% of tumor cells) underwent next-generation sequencing (NGS). Immunohistochemical staining was observed in 16 (3.2%) cases. Eleven cases with focal weak and moderate cytoplasmic/membranous or focal moderate to strong nuclear staining did not harbor an NTRK-fusion (three synovial sarcomas, three leiomyosarcomas, two extraskeletal myxoid chondrosarcomas, and one each: dedifferentiated liposarcoma, pleomorphic liposarcoma, and myxofibrosarcoma). Four cases showed strong diffuse nuclear and/or cytoplasmatic staining, and one case showed diffuse, but weak cytoplasmic staining. All these cases demonstrated an NTRK-fusion (LMNA-NTRK1, IRF2BP2-NTRK1, TMB3-NTRK1, ETV6-NTRK3, RBPMS-NTRK3). Pan-TRK assay (clone EPR17341, RTU, Roche, Ventana) immunohistochemistry serves as a reliable diagnostic marker that can also be expressed in non-NTRK-rearranged mesenchymal neoplasms. It can be used as a surrogate marker for identification of NTRK fusion, nevertheless, an RNA-based NGS for detection of the specific fusion should be performed to confirm the rearrangement, if patients are undergoing targeted therapy. Additionally, we identified NTRK-fusion-positive, primary mesenchymal tumors of the lung and the skin.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. The majority are sporadic, solitary tumors that harbor mutually exclusive KIT or PDGFRA gain-of-function mutations. The type of mutation in addition to risk stratification corresponds to the biological behavior of GIST and response to treatment. Up to 85% of pediatric GISTs and 10–15% of adult GISTs are devoid of these (KIT/PDGFRA) mutations and are referred to as wild-type GISTs (wt-GIST). It has been shown that these wt-GISTs are a heterogeneous tumor group with regard to their clinical behavior and molecular profile. Recent advances in molecular pathology helped to further sub-classify the so-called “wt-GISTs”. Based on their significant clinical and molecular heterogeneity, wt-GISTs are divided into a syndromic and a non-syndromic (sporadic) subgroup. Recently, the use of succinate dehydrogenase B (SDHB) by immunohistochemistry has been used to stratify GIST into an SDHB-retained and an SDHB-deficient group. In this review, we focus on GIST sub-classification based on clinicopathologic, and molecular findings and discuss the known and yet emerging prognostic and predictive genetic alterations. We also give insights into the limitations of targeted therapy and highlight the mechanisms of secondary resistance.

Pre-analytical factors, such as fixation time, influence morphology of diagnostic and predictive immunohistochemical staining, which are increasingly used in the evaluation of lung cancer. Our aim was to investigate if variations in fixation time influence the outcome of immunohistochemical staining in lung cancer. From lung resections, specimen with tumor size bigger than 4 cm, 10 samples were obtained: 2 were put through the standard fixation protocol, 5 through the delayed, and 3 through the prolonged fixation protocol. After paraffin embedding, tissue microarrays (TMAs) were made. They were stained with 20 antibodies and scored for quality and intensity of staining. Samples with delay in fixation showed loss of TMA cores on glass slides and deterioration of tissue quality leading to reduction in the expression of CK 7, Keratin MNF116, CAM 5.2, CK 5/6, TTF-1, C-MET, Napsin A, D2-40, and PD-L1. Prolonged fixation had no influence on the performance of immunohistochemical stains. Delay of fixation negatively affects the expression of different immunohistochemical markers, influencing diagnostic (cytokeratins) and predictive (PD-L1) testing. These results emphasize the need for adequate fixation of resection specimen.