Metastatic Specimens Research Articles

Immunogenic cell death signatures from on-treatment tumor specimens predict immune checkpoint therapy response in metastatic melanoma

Melanoma is a highly malignant form of skin cancer that typically originates from abnormal melanocytes. Despite significant advances in treating metastatic melanoma with immune checkpoint blockade (ICB) therapy, a substantial number of patients do not respond to this treatment and face risks of recurrence and metastasis. This study collected data from multiple datasets, including cohorts from Riaz et al., Gide et al., MGH, and Abril-Rodriguez et al., focusing on on-treatment samples during ICB therapy. We used the single-sample gene set enrichment analysis (ssGSEA) method to calculate immunogenic cell death scores (ICDS) and employed an elastic network algorithm to construct a model predicting ICB efficacy. By analyzing 18 ICD gene signatures, we identified 9 key ICD gene signatures that effectively predict ICB treatment response for on-treatment metastatic melanoma specimens. Results showed that patients with high ICD scores had significantly higher response rates to ICB therapy compared to those with low ICD scores. ROC analysis demonstrated that the AUC values for both the training and validation sets were around 0.8, indicating good predictive performance. Additionally, survival analysis revealed that patients with high ICD scores had longer progression-free survival (PFS). This study used an elastic network algorithm to identify 9 ICD gene signatures related to the immune response in metastatic melanoma. These gene features can not only predict the efficacy of ICB therapy but also provide references for clinical decision-making. The results indicate that ICD plays an important role in metastatic melanoma immunotherapy and that expressing ICD signatures can more accurately predict ICB treatment response and prognosis for on-treatment metastatic melanoma specimens, thus providing a basis for personalized treatment.

Spatial multiplexed immunofluorescence analysis reveals coordinated cellular networks associated with overall survival in metastatic osteosarcoma

Patients diagnosed with advanced osteosarcoma, often in the form of lung metastases, have abysmal five-year overall survival rates. The complexity of the osteosarcoma immune tumor microenvironment has been implicated in clinical trial failures of various immunotherapies. The purpose of this exploratory study was to spatially characterize the immune tumor microenvironment of metastatic osteosarcoma lung specimens. Knowledge of the coordinating cellular networks within these tissues could then lead to improved outcomes when utilizing immunotherapy for treatment of this disease. Importantly, various cell types, interactions, and cellular neighborhoods were associated with five-year survival status. Of note, increases in cellular interactions between T lymphocytes, positive for programmed cell death protein 1, and myeloid-derived suppressor cells were observed in the 5-year deceased cohort. Additionally, cellular neighborhood analysis identified an Immune-Cold Parenchyma cellular neighborhood, also associated with worse 5-year survival. Finally, the Osteosarcoma Spatial Score, which approximates effector immune activity in the immune tumor microenvironment through the spatial proximity of immune and tumor cells, was increased within 5-year survivors, suggesting improved effector signaling in this patient cohort. Ultimately, these data represent a robust spatial multiplexed immunofluorescence analysis of the metastatic osteosarcoma immune tumor microenvironment. Various communication networks, and their association with survival, were described. In the future, identification of these networks may suggest the use of specific, combinatory immunotherapeutic strategies for improved anti-tumor immune responses and outcomes in osteosarcoma.

Inhibition of osteosarcoma metastasis in vivo by targeted downregulation of MMP1 and MMP9

Osteosarcoma (OS) mortality stems from lung metastases. Matrix metalloproteinases (MMPs) facilitate metastatic dissemination by degrading extracellular matrix components. Herein we studied the impact of targeted MMP downregulation on OS metastasis. Differential gene expression analysis of human OS cell lines revealed high MMP9 expression in the majority of OS cell lines. Furthermore, 143B, a metastatic OS cell line, exhibited increased MMP1 and MMP9 mRNA levels. Gene set enrichment analysis on metastatic and non-metastatic OS patient specimens indicated epithelial-mesenchymal transition as the most enriched gene set, with MMP9 displaying strong association to genes in this network. Using the same dataset, Kaplan-Meier analysis revealed a correlation between MMP1 expression and dismal patient survival. Hence, we undertook targeted suppression of MMP1 and MMP9 gene expression in OS cell lines. The ability of OS cells to migrate and form colonies was markedly reduced upon MMP1 and MMP9 downregulation, whereas their cell proliferation capacity remained intact. MMP9 downregulation decreased tumor growth and lung metastases area in an orthotopic mouse OS model. Consistently, human OS lung metastasis specimens displayed marked MMP9 protein expression. Our findings highlight the role of MMP1 and MMP9 in OS metastasis, warranting further exploration of simultaneous inhibition of MMPs for future OS therapeutics.

Identification of a biomarker to predict doxorubicin/cisplatin chemotherapy efficacy in osteosarcoma patients using primary, recurrent and metastatic specimens

BackgroundDoxorubicin and cisplatin are both first-line chemotherapeutics for osteosarcoma (OS) treatment. However, the efficacy of doxorubicin/cisplatin chemotherapy varies considerably. Thus, identifying an efficient diagnostic biomarker to distinguish patients with good and poor responses to doxorubicin/cisplatin chemotherapy is of paramount importance. MethodsTo predict the efficacy of doxorubicin/cisplatin chemotherapy, we analyzed the differentially expressed proteins in 37 resected OS samples, which were categorized into the primary group (PG), the recurrent group (RG) and the metastatic group (MG). The characteristics of the enriched differentially expressed proteins were assessed via GO and KEGG analyses. Protein‒protein interactions were identified to determine the relationships among the differentially expressed proteins. Receiver operating characteristic (ROC) curve analyses were performed to explore the clinical significance of the differentially expressed proteins. Parallel reaction monitoring (PRM) was used to validate the candidate proteins. Immunohistochemical (IHC) staining was performed to confirm the expression of cathepsin (CTSG) in patients with good and poor response to doxorubicin/cisplatin. ResultsA total of 9458 proteins were identified and quantified, among which 143 and 208 exhibited significant changes (|log2FC|>1, p < 0.05) in the RG and MG compared with the PG, respectively. GO and KEGG enrichment led to the identification of neutrophil extracellular traps (NETs). ROC curve analyses revealed 74 and 86 proteins with areas under the curve greater than 0.7 in the RG and MG, respectively. PRM validation revealed the statistical significance of CTSG, which is involved in NET formation, at the protein level in both the RG and MG. IHC staining of another cohort revealed that CTSG was prominently upregulated in the poor response group after treatment with doxorubicin/cisplatin. ConclusionCTSG and its associated NETs are potential biomarkers with which the efficacy of doxorubicin/cisplatin chemotherapy could be predicted in OS patients.

Study Protocol for a Prospective Self-Controlled Trial on Success in Meeting Comprehensive Genomic Profiling Analysis Criteria for Specimens Obtained by Endoscopic Ultrasound-Guided Tissue Acquisition Using a 19G Needle from Primary and Metastatic Lesions in Pancreatic Cancer with Metastatic Lesions: The PRIMATE Study.

EUS-TA in unresectable pancreatic cancer requires not only a tissue diagnosis but also tissue collection in anticipation of comprehensive genomic profiling. However, the optimal puncture target remains controversial. Therefore, the Primary and Metastatic Lesions in Pancreatic Cancer (PRIMATE) study was designed to clarify the optimal target by comparing the success rates for meeting OncoGuide NCC Oncopanel (NOP) analysis criteria on pre-check primary and metastatic lesion specimens obtained during the same EUS-TA session in patients with invasive pancreatic ductal adenocarcinoma. In this ongoing prospective study, two specimens, each from primary and metastatic lesions, are obtained by EUS-TA (typically using a 19G fine-needle biopsy needle) in patients with invasive pancreatic ductal adenocarcinoma. The primary endpoint is the proportion of EUS-TA specimens that meet NOP analysis criteria during pre-check (i.e., tumor cellularity of ≥20% and a tissue area of ≥4 mm2), which are then compared between primary and metastatic lesions. This study has been approved by the National Cancer Center Institutional Review Board (Research No. 2022-168). The results of this study will be reported at an international conference and published in an international peer-reviewed journal. The trial registration number is UMIN 000048966.

TIGIT expression in renal cell carcinoma infiltrating T cells is variable and inversely correlated with PD-1 and LAG3

PurposeImmune checkpoint inhibitors have revolutionized the treatment of renal cell carcinoma (RCC), but many patients do not respond to therapy and the majority develop resistant disease over time. Thus, there is increasing need for alternative immunomodulating agents. The co-inhibitory molecule T-cell immunoglobulin and ITIM domain (TIGIT) may play a role in resistance to approved immune checkpoint inhibitors and is being investigated as a potential therapeutic target. The purpose of this study was to quantify TIGIT positivity in tumor-infiltrating T cells in RCC.MethodsWe employed tissue microarrays containing specimens from primary RCC tumors, adjacent normal renal tissue, and RCC metastases to quantify TIGIT within tumor-infiltrating CD3+ T cells using quantitative immunofluorescent analysis. We also compared these results to TIGIT+ CD3+ levels in four other tumor types (melanoma, non-small cell lung, cervical, and head and neck cancers).ResultsWe did not observe significant differences in TIGIT positivity between primary RCC tumors and patient-matched metastatic samples. We found that the degree of TIGIT positivity in RCC is comparable to that in lung cancer but lower than that in melanoma, cervical, and head and neck cancers. Correlation analysis comparing TIGIT positivity to previously published, patient-matched spatial proteomic data by our group revealed a negative association between TIGIT and the checkpoint proteins PD-1 and LAG3.ConclusionOur findings support careful evaluation of TIGIT expression on T cells in primary or metastatic RCC specimens for patients who may be treated with TIGIT-targeting antibodies, as increased TIGIT positivity might be associated with a greater likelihood of response to therapy.

Heterogeneity of claudin 18.2 expression in metastatic gastric cancer

Claudin 18.2 has emerged as a viable therapeutic target in gastric cancer (GC), but little is known about the heterogeneity of its expression in GC. This study investigated the heterogeneity of claudin 18.2 expression in 166 patients with metastatic GC whose surgical or paired primary–metastatic specimens were available. The prevalence of claudin 18.2 positivity (moderate-to-strong expression in ≥ 75% by the 43-14A clone) was 47.0%. Claudin 18.2-positive tumors exhibited more frequent peritoneal metastasis and a lower incidence of hepatic and distant lymph node involvement. Survival outcomes were comparable between patients with claudin 18.2-positive and -negative tumors. Intratumoral heterogeneity was noted in 38.5% of surgical specimens. Paired primary-metastatic site analysis revealed that 25.2% of patients had discordant results for claudin 18.2 positivity. Across different metastatic organ categories, peritoneal lesions showed the highest positivity rate (44.3%) and positive concordance rate (31.4%), whereas liver lesions had the lowest positivity rate (17.9%) and concordance rate (12.8%). In conclusion, claudin 18.2 expression exhibits intratumoral and intrapatient spatial heterogeneity in metastatic GC. Claudin 18.2 positivity is associated with more frequent peritoneal metastasis, and peritoneal lesions are more likely to have positively concordant claudin 18.2 results with the primary site than other metastatic sites.

Real-world data on ALK rearrangement test in Chinese advanced non-small cell lung cancer (RATICAL): a nationwide multicenter retrospective study.

Anaplastic lymphoma kinase (ALK) test in advanced non-small cell lung cancer (NSCLC) can help physicians provide target therapies for patients harboring ALK gene rearrangement. This study aimed to investigate the real-world test patterns and positive rates of ALK gene rearrangements in advanced NSCLC. In this real-world study (ChiCTR2000030266), patients with advanced NSCLC who underwent an ALK rearrangement test in 30 medical centers in China between October 1, 2018 and December 31, 2019 were retrospectively analyzed. Interpretation training was conducted before the study was initiated. Quality controls were performed at participating centers using immunohistochemistry (IHC)-VENTANA-D5F3. The positive ALK gene rearrangement rate and consistency rate were calculated. The associated clinicopathological characteristics of ALK gene rearrangement were investigated as well. The overall ALK gene rearrangement rate was 6.7% in 23,689 patients with advanced NSCLC and 8.2% in 17,436 patients with advanced lung adenocarcinoma. The quality control analysis of IHC-VENTANA-D5F3 revealed an intra-hospital consistency rate of 98.2% (879/895) and an inter-hospital consistency rate of 99.2% (646/651). IHC-VENTANA-D5F3 was used in 53.6%, real-time polymerase chain reaction (RT-PCR) in 25.4%, next-generation sequencing (NGS) in 18.3%, and fluorescence in-situ hybridization (FISH) in 15.9% in the adenocarcinoma subgroup. For specimens tested with multiple methods, the consistency rates confirmed by IHC-VENTANA-D5F3 were 98.0% (822/839) for FISH, 98.7% (1,222/1,238) for NGS, and 91.3% (146/160) for RT-PCR. The overall ALK gene rearrangement rates were higher in females, patients of ≤ 35 years old, never smokers, tumor cellularity of > 50, and metastatic specimens used for testing in the total NSCLC population and adenocarcinoma subgroup (all P < 0.05). This study highlights the real-world variability and challenges of ALK test in advanced NSCLC, demonstrating a predominant use of IHC-VENTANA-D5F3 with high consistency and distinct clinicopathological features in ALK-positive patients. These findings underscore the need for a consensus on optimal test practices and support the development of refined ALK test strategies to enhance diagnostic accuracy and therapeutic decision-making in NSCLC.

SERPINH1 modulates apoptosis by inhibiting P62 ubiquitination degradation to promote bone metastasis of prostate cancer

SERPINH1 modulates apoptosis by inhibiting P62 ubiquitination degradation to promote bone metastasis of prostate cancer

Baseline tumor-infiltrating lymphocyte patterns and response to immune checkpoint inhibition in metastatic cutaneous melanoma

IntroductionThe presence of tumor-infiltrating lymphocytes (TILs) in melanoma has been linked to survival. Their predictive capability for immune checkpoint inhibition (ICI) response remains uncertain. Therefore, we investigated the association between treatment response and TILs in the largest cohort to date and analyzed if this association was independent of known clinical predictors. MethodsIn this multicenter cohort study, patients who received first-line anti-PD1 ± anti-CTLA4 for advanced melanoma were identified. TILs were scored on hematoxylin and eosin (H&E) slides of primary melanoma and pre-treatment metastases using the validated TILs-WG, Clark and MIA score. The primary outcome was objective response rate (ORR), with progression free survival and overall survival being secondary outcomes. Univariable and multivariable logistic regression and Cox proportional hazard were performed, adjusting for known clinical predictors. ResultsMetastatic melanoma specimens were available for 650 patients and primary specimens for 565 patients. No association was found in primary melanoma specimens. In metastatic specimens, a 10-point increase in the TILs-WG score was associated with a higher probability of response (aOR 1.17, 95 % CI 1.07–1.28), increased PFS (HR 0.93, 95 % CI 0.87–0.996), and OS (HR 0.94, 95 % CI 0.89–0.99). When categorized, patients in the highest tertile TILs-WG score (15–100 %) compared to the lowest tertile (0 %) had a longer median PFS (13.1 vs. 7.3 months, p = 0.04) and OS (49.4 vs. 19.5 months, p = 0.003). Similar results were noted using the MIA and Clark scores. ConclusionIn advanced melanoma patients, TIL patterns on H&E slides of pre-treatment metastases, regardless of measurement method, are independently associated with ICI response. TILs are easily scored on readily available H&Es, facilitating the use of this biomarker in clinical practice.

Differential predictive value of tissue-specific PD-L1 expression scores in adjuvant immunotherapy of melanoma.

Adjuvant treatment of stage II-IV melanoma with PD-1-based immune checkpoint inhibitors (ICI) has improved relapse-free survival (RFS) and has therefore become a standard-of-care treatment option. Approximately 25%-30% of patients still recur within 1 year. Predictive biomarkers reflecting real-world data are desired. The predictive relevance of tumour tissue PD-L1 expression in the adjuvant setting remains inconclusive. This retrospective, observational study was conducted to evaluate the value of PD-L1 expression scores in different tumour tissue locations in predicting response towards adjuvant immunotherapeutic treatment. Tumour tissue taken prior to anti-PD-1 adjuvant ICI in 243 stage II-IV melanoma patients was collected at University Skin Cancer Center Hamburg. PD-L1 expression was evaluated on immune cells (ICS), tumour cells (TPS) and combined (CPS). Scores were determined by independent pathological physician quantification and correlated with therapy outcome at different cut-off (CO) levels (relapse-free survival, RFS) for different tumour tissue locations (primary tumour, metastases). A total of 104 patients were eligible for analysis. Positivity of ICS, TPS and CPS showed no predictive RFS outcome association at different CO levels when analysed irrespective of tissue origin. In primary tumours, ICS at CO 1% showed a significantly improved RFS upon positivity (HR 0.22). In contrast, positivity to TPS (CO 1%) correlated significantly and independently with improved RFS when evaluated in metastatic tumour tissue specimens (HR 0.37). PD-L1 tumour tissue expression may serve as a predictive biomarker for adjuvant ICI treatment response stratification in melanoma, but caution should be spent on the origin of tumour tissue analysed. The cell-type relevant for the predictive value of PD-L1 expression is tissue-specific with immune cells being important in primary tumours while tumour cells are key in metastases. The present results should be validated in a multicentre cohort.

Tumor-infiltrating lymphocytes and immune-related adverse events in advanced melanoma

The predictive value of tumor-infiltrating lymphocytes (TILs) in immune-related adverse event (irAE) development remains unknown, although an association between tumor immunogenicity and irAEs has been suggested. We investigated the association between TIL abundance in pretreatment primary and metastasis specimens and the subsequent development of severe irAEs. We retrospectively identified patients with advanced cutaneous melanoma who received first-line anti-programmed cell death protein 1 (PD-1) with or without anti-cytotoxic T-lymphocyte associated protein 4 (anti-CTLA-4) from 10 hospitals in the Netherlands. TILs were scored on representative hematoxylin and eosin (H&E) stains of the primary melanoma and pretreatment melanoma metastasis as 'absent', 'nonbrisk', or 'brisk'. A univariable logistic regression analysis was carried out to assess the association between the TIL scores and the development of severe irAEs. Fine and Gray subdistribution hazard models were used to estimate the cumulative incidence of severe irAEs. Of the 1346 eligible patients, 536 patients had primary melanoma specimens available, and 613 patients had metastasis specimens available. Severe irAEs occurred in 15% of anti-PD-1-treated patients and 49% of anti-PD-1+ anti-CTLA-4-treated patients. The presence of TILs was not associated with the occurrence of grade ≥3 irAEs in primary melanoma specimens (P= 0.70) nor pretreatment metastasis specimens (P= 0.91). In the univariable analysis, patients with brisk TILs did not have a higher chance of developing severe irAEs compared with patients with absent TILs, for both primary specimen (odds ratio 1.15, 95% confidence interval 0.60-2.18) and metastasis specimen (odds ratio 0.77, 95% confidence interval 0.37-1.59). There was also no significant difference in the lifetime risk or timing of the development of severe irAEs in patients with TILs present compared with patients with TILs absent. There was no association between the TIL scores on H&E-stained slides from the primary melanoma or pretreatment metastasis and the development of grade 3 or higher irAEs. Additionally, no correlation was found between the presence of TILs and the timing of irAEs.

Exosomal HMGB1 educate PD-L1+-tumor associated macrophages via glycometabolic reprogramming to induce lung-tropic metastasis of triple negative breast cancer.

1090 Background: Lung metastasis occurs in one third of triple negative breast cancer (TNBC), the underlying molecular mechanism remains largely unclear. Mounting evidence suggests that PD-L1+-tumor associated macrophages (PD-L1+-TAMs) are closely associated with immune suppression, invasion, and metastasis. Tumor-derived exosomes have been reported to remodel tumor microenvironment and accelerate metastasis through packing and delivering a variety of biologically active molecules. The aim of this study was to investigate whether TNBC-derived exosomes were able to polarize lung macrophages towards PD-L1+-TAMs and promote a pre-metastatic immunosuppressive niche, therefore discovering the underlying mechanism of lung-tropic metastasis. Methods: Exosomes obtained from EO771 TNBC cell line (EO771/exo) and HC11 normal breast epithelial cell line (HC11/exo) were characterized. Absorption of exosomes by macrophages was observed by confocal microscopy. Mass spectrometry was used to identify the significantly highly expressed HMGB1 protein in EO771/exo, and ligands of HMGB1 on lung macrophages were detected by co-immunoprecipitation. Significance of EO771/exo and HMGB1 in inducing PD-L1+-TAMs and generating a pre-metastatic immunosuppressive niche were confirmed by both in vitro and in vivo studies. Functions of EO771/exo and HMGB1 on glycolysis and lactate production were evaluated by metabolism assays. Results: EO771/exo could polarize macrophages toward PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche. Mass spectrometry, clinical samples, and bioinformatics analysis showed that HMGB1 was significantly expressed in EO771/exo and was involved in cellular communication and metabolism processes. Co-immunoprecipitation and 3-dimensional modeling suggested a strong binding of HMGB1 with Tim-3 ligand on lung macrophages. Macrophages incubated with EO771/exo exhibited an enhanced glycolysis and lactate production. Flow cytometry demonstrated that green fluorescent protein (GFP)-EO771 tumor-bearing mice had more lung micro-metastases than brain and liver. Immunofluorescence and laser confocal microscopy demonstrated that EO771/exo were able to increase lung micro-metastases and escalate PD-L1 expression on lung macrophages. Moreover, elevated PD-L1+-TAMs and reduced CD3+CD8+ T lymphocytes were detected in TNBC positive axillary lymph nodes and lung metastasis specimens than TNBC negative axillary lymph nodes. Conclusions: Exosomal HMGB1 from TNBC could target Tim-3 ligand on lung macrophages and accelerate glycolysis and lactate production to induce PD-L1+-TAMs, thereby suppressing CD3+CD8+ T lymphocytes immunity and generating a pre-metastasis immune-suppressive niche. Our research would uncover a novel mechanism of lung-tropic metastasis.

Clinical significance of HER2 expression between primary tumors and metastatic lymph nodes in patients with extramammary Paget's disease.

e21568 Background: HER2 has been reported to be a potential oncogene and therapeutic target in extramammary Paget's disease (EMPD). However, the expression of HER2 in EMPD, especially in matched metastatic lymph nodes (LNs), is limited. Here, we investigated the heterogeneity of HER2 expression between primary tumors and positive nodes and its clinical significance in advanced EMPD patients and detected the efficacy of HER2-targeted antibody drug conjugates (ADCs) in EMPD. Methods: A total of 170 patients with pathologically confirmed EMPD of the scrotum and/or penis treated at SYSUCC from 2003 to 2023 were included. HER2 IHC staining (anti-HER2/neu (4B5), Ventana) and scoring were performed according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2023 guidelines. Outcome relationships were explored using Cohen's kappa coefficient, multivariate Cox regression and log-rank analysis. Results: A total of 102 primary tumor and 32 metastatic specimens were ultimately subjected to HER2 IHC staining. HER2 high-expression (IHC: 2/3+) was detected in 71.6% (0: 9.8%, 1+: 18.6%, 2+: 40.2% and 3+: 31.4%) and 68.8% (0: 21.9%, 1+: 9.4%, 2+: 28.1% and 3+: 40.6%) of the primary tumors and metastatic LNs, respectively. HER2-high EMPD patients experienced poorer outcomes, with a 5-year OS of 56.5%, than did HER2-low patients (P = 0.032). A multivariate Cox regression model showed that a positive lymph node (HR 15.39, 95% CI 3.79~62.43; P &lt; 0.001) and HER2 high-expression in primary tumors (HR 3.68, 95% CI 1.49~9.09; P = 0.005) were independent poor prognostic factors. Notably, in 31 paired samples, 35.5% (n = 11) of patients had inconsistent HER2 expression between primary tumors and corresponding lymph node metastases. Six (19.4%) patients exhibited a shift from high expression of HER2 in primary tumors to low expression of HER2 in the lymph nodes, and 5 (16.1%) patients exhibited the reverse change. Advanced EMPD patients with HER2 high-expression were treated with Disitamab Vedotin (RC48), for which the ORR was 80% and the DCR was 100% (n = 5). No adverse events (AEs) above grade 3-4 were observed. Conclusions: HER2 was highly expressed in both primary and metastatic LNs in EMPD patients. However, there was some heterogeneity in HER2 expression between paired primary and corresponding metastatic LNs. The expression status of HER2 should be evaluated in both primary tumors and corresponding lymph node metastases. A positive lymph node and HER2 high-expression are poor prognostic factors in patients with EMPD. RC48 demonstrated significant efficacy in treating patients with HER2-high advanced EMPD and has promising potential that requires further validation.

Abstract PO1-06-11: Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer

Abstract Background: Lung metastasis occurs in 30% of triple negative breast cancer (TNBC), the detailed molecular mechanism remains largely unexplored. Accumulating evidence suggests that PD-L1+-tumor associated macrophages (PD-L1+-TAMs) are closely related to immune suppression, invasion, and metastasis. Tumor-derived exosomes have been reported to remodel tumor microenvironment and accelerate metastasis via packing and delivering a variety of biologically active molecules. Our previous studies found that exosomes from breast cancer cells polarized macrophages toward TAMs within the primary tumor nest. The aim of this study was to reveal whether TNBC-derived exosomes were able to polarize lung macrophages towards PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, therefore discovering the underlying mechanism of lung-tropic metastasis. Methods: Exosomes isolated from EO771 TNBC cell line (EO771/exo) and HC11 normal breast epithelial cell line (HC11/exo) were characterized. Uptake of exosomes by macrophages was visualized by confocal microscopy. Mass spectrometry was applied to identify the significantly highly expressed HMGB1 protein in EO771/exo, and ligands of HMGB1 on lung macrophages were detected by co-immunoprecipitation. Functions of EO771/exo and HMGB1 in inducing PD-L1+-TAMs and generating a pre-metastatic immunosuppressive niche were confirmed by both in vitro and in vivo studies. Roles of EO771/exo and HMGB1 on glycolysis and lactate production were evaluated by metabolism assays. Associations between PD-L1+-TAMs and CD3+CD8+ T lymphocytes within TNBC axillary lymph nodes and lung metastasis specimens were analyzed by immunofluorescence and immunohistochemistry. Results: Confocal microscopy showed constant internalization and absorption of EO771/exo by macrophages. EO771/exo could polarize macrophages toward PD-L1+-TAMs and generate a pre-metastatic immunosuppressive niche, with respect to HC11/exo. Mass spectrometry, clinical samples, and bioinformatics analysis revealed that HMGB1 was highly expressed in EO771/exo and was involved in cellular communication and metabolism processes. Co-immunoprecipitation and 3-dimensional modeling indicated a strong binding of HMGB1 with Tim-3 ligand on lung macrophages. Macrophages treated with EO771/exo displayed an enhanced glycolysis and lactate production. Flow cytometry demonstrated that green fluorescent protein (GFP)-EO771 tumor-bearing mice had more lung micro-metastases than brain and liver. Immunofluorescence and laser confocal microscopy confirmed that EO771/exo, with respect to HC11/exo, were able to increase lung micro-metastases and escalate PD-L1 expression on lung macrophages. Moreover, elevated PD-L1+-TAMs and reduced CD3+CD8+ T lymphocytes were found in TNBC positive axillary lymph nodes and lung metastasis specimens than TNBC negative axillary lymph nodes. Conclusions: Exosomal HMGB1 from TNBC could target Tim-3 ligand on lung macrophages and accelerate glycolysis and lactate production to induce PD-L1+-TAMs, thereby suppressing CD3+CD8+ T lymphocytes immunity and generating a pre-metastasis immune-suppressive niche. Our research would uncover a novel mechanism of lung-tropic metastasis and provide a new therapeutic approach for TNBC treatment. Keywords: breast cancer; lung metastasis; exosomes; tumor-associated macrophages; metabolic reprogramming Citation Format: Wei-Xian Chen, Jia-Hao Wu, Tian-Cheng Cheng, Sujin Yang. Exosomal HMGB1 induce PD-L1+-tumor associated macrophages through glycometabolic reprogramming to promote lung-tropic metastasis of triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-11.

GLI1 Coamplification in Well-Differentiated/Dedifferentiated Liposarcomas: Clinicopathologic and Molecular Analysis of 92 Cases

GLI1(12q13.3) amplification is identified in a subset of mesenchymal neoplasms with a distinct nested round cell/epithelioid phenotype. MDM2 and CDK4 genes are situated along the oncogenic 12q13-15 segment, amplification of which defines well-differentiated liposarcoma (WDLPS)/dedifferentiated liposarcoma (DDLPS). The 12q amplicon can occasionally include GLI1, a gene in close proximity to CDK4. We hereby describe the first cohort of GLI1/MDM2/CDK4 coamplified WD/DDLPS. The departmental database was queried retrospectively for all cases of WD/DDLPS having undergone next-generation (MSK-IMPACT) sequencing with confirmed MDM2, CDK4, and GLI1 coamplification. Clinicopathologic data was obtained from a review of the medical chart and available histologic material. Four hundred eighty-six WD/DDLPS cases underwent DNA sequencing, 92 (19%) of which harbored amplification of the GLI1 locus in addition to that of MDM2 and CDK4. These included primary tumors (n = 60), local recurrences (n = 29), and metastases (n = 3). Primary tumors were most frequently retroperitoneal (47/60, 78%), mediastinal (4/60, 7%), and paratesticular (3/60, 5%). Average age was 63 years, with a male:female ratio of 3:2. The cohort was comprised of DDLPS (86/92 [93%], 6 of which were WDLPS with early dedifferentiation) and WDLPS without any longitudinal evidence of dedifferentiation (6/92, 7%). One-fifth (13/86, 17%) of DDLPS cases showed no evidence of a well-differentiated component in any of the primary, recurrent, or metastatic specimens. Dedifferentiated areas mostly showed high-grade undifferentiated pleomorphic sarcoma-like (26/86,30%) and high-grade myxofibrosarcoma-like (13/86,16%) morphologies. A disproportionately increased incidence of meningothelial whorls with/without osseous metaplasia was observed as the predominant pattern in 16/86 (19%) cases, and GLI1-altered morphology as described was identified in a total of 10/86 (12%) tumors. JUN (1p32.1), also implicated in the pathogenesis of WD/DDLPS, was coamplified with all 3 of MDM2, CDK4, and GLI1 in 7/91 (8%) cases. Additional loci along chromosomal arms 1p and 6q, including TNFAIP3, LATS1, and ESR1, were also amplified in a subset of cases. In this large-scale cohort of GLI1 coamplified WD/DDLPS, we elucidate uniquely recurrent features including meningothelial whorl-like and GLI-altered morphology in dedifferentiated areas. Assessment of tumor location (retroperitoneal or mediastinal), identification of a well-differentiated liposarcoma component, and coamplification of other spatially discrete genomic segments (1p and 6q) might aid in distinction from tumors with true driver GLI1 alterations.

DNA variants detected in primary and metastatic lung adenocarcinoma: a case report and review of the literature.

Non-small cell lung cancer (NSCLC) has been found to have recurrent genetic abnormalities, and novel therapies targeting these aberrations have improved patient survival. In this study, specimens from benign tissue, primary tumors, and brain metastases were obtained at autopsy from a 55-year-old White female patient diagnosed with NSCLC and were examined using next-generation sequencing (NGS) and chromosomal microarray assay (CMA). No genetic aberrations were noted in the benign tissue; however, NGS identified a mutation in the KRAS proto-oncogene, GTPase (KRAS): KRAS exon 2 p.G12D in primary and metastatic tumor specimens. We observed 7 DNA copy number aberrations (CNAs) in primary and metastatic tumor specimens; an additional 7 CNAs were exclusively detected in the metastatic tumor specimens. These DNA alterations may be genetic drivers in the pathogenesis of the tumor specimen from our patient and may serve as biomarkers for the classification and prognosis of NSCLC.

Abstract 2522: Predictive value of tumor mutational burden (TMB) in patients with metastatic microsatellite-stable (MSS) colorectal cancer (CRC) given first-line oxaliplatin-based chemotherapy and immune checkpoint blockade (ICB)

Abstract Purpose: Most CRC patients harbor primarily non-immunogenic MSS tumor. The randomized METIMMOX trial (NCT03388190) examined if patients with previously untreated, unresectable abdominal metastases from MSS-CRC may achieve therapeutic efficacy from short-course oxaliplatin-based chemotherapy (FLOX) and sequential ICB (nivolumab) repeatedly. Half of study patients assigned to this experimental treatment had significantly extended progression-free survival (PFS) while the other half had shortened PFS, compared to the control-group patients given standard FLOX chemotherapy with median PFS 9.3 months. Here we explored if somatic mutations (mut) unveiled by next-generation sequencing might provide insights into responsiveness to the METIMMOX regimen. Procedures: Patients had MSS-CRC with infradiaphragmatic metastases deemed unresectable and were eligible for first-line oxaliplatin-based therapy. They were randomly assigned to the control group of FLOX (oxaliplatin, 5-fluorouracil, folinic acid) Q2W or the experimental group of alternating 2 cycles each of FLOX Q2W and nivolumab Q2W, with prespecified break periods. Radiologic response assessment was done every 8 weeks with PFS as the primary endpoint. Diagnostic tumor biopsies and baseline metastasis biopsies were sequenced with the TruSight Oncology 500 assay. TMB was calculated using only coding, non-synonymous single-nucleotide variants and insertions/deletions with variant allele frequency ≥5% and sequencing depth ≥50 ×, applying the effective panel size as the megabases (Mb) denominator. Results: Sequencing data were acquired from 20 experimental-group patients with median PFS 7.9 months (minimum 2.0, maximum 41.6). Median TMB was 5.1 mut/Mb (minimum 0.8, maximum 11.8), concordant in patient-paired primary tumor and metastasis specimens. The TMB was associated with treatment outcome; the higher TMB, the better PFS with hazard ratio 0.83 (95% confidence interval (CI) 0.70-0.98; p = 0.026, Cox regression) for a PFS event with increasing TMB. TMB cut-off as low as 5.0 mut/Mb distinguished between patient groups (p = 0.007, log-rank test) with median PFS 34.9 months (95% CI 10.6-59.2; TMB&amp;gt;5, n = 10) and median PFS 4.6 months (95% CI 2.7-6.5; TMB≤5, n = 10). In the TMB&amp;gt;5 group, 8/10 were KRAS- or BRAF-mutant cases. In the TMB≤5 group, 9/10 metastasis specimens were RAS/BRAF-wildtype cases. Conclusions: TMB &amp;gt;5 mut/Mb was associated with tumor KRAS or BRAF driver mutation and remarkably long PFS in first-line treatment of patients with abdominal metastases from MSS-CRC with alternating short-course oxaliplatin-based chemotherapy and ICB. Citation Format: Anne Hansen Ree, Paula A. Bousquet, Hilde L. Nilsen, Torben Lüders, Shixiong Wang, Tina Visnovska, Diana L. Bordin, Eirik Høye, Hanne M. Hamre, Christian Kersten, Eva Hofsli, Marianne G. Guren, Halfdan Sorbye, Kjersti Flatmark, Sebastian Meltzer. Predictive value of tumor mutational burden (TMB) in patients with metastatic microsatellite-stable (MSS) colorectal cancer (CRC) given first-line oxaliplatin-based chemotherapy and immune checkpoint blockade (ICB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2522.

Abstract 3629: Prevalence of claudin18.2 expression in gastric/gastroesophageal junction adenocarcinoma among patients in TranStar101 and TranStar102 clinical trials

Abstract Background: Claudin18.2 (CLDN18.2) is a tight junction protein highly specific to gastric mucosa, and a validated target for gastric cancer (GC) treatment. Immune checkpoint therapy targeting PD-1 combined with chemotherapy has been approved as the first line therapy of GC. Understanding the expression profiles of CLDN18.2 and PD-L1 could guide the development of combination therapies to maximize the benefits of these two agents. This study investigated the prevalence of CLDN18.2 expression in gastric/gastroesophageal junction adenocarcinoma (G/GEJC) screening samples from studies Transtar101 (NCT04396821 in US) and TranStar102 (NCT04495296 in China), and its correlation with various clinical characteristics and PD-L1 expression. Methods: CLDN18.2 expression in formalin-fixed, paraffin-embedded (FFPE) G/GEJC tissue samples was prospectively detected by an immunohistochemistry-based LDT using an in-house anti-CLDN18.2 antibody (Clone14G11) on the Leica Bond III stainer. CLDN18.2 expression was assessed by scoring the staining intensity (0, 1+, 2+, 3+) and the percentage of positive tumor cells. Positive CLDN18.2 expression is defined as the cutoff at ≥10% of tumor cell with ≥1+ staining intensity for this evaluation. PD-L1 expression was assessed by combined positive score (CPS) using Agilent’s PD-L1 IHC 28-8 pharmDx. Both assays were conducted in CAP/CLIA certified LabCorp central lab. Results: 547 G/GEJC patient samples had CLDN18.2 results as part of the screening procedures for the clinical trials. Of these patients (454 GC/93 GEJC), 439 (80%) were Asian, 29 (5%) were Caucasian, 2 (0.4%) were other ethnic group, and 77 (14%) were not recorded; 434 (79%) were primary tumors and 113 (21%) were metastasis; 201 (37%) were core needle biopsies (CNB), 108 (20%) were surgical resections (SR) and 238 (44%) had no information. 311 (57%) samples had positive CLDN18.2 expressions (≥10%/≥1+). No significant difference in CLDN18.2 positive rates were found between TranStar101 and TranStar102 studies (p=0.801), Asian and Caucasian (p=0.612), GC and GEJC (p=0.373), primary and metastatic specimens (p=0.055) or CNBs and SRs (p=0.715). Out of 83 TranStar102 specimens that had both CLDN18.2 and PD-L1 results, 15 (18%) had PD-L1 CPS≥5, 59 (71%) had positive CLDN18.2, and 10 (15%) had both positive CLDN18.2 and PD-L1 CPS≥5. No correlation (p=0.393) was observed between PD-L1 scores (CPS&amp;lt;5 or CPS≥5) and CLDN18.2 expression (≥10%/≥1+ or &amp;lt;10%/≥1+). Conclusions: Data suggested CLDN18.2 expression levels were independent of PD-L1 status, and support the use of Transcenta 14G11 antibody for CLDN18.2 detection regardless of sample collection methods, location, and patient demographics. An anti-CLDN18.2 companion diagnostic device based on 14G11 is being developed (CLDN18.2 IHC 14G11 pharmDx, Agilent Technologies, Inc.). Citation Format: Li Shen, Carol Mao, Alan Lin, Ying Gu, Jenny Milata, Lijuan Zhang, Chuan Qi, Jenny Yao, Steven Yu, Changjun Yue, Lin Shen, Yelena Janjigian, Xueming Qian, Wen-I Chang, Caroline Germa. Prevalence of claudin18.2 expression in gastric/gastroesophageal junction adenocarcinoma among patients in TranStar101 and TranStar102 clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3629.

Abstract 3779: Genomic and clinical characterization of metastatic patterns using real-word data from a large cohort of colorectal cancer patients

Abstract Background: Colorectal cancers (CRCs) can exhibit disparate metastatic patterns that affect outcomes. We investigated associations between genomic features and organ-specific metastatic tropism and we compared the frequency of actionable alterations detected in patients undergoing longitudinal clinical sequencing. Methods: We analyzed data for 6574 tumors from 6153 CRC patients, including 4510 primary and 2064 metastatic specimens. All patients had microsatellite stable (MSS) disease and were treated at Memorial Sloan Kettering. Paired primary and metastatic samples were available for 203 patients. Tumors were sequenced with MSK-IMPACT, a targeted DNA sequencing assay that identifies genomic alterations in 341-505 genes. Clinical annotations included sex, stage at diagnosis (Dx), and primary tumor location. History of metastatic disease was automatically retrieved from radiology reports using validated natural language processing methods; time to specific-site metastasis was modeled with Cox regression. False discovery rate (FDR) was used to correct for multiple hypothesis testing. The OncoKB knowledge base was used to identify clinically actionable alterations. Results: Liver (n=1068, 51.7%), peritoneal (n=269, 13.0%) and lung metastases (n=263, 12.7%) accounted for the majority of non-primary sequenced specimens. The frequency of actionable alterations (OncoKB Level≥3A) did not vary between unmatched metastatic and primary samples (12.3% vs 11.4%, p=0.302). In primary tumor samples from patients with localized disease at the time of diagnosis, TGFB pathway alterations were associated with increased risk of metastasis to the brain (HR=2.41, p=0.028), bone (HR=2.10, p&amp;lt;0.001), peritoneum (HR=1.82, p=0.001) and liver (HR=1.42, p=0.049). Alterations in the RAS pathway were associated with increased risk of lung (HR=1.47, p=0.009), peritoneum (HR=1.54, p=0.021) and liver (HR=1.44, p=0.026) metastases. By contrast, WNT pathway alterations were associated with a lower risk of developing lung (HR=0.69, p=0.033) and peritoneal metastasis (HR=0.68, p=0.081). Among patients with paired primary and metastatic samples, no significant difference was observed between the frequency of clinically actionable alterations in primary vs. metastatic specimens (16.7% vs. 18.2%, p=0.794). Presence of metastases-private or primary-private alterations did not correlate with site-specific metastasis risk. Conclusions: The risk of developing metastatic disease and organ-specific dissemination might be linked to genomic abnormalities detectable at the localized stage. No significant difference in the frequency of actionable alterations was observed between primary and metastatic CRC samples. Citation Format: Paolo Manca, Ayush V. Kris, Henry Walch, Christopher Fong, Justin Jee, Karl Pichotta, Nikolaus Schultz, Walid K. Chatila, Rona Yaeger, Francisco Sanchez-Vega. Genomic and clinical characterization of metastatic patterns using real-word data from a large cohort of colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3779.