The prognostic significance of the IASLC grading system in ALK\u2010positive invasive non\u2010mucinous adenocarcinoma of the lung: correlation with clinicopathological features

The tumor immune microenvironment of anaplastic lymphoma kinase (ALK)-rearranged lung adenocarcinoma (LUAD) stratified by ALK fusion variants is poorly pictured. Hence, in this study, we aim to explore the immune heterogeneity of ALK+ LUAD across different ALK fusion variants and further investigate their significance on clinical prognosis. A retrospective analysis was conducted on ALK+ LUAD patients (N=68). DNA and RNA-based next-generation sequencing (NGS) was performed to clarify the specific ALK fusion variants. Clinical and pathological characteristics were compared between long and short ALK variants. To research the immune heterogeneity, multi-fluorescence was carried out to explore the differences in immune properties, such as tumor-infiltrating lymphocyte (TIL) number, TIL subset, and tertiary lymphoid structures (TLS) development, between long and short ALK variants. Furthermore, the prognostic value of these characteristics was analyzed. Finally, the expression of lymphocyte-activation gene-3 (LAG3), one novel immune therapy target, was assessed across ALK+ LUAD. LUAD patients with short ALK fusion variant-driven tumors exhibited higher American Joint Committee on Cancer (AJCC) stage as well as larger tumor size than those with long ALK fusion variant-driven tumors. Compared to long ALK fusion variants, there were more TILs, especially natural killer (NK) cells, within short ALK variants. However, fewer TLS were established in cancers harboring short ALK variants than those with long ALK variants. In advanced-stage LUAD patients with ALK fusion, short ALK variants, hot immune status, and high-level NK cells were identified to be adverse prognostic factors, while high-level B cells, as well as the development of TLS, served as positive prognostic factors. As for LAG3 expression, LAG3+ immune cells were more enriched in short ALK variants than in long ALK variants. LUAD patients with short ALK fusion variant-driven tumors exhibited worse prognosis than those with long ALK fusion variant-driven tumors. The tumor immune microenvironments are heterogeneous across different ALK fusion variants with short variants characterized by higher levels of TIL, especially NK cells, but by less TLS development than long variants ALK+ LUAD, which disfavor disease outcomes.

Objective: The aim of this study was to investigate how the tumor immune microenvironment differs regarding tumor genomics, as well as its impact on prognoses and responses to immunotherapy in East Asian patients with non-small cell lung cancer (NSCLC).Methods: We performed an integrated analysis using publicly available data to identify associations between anti-programmed death 1 (PD-1)/ programmed death-ligand 1 (PD-L1) immunotherapy efficacy and classic driver oncogene mutations in East Asian NSCLC patients. Four pooled and clinical cohort analyses were used to correlate driver oncogene mutation status and tumor microenvironment based on PD-L1 and CD8+ tumor-infiltrating lymphocytes (TILs). Immune infiltrating patterns were also established for genomic NSCLC subgroups using the CIBERSORT algorithm.Results: Based on East Asian NSCLC patients, TIDE analyses revealed that for anti-PD-1/PD-L1 immunotherapy, epidermal growth factor receptor (EGFR)-mutant and anaplastic lymphoma kinase (ALK)-rearranged tumors yielded inferior responses; however, although Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutant tumors responded better, the difference was not statistically significant (EGFR: P = 0.037; ALK: P < 0.001; KRAS: P = 0.701). Pooled and clinical cohort analyses demonstrated tumor immune microenvironment heterogeneities correlated with oncogenic patterns. The results showed remarkably higher PD-L1- and TIL-positive KRAS-mutant tumors, suggesting KRAS mutations may drive an inflammatory phenotype with adaptive immune resistance. However, the EGFR-mutant or ALK-rearranged groups showed a remarkably higher proportion of PD-L1-/TIL-tumors, suggesting an uninflamed phenotype with immunological ignorance. Notably, similar to triple wild-type NSCLC tumors, EGFR L858R-mutant tumors positively correlated with an inflammatory phenotype, suggesting responsiveness to anti-PD-1/PD-L1 immunotherapy (P < 0.05). Furthermore, the CIBERSORT algorithm results revealed that EGFR-mutant and ALK-rearranged tumors were characterized by an enriched resting memory CD4+ T cell population (P < 0.001), as well as a lack of CD8+ T cells (P < 0.01), and activated memory CD4+ T cells (P = 0.001).Conclusions: Our study highlighted the complex relationships between immune heterogeneity and immunotherapeutic responses in East Asian NSCLC patients regarding oncogenic dependence.

BACKGROUD The aim of this study is to evaluate prevalence of ALK and MET copy number change and its relation to clinicopathologic characteristics and prognosis of patients with IBC. METHODS This study included 32 patients diagnosed as IBC between August 2000 and December 2011 from three cancer centers in Republic of Korea. ALK gene copy number change and rearrangement were assessed using FISH technique and ALK expression was assessed by IHC assay from biopsy or surgical pathology of IBC patients. FISH analysis of MET was also performed in same patient group. We determined disomy as ALK CNG (-); trisomy (3 fusion signals in ≥30% of cells) and polysomy (≥4 fusion signals in ≥ 10% of cells) as ALK CNG (+). Clinicopathologic characteristics, expression status of ER, PR, HER-2, progression-free survival (PFS) and overall survival (OS) were compared according to ALK gene copy number status. RESULTS The median PFS and the median OS of IBC patients were 22.5 months and 37.2 months, and 50% and 34.4% of patients were HER-2 positive and triple negative breast cancer (TNBC), respectively. ALK CNG was observed in 17 patients (53%) out of 32 IBC patients and none had EML4-ALK rearrangement. IHC assay of ALK revealed moderate to strong cytoplasmic staining in majority of tumor cells in all IBC patients, but H-score was not correlated to ALK copy number status (p=0.417). The clinical characteristics of the patients were similar between ALK CNG (-) and ALK CNG (+) group, and proportion of TNBC is higher in ALK CNG (+) patients, but without statistical significance. (47.1% in ALK CNG (+) vs 20.0% in ALK CNG (-), p=0.147) All brain metastasis during follow up period occurred only in ALK CNG (+) patients. (4 patients) At a median follow-up of 17.6 months (range, 1.6-47.0), ALK CNG (+) patients showed worse PFS than patients with ALK CNG (-) patients (median PFS; 12.7months vs 34.3 months), but without statistical significance. (p = 0.274) Overall survival was worse in ALK CNG (+) patients compared to ALK CNG (-) patients (median OS; 24.9 months vs 60.6 months) with marginal statistical significance. (p= 0.074) Multivariate analysis of OS with adjustment for factors including cancer stage, mastectomy, HER2 positivity also revealed worse OS of ALK CNG (+) patients with marginal significance. (HR, 3.60; 95% CI, 0.962-13.463; p = 0.057). Increased MET copy number observed in 42.9% of IBC patients, but it was not related to PFS and OS of IBC patients (p=0.402, p=0.249), and there was no correlation between ALK and MET copy number change. CONCLUSION This study showed significant frequency of ALK CNG in IBC patients. ALK CNG was associated to poor overall survival in IBC patients in this study. Our finding suggests that ALK CNG may have a prognostic significance in IBC patients and their biological significance and relation to susceptibility to ALK inhibitor need to be elucidated. Citation Format: Min Hwan Kim, Joo Hyuk Sohn, Ja Seung Koo, In Hae Park, Kyung Hae Jung, Soohyeon Lee. Anaplastic lymphoma kinase (ALK) gene copy number gain in inflammatory breast cancer (IBC): frequency, clinicopathologic features and prognostic implication . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1212. doi:10.1158/1538-7445.AM2013-1212

Next-generation ALK inhibitors including alectinib, brigatinib, and lorlatinib are the preferred first systemic treatment for individuals with advanced ALK-rearranged NSCLC. Further trials are ongoing including investigation of first-line ensartinib. Next-generation inhibitors have not been compared to each other, and it is unknown which should be used first and what subsequent treatment sequence is optimal.

Next-Generation Sequencing Identified a Novel Crizotinib-Sensitive PLB1-ALK Rearrangement in Lung Large-Cell Neuroendocrine Carcinoma

The concept of “oncogene addiction” led to the realization that not all patients respond equally to each targeted therapy, and best therapeutic results are obtained when the therapy is given to patients whose tumor harbors the “driver mutation” that is sensitive to the drug. In lung cancer, two currently approved new therapies require patient selection based on the presence of sensitizing epidermal growth factor (EGFR) tyrosine kinase domain (TKD) mutation or the anaplastic lymphoma kinase (ALK) gene rearrangement. EGFR mutations occur almost always in lung cancer of adenocarcinoma histology, and uncommonly in other histologies except when they also contain adenocarcinoma component. They are more common in East Asian (~45%) compared to Caucasian (~15-25%) patients, in never/light smokers and in female patients. Ninety percent of EGFR sensitizing mutations involve the 3-5 amino acid deletions on exon 19 and the L858R mutation on exon 21. Although ALK rearrangement also occurs mainly in lung adenocarcinoma and never/light smokers, it is not associated with sex or ethnicity. A great majority of the ALK gene rearrangements that occur in lung cancer involve a fusion between the C-terminal of ALK containing the kinase domain and the N-terminal of echinoderm microtubule-associated protein-like 4 (EML4), which results from an inversion on the short arm of chromosome 2. While EGFR mutations are detected by mainly by sequencing or PCR-based techniques, ALK rearrangement is best detected by the fluorescent in situ hybridization (FISH). However, recent data suggest that the oncogenic ALK fusion proteins can be detected with close to 100% sensitivity and specificity in lung cancer by immunohistochemistry using commercially available ALK specific antibodies, while the native ALK protein is not expressed in normal lung cells. Based on a systematic review of the published literatures, experts representing the International Association for the Study of Lung Cancer (IASLC), in collaboration with the College of American Pathologists (CAP) and the Association of Molecular Pathologists (AMP) recently issued a guideline on Molecular Testing of EGFR and ALK in Lung Cancer. The testing algorithm reported in this guideline will be discussed. Citation Format: Ming Sound Tsao. Molecular testing to personalized EGFR and ALK inhibitor therapies in lung cancer. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr IA10.

Prognostic Factors and Long Term Outcome of 138 Adults with Systemic Anaplastic Large-Cell Lymphoma: a Retrospective Study by the Groupe d'Etude Des Lymphomes De l'Adulte (GELA)

Molecular Testing for Stage IV Non-Small Cell Lung Cancer Patients With Targetable Mutations Following Disease Progression.

Anaplastic large cell lymphomas (ALCL) encompass several subgroups that differ in their clinical presentation, genetic features, and prognosis. We characterized the genetic subgroups of 74 patients with ALCL and correlated programmed death ligand 1 (PD-L1) protein expression and compared the densities and ratios of FOXP3+ T regulatory cells and CD8+ tumor-infiltrating lymphocytes (TILs) in tumor cells and the immune microenvironment. The subgroups included anaplastic lymphoma kinase (ALK)-positive (ALK+) ALCL and ALK-negative (ALK-) ALCL and DUSP22-rearranged and nonrearranged ALK- ALCL. None of our cases represented the TP63-rearrangement ALK- ALCL subgroup. Our results showed that ALK+ ALCL had a higher expression of PD-L1 in the tumor cells, in contrast to ALK- ALCL, which expressed high PD-L1 in tumor-associated macrophages (TAMs). DUSP22-rearranged ALK- ALCL lacked PD-L1 expression in the tumor cells and instead expressed PD-L1 only in TAMs. There was a significant positive correlation of PD-L1 expression between tumor and TAMs in ALK+ ALCL with a negative correlation in ALK- ALCL. Systemic ALCL subgroups had similar densities of CD8+ tumor-infiltrating lymphocytes and FOXP3 T regulatory cells, but differences were observed in the ratio of CD8/FOXP3. Our results suggest that alterations in tumor microenvironment and immune responses exist among systemic ALCL subgroups and these features may account for different clinical behavior and prognosis.

PD-L1 expression in colorectal cancer is associated with microsatellite instability, BRAF mutation, medullary morphology and cytotoxic tumor-infiltrating lymphocytes

GR01.05 Site of Oligometastases

The International Association for the Study of Lung Cancer (IASLC) pathology panel has proposed a new grading system for invasive lung adenocarcinoma (LADC). This study aims to validate this novel grading system for invasive LADC using propensity score matching (PSM), with a specific focus on patients exhibiting spread through air space (STAS). We retrospectively analyzed the clinicopathologic features of a large cohort of 910 non-mucinous LADCs with STAS from 2017 to 2020 and classified them according to the novel grading system. We applied PSM to adjust for potential confounders between the grading groups. Kaplan-Meier and Cox proportional hazards models were adopted for prognostic evaluation. The results showed that the IASLC grading system (grades 2 and 3) stratified well in terms of recurrence-free survival (RFS) and overall survival (OS) (P=0.02 and P=0.02, respectively) after matching, with Grade 3 being an independent predictor of RFS [hazard ratio (HR), 1.533; P=0.02] and OS (HR, 2.765; P=0.02) in multivariable models. The concordance index (C-index) and area under the curve (AUC) of the IASLC system were 0.719 and 0.754 for recurrence and 0.844 and 0.891 for death, respectively. In addition, anaplastic lymphoma kinase (ALK) fusion and tumor protein p53 (TP53) mutations were detected more frequently in grade 3 tumors, while epidermal growth factor receptor (EGFR) mutations were more prevalent in grade 2 tumors. The IASLC grade did not predict the benefit of adjuvant chemotherapy (ACT). This study suggests that the new IASLC grading system is a valuable prognostic tool for patients with STAS-positive LADC.

Introduction: The immune tumor microenvironment (iTME) of soft-tissue sarcomas (STS) is not well characterized. The use of tissue microarray (TMA) methods is an attractive means of high-throughput immunohistochemistry-based immune profiling, Typically, between 1-3 tumor cores per case are included on conventional TMAs that are well validated for many protein markers across multiple tumor types. However, the reproducibility/representativeness of TMA for sarcomas is not fully established, and a significant factor is the marked intratumoral morphologic heterogeneity in STS. Here, we examine the variability of lymphoid infiltrate between different regions of the same tumor and how this might be optimally represented on a TMA. Methods: Multiple FFPE blocks, representing spatially discrete tumor areas, were selected from primary surgical specimen of 46 cases of localized leiomyosarcoma (LMS) and 9 cases of localized synovial sarcoma (SS) identified from institutional diagnostic archive. Following sectioning and immunohistochemistry for lymphocyte markers, average tumor-infiltrating lymphocytes (TIL) number per section was manually assessed and scored as a numerical variable. Variability of TIL numbers within and between cases was then statistically assessed through two-way ANOVA and intraclass correlation To determine the optimal number of 1-mm TMA cores that need to be sampled to provide representative overview of immune microenvironment, we performed iterative sampling of 1 to 20 virtual cores from digital microscopy images taken from representative blocks from 46 LMS cases. TILs within each virtual core area were digitally counted, with the average TIL number from the combination of increasing number of cores compared to the tumor overall TIL value. Results: We found a dynamic range in TIL numbers between individual cases of LMS but little variability in low TIL numbers seen between SS cases. Focusing on infiltrating T-lymphocytes in LMS, we found marked variation of TIL numbers between discrete areas of highly infiltrated tumors. However, 2-way ANOVA showed that intratumoral variance only accounted for 0.2% of variation in TILs, whilst intertumoral variation accounted for 54.1% of variance, suggesting greater intertumor than intratumor heterogeneity in lymphocyte infiltration in the LMS cohort. In our virtual core experiment, many more TMA cores (median = 11) than the conventional 1-3 were required in a sample to provide an accurate picture of the degree of an individual tumor's lymphocyte infiltration in an individual tumor (within +/-20% of mean TIL number). However, when the 46-case cohort was divided into high or low lymphocyte-infiltrated tumors around the cohort median TIL value, a single core was sufficient to correctly identify a tumor as being TIL high or TIL low in the majority of cases. Conclusion: The variability in TIL number between LMS cases (intertumor variation) in our cohort was much greater than the variability between different tissue blocks from the same individual tumor (intratumor variation), indicating that choice of tumor-containing block from those available for a given specimen may not be an important source of variation when studying the immune microenvironment of STS cohorts. We found that using a conventional number of TMA cores was sufficient to distinguish between tumors with high and low degrees of lymphocyte infiltration. However, in many cases, far more than 3 cores were required to accurately recapitulate the precise degree of infiltration in a tumor. Researchers investigating the iTME of STS should be wary of the apparent limitations of conventional TMA approaches in this field. The extent and biologic significance in the variability of lymphocyte distribution in STS lesions requires further investigation. Citation Format: Alex Lee, Khin Thway, Seth Pollack, Ian Judson, Paul Huang, Robin Jones. The accuracy of tissue microarrays in the study of the sarcoma immune microenvironment is dependent on the number of sampled cores [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B28.

Background: Involvement of tumor immune microenvironment including programmed death 1 (PD-1) and PD-ligand 1 (PD-L1) in biological properties of EGFR-mutant non-small cell lung cancer (NSCLC) remains elusive. Correlation of EGFR mutational status and PD-L1 expression and its impact on anti-PD-1/PD-L1 therapies have been intensively investigated but the relationship between pretreatment EGFR T790M (preT790M) and PD-L1 status has not yet been tackled. We have previously reported that EGFR-TKI-naïve patients with high preT790M had significantly shorter progression-free survival (PFS) upon EGFR-TKI treatment. In this study we investigated the association between preT790M status of tumor and immune microenvironment and its impact on efficacy of EGFR-TKI. Materials and methods: We had previously assessed preT790M by droplet digital PCR in 44 advanced NSCLC patients harboring activating EGFR mutations treated with first-line EGFR-TKIs at Osaka City University Hospital between August 2013 and July 2016. For the current study, tumor proportion score (TPS) for PD-L1 and PD-L2 expression was assessed immunohistochemically using 28-8 antibody and D7U8C antibody, respectively. CD8-positive tumor-infiltrating lymphocytes (TILs) in tumor specimens were also evaluated. Results: PD-L1/PD-L2 TPS and CD8-positive TILs were evaluable in 39 of 44 pretreatment tumor specimens. PD-L1 TPS of ≥50%, 1-49% and &amp;lt;1% were observed in 9 (23.1%), 10 (25.6%) and 20 patients (51.3%), respectively. PD-L2 TPS of ≥50%, 1-49% and &amp;lt;1% were observed in 1 (2.6%), 8 (20.5%) and 30 patients (76.9%), respectively. Significantly more PD-L1 high cases (TPS of ≥50%) were detected in subgroup of tumor with preT790M (41.2%, n=7/17) than in that without preT790M (9.1%, n=2/22) (p=0.026). In non-preT790M subgroup, CD8-positive TILs density was significantly higher in PD-L1 high than in PD-L1 low (median: 1298/mm2 vs. 331/mm2, p=0.035), while in preT790M subgroup, there was no significant difference regardless of PD-L1 level (median: 329/mm2 vs. 226/mm2, p=0.19). The median PFS and response rate for initial EGFR-TKIs were 13.6 months and 95% in non-preT790M/PD-L1 low, 1.6 months and 0% in non-preT790M/PD-L1 high, 11.4 months and 80% in preT790M/PD-L1 low, and 6.7 months and 57% in preT790M/PD-L1 high, respectively. No correlation was observed between preT790M and PD-L2 expression levels. Conclusion: Results of our study suggest that there may exist the subtypes in EGFR-mutated NSCLC according to PD-L1 status and tumor immune microenvironment. Differential efficacy of EGFR-TKIs based on these subtypes further implies the potential impact of PD-L1 status and tumor immune microenvironment in EGFRblockade along with preT790M. Citation Format: Yoshiya Matsumoto, Kenji Sawa, Mitsuru Fukui, Jun Oyanagi, Motohiro Izumi, Koichi Ogawa, Tomohiro Suzumura, Tetsuya Watanabe, Hiroyasu Kaneda, Shigeki Mitsuoka, Kazuhisa Asai, Tatsuo Kimura, Nobuyuki Yamamoto, Tomoya Kawaguchi, Yasuhiro Koh. Association between low-frequency pretreatment EGFR T790M mutation and tumor immune microenvironment in patients with EGFR-mutated non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4906.

Validation Study of New IASLC Histology Grading System in Stage I Non-Mucinous Adenocarcinoma Comparing With Minimally Invasive Adenocarcinoma