The multimodality cell segmentation challenge: toward universal solutions.

Cell segmentation is a critical step for quantitative single-cell analysis in microscopy images. Existing cell segmentation methods are often tailored to specific modalities or require manual interventions to specify hyper-parameters in different experimental settings. Here, we present a multimodality cell segmentation benchmark, comprising more than 1,500 labeled images derived from more than 50 diverse biological experiments. The top participants developed a Transformer-based deep-learning algorithm that not only exceeds existing methods but can also be applied to diverse microscopy images across imaging platforms and tissue types without manual parameter adjustments. This benchmark and the improved algorithm offer promising avenues for more accurate and versatile cell analysis in microscopy imaging.

Computational textural mapping harmonises sampling variation and reveals multidimensional histopathological fingerprints.

BACKGROUND: Technical factors can bias H&E digital slides potentially compromising computational histopathology studies. Here, we hypothesised that sample quality and sampling variation can introduce even greater and undocumented technical fallacy. METHODS: Using The Cancer Genome Atlas (TCGA) clear-cell renal cell carcinoma (ccRCC) as a model disease, we annotated ~78,000 image tiles and trained deep learning models to detect histological textures and lymphocyte infiltration at the tumour core and its surrounding margin and correlated these with clinical, immunological, genomic, and transcriptomic profiles. RESULTS: The models reached 95% validation accuracy for classifying textures and 95% for lymphocyte infiltration enabling reliable profiling of ccRCC samples. We validated the lymphocyte-per-texture distributions in the Helsinki dataset (n = 64). Texture analysis indicated constitutive sampling bias by TCGA clinical centres and technically suboptimal samples. We demonstrate how computational texture mapping (CTM) can abrogate these issues by normalising textural variance. CTM-harmonised histopathological architecture resonated with both expected associations and novel molecular fingerprints. For instance, tumour fibrosis associated with histological grade, epithelial-to-mesenchymal transition, low mutation burden and metastasis. CONCLUSIONS: This study highlights texture-based standardisation to resolve technical bias in computational histopathology and understand the molecular basis of tissue architecture. All code, data and models are released as a community resource.

Real-world evidence of multiple myeloma treated from 2013 to 2019 in the Hospital District of Helsinki and Uusimaa, Finland.

Background: The rapid development of multiple myeloma (MM) management underscores the value of real-world data. In our study we examined 509 adult MM patients treated with immunochemotherapy (ICT) with/without stem cell transplantation (SCT) from 2013 to 2019 in the Hospital District of Helsinki and Uusimaa, Finland. Materials & methods: Our study was based on computational analyses of data integrated into the hospital data lake. Results: After 2017, treatment pattern diversity increased with improved access to novel treatments. 5-year survivals were 74.4% (95% CI: 65.5-84.5) in SCT-eligible and 44.0% (95% CI: 37.6-51.4) in non-SCT subgroups. In the SCT-eligible subgroup, high first-year hospitalization costs were followed by stable resource requirements. Conclusion: Hospital data lakes can be adapted to carry out complex analysis of large MM cohorts.

To better understand how multiple myeloma (a type of blood cancer) is clinically managed, we examined 509 adult patients using advanced computer analysis and data stored in the Hospital District of Helsinki and Uusimaa information system. Our study found that after 2017, there was more variety in treatments due to better access to new therapies. Compared with a nontransplant group (44.0%), patients eligible for stem cell transplantation had a better 5-year survival rate (74.4%) and used higher levels of healthcare resources. Our study highlights the potential of hospital data systems to study large groups of multiple myeloma patients and inform strategies to tackle the burden associated with the treatment costs of multiple myeloma.

Prognostic implications of tumor-infiltrating T cells in early-stage endometrial cancer.

Patients with endometrial cancer differ in terms of the extent of T-cell infiltration; however, the association between T-cell subpopulations and patient outcomes remains unexplored. We characterized 285 early-stage endometrial carcinoma samples for T-cell infiltrates in a tissue microarray format using multiplex fluorescent immunohistochemistry. The proportion of T cells and their subpopulations were associated with clinicopathological features and relapse-free survival outcomes. CD3+ CD4+ infiltrates were more abundant in the patients with higher grade or non-endometrioid histology. Cytotoxic T cells (CD25+, PD-1+, and PD-L1+) were strongly associated with longer relapse-free survival. Moreover, CD3+ PD-1+ stromal cells were independent of other immune T-cell populations and clinicopathological factors in predicting relapses. Patients with high stromal T-cell fraction of CD3+ PD-1+ cells were associated with a 5-year relapse-free survival rate of 93.7% compared to 79.0% in patients with low CD3+ PD-1+ fraction. Moreover, in patients classically linked to a favorable outcome (such as endometrioid subtype and low-grade tumors), the stromal CD3+ PD-1+ T-cell fraction remained prognostically significant. This study supports that T-cell infiltrates play a significant prognostic role in early-stage endometrial carcinoma. Specifically, CD3+ PD-1+ stromal cells emerge as a promising novel prognostic biomarker.

Spatial immunoprofiling of the intratumoral and peritumoral tissue of renal cell carcinoma patients.

While the abundance and phenotype of tumor-infiltrating lymphocytes are linked with clinical survival, their spatial coordination and its clinical significance remain unclear. Here, we investigated the immune profile of intratumoral and peritumoral tissue of clear cell renal cell carcinoma patients (n = 64). We trained a cell classifier to detect lymphocytes from hematoxylin and eosin stained tissue slides. Using unsupervised classification, patients were further classified into immune cold, hot and excluded topographies reflecting lymphocyte abundance and localization. The immune topography distribution was further validated with The Cancer Genome Atlas digital image dataset. We showed association between PBRM1 mutation and immune cold topography, STAG1 mutation and immune hot topography and BAP1 mutation and immune excluded topography. With quantitative multiplex immunohistochemistry we analyzed the expression of 23 lymphocyte markers in intratumoral and peritumoral tissue regions. To study spatial interactions, we developed an algorithm quantifying the proportion of adjacent immune cell pairs and their immunophenotypes. Immune excluded tumors were associated with superior overall survival (HR 0.19, p = 0.02) and less extensive metastasis. Intratumoral T cells were characterized with pronounced expression of immunological activation and exhaustion markers such as granzyme B, PD1, and LAG3. Immune cell interaction occurred most frequently in the intratumoral region and correlated with CD45RO expression. Moreover, high proportion of peritumoral CD45RO+ T cells predicted poor overall survival. In summary, intratumoral and peritumoral tissue regions represent distinct immunospatial profiles and are associated with clinicopathologic characteristics.

Immune cell constitution in the tumor microenvironment predicts the outcome in diffuse large B-cell lymphoma.

The tumor microenvironment (TME) and limited immune surveillance play important roles in lymphoma pathogenesis. Here we aimed to characterize immunological profiles of diffuse large B-cell lymphoma (DLBCL) and predict the outcome in response to immunochemotherapy. We profiled the expression of 730 immune-related genes in tumor tissues of 81 patients with DLBCL utilizing the Nanostring platform, and used multiplex immunohistochemistry to characterize T-cell phenotypes, including cytotoxic T cells (CD8, Granzyme B, OX40, Ki67), T-cell immune checkpoint (CD3, CD4, CD8, PD1, TIM3, LAG3), as well as regulatory T-cells and Th1 effector cells (CD3, CD4, FOXP3, TBET) in 188 patients. We observed a high degree of heterogeneity at the transcriptome level. Correlation matrix analysis identified gene expression signatures with highly correlating genes, the main cluster containing genes for cytolytic factors, immune checkpoint molecules, T cells and macrophages, together named a TME immune cell signature. Immunophenotyping of the distinct cell subsets revealed that a high proportion of immune checkpoint positive T cells translated to unfavorable survival. Together, our results demonstrate that the immunological profile of DLBCL TME is heterogeneous and clinically meaningful. This highlights the potential impact of T-cell immune checkpoint in regulating survival and resistance to immunochemotherapy. (Registered at clinicaltrials.gov identifiers: NCT01502982 and NCT01325194.)

Age-associated changes in the immune system may influence the response to anti-PD1 therapy in metastatic melanoma patients.

Anti-PD1 treatment has improved the survival of metastatic melanoma patients, yet it is unknown which patients benefit from the treatment. In this exploratory study, we aimed to understand the effects of anti-PD1 therapy on the patients' immune system and discover the characteristics that would result in successful treatment. We collected peripheral blood (PB) samples from 17 immuno-oncology-naïve metastatic melanoma patients before and after 1 and 3 months of anti-PD1 therapy. In addition, matching tumor biopsies at the time of diagnosis were collected for tissue microarray. The complete blood counts, PB immunophenotype, serum cytokine profiles, and tumor-infiltrating lymphocytes were analyzed and correlated with the clinical data. Patients were categorized based on their disease control into responders (complete response, partial response, stable disease > 6 months, N = 11) and non-responders (progressive disease, stable disease ≤ 6 months, N = 6). During therapy, the PB natural killer T (NKT) cell frequency, expression of CD25 and CD45RO on cytotoxic natural killer (NK) cells, and serum CXC chemokine levels were significantly increased in responders. Furthermore, higher age together with age-associated characteristics from PB, lower frequency of PB-naïve CD8+ T cells, and elevated levels of serum MCP-4 and OPG were discovered as baseline predictors of treatment response. We therefore propose that in addition to T cells, anti-PD1 treatment is associated with NK- and NKT-cell population dynamics, and that the age-associated characteristics from PB together with older age may contribute to prolonged PFS in anti-PD1-treated melanoma patients.

T-cell inflamed tumor microenvironment predicts favorable prognosis in primary testicular lymphoma.

Primary testicular lymphoma is a rare lymphoid malignancy, most often, histologically, representing diffuse large B-cell lymphoma. The tumor microenvironment and limited immune surveillance have a major impact on diffuse large B-cell lymphoma pathogenesis and survival, but the impact on primary testicular lymphoma is unknown. Here, the purpose of the study was to characterize the tumor microenvironment in primary testicular lymphoma, and associate the findings with outcome. We profiled the expression of 730 immune response genes in 60 primary testicular lymphomas utilizing the Nanostring platform, and used multiplex immunohistochemistry to characterize the immune cell phenotypes in the tumor tissue. We identified a gene signature enriched for T-lymphocyte markers differentially expressed between the patients. Low expression of the signature predicted poor outcome independently of the International Prognostic Index (progression-free survival: HR=2.810, 95%CI: 1.228-6.431, P=0.014; overall survival: HR=3.267, 95%CI: 1.406-7.590, P=0.006). The T-lymphocyte signature was associated with outcome also in an independent diffuse large B-cell lymphoma cohort (n=96). Multiplex immunohistochemistry revealed that poor survival of primary testicular lymphoma patients correlated with low percentage of CD3+CD4+ and CD3+CD8+ tumor-infiltrating lymphocytes (P<0.001). Importantly, patients with a high T-cell inflamed tumor microenvironment had a better response to rituximab-based immunochemotherapy, as compared to other patients. Furthermore, loss of membrane-associated human-leukocyte antigen complexes was frequent and correlated with low T-cell infiltration. Our results demonstrate that a T-cell inflamed tumor microenvironment associates with favorable survival in primary testicular lymphoma. This further highlights the importance of immune escape as a mechanism of treatment failure.

PD-L1+ tumor-associated macrophages and PD-1+ tumor-infiltrating lymphocytes predict survival in primary testicular lymphoma.

Primary testicular lymphoma is a rare and aggressive lymphoid malignancy, most often representing diffuse large B-cell lymphoma histologically. Tumor-associated macrophages and tumor-infiltrating lymphocytes have been associated with survival in diffuse large B-cell lymphoma, but their prognostic impact in primary testicular lymphoma is unknown. Here, we aimed to identify macrophages, their immunophenotypes and association with lymphocytes, and translate the findings into survival of patients with primary testicular lymphoma. We collected clinical data and tumor tissue from 74 primary testicular lymphoma patients, and used multiplex immunohistochemistry and digital image analysis to examine macrophage markers (CD68, CD163, and c-Maf), T-cell markers (CD3, CD4, and CD8), B-cell marker (CD20), and three checkpoint molecules (PD-L1, PD-L2, and PD-1). We demonstrate that a large proportion of macrophages (median 41%, range 0.08-99%) and lymphoma cells (median 34%, range 0.1-100%) express PD-L1. The quantity of PD-L1+ CD68+ macrophages correlates positively with the amount of PD-1+ lymphocytes, and a high proportion of either PD-L1+ CD68+ macrophages or PD-1+ CD4+ and PD-1+ CD8+ T cells translates into favorable survival. In contrast, the number of PD-L1+lymphoma cells or PD-L1- macrophages do not associate with outcome. In multivariate analyses with IPI, PD-L1+ CD68+ macrophage and PD-1+ lymphocyte contents remain as independent prognostic factors for survival. In conclusion, high PD-L1+ CD68+ macrophage and PD-1+ lymphocyte contents predict favorable survival in patients with primary testicular lymphoma. The findings implicate that the tumor microenvironment and PD-1 - PD-L1 pathway have a significant role in regulating treatment outcome. They also bring new insights to the targeted thera py of primary testicular lymphoma.

Somatic STAT3 mutations in Felty syndrome: an implication for a common pathogenesis with large granular lymphocyte leukemia.

Felty syndrome is a rare disease defined by neutropenia, splenomegaly, and rheumatoid arthritis. Sometimes the differential diagnosis between Felty syndrome and large granular lymphocyte leukemia is problematic. Recently, somatic STAT3 and STAT5B mutations were discovered in 30-40% of patients with large granular lymphocyte leukemia. Herein, we aimed to study whether these mutations can also be detected in Felty syndrome, which would imply the existence of a common pathogenic mechanism between these two disease entities. We collected samples and clinical information from 14 Felty syndrome patients who were monitored at the rheumatology outpatient clinic for Felty syndrome. Somatic STAT3 mutations were discovered in 43% (6/14) of Felty syndrome patients with deep amplicon sequencing targeting all STAT3 exons. Mutations were located in the SH2 domain of STAT3, which is a known mutational hotspot. No STAT5B mutations were found. In blood smears, overrepresentation of large granular lymphocytes was observed, and in the majority of cases the CD8+ T-cell receptor repertoire was skewed when analyzed by flow cytometry. In bone marrow biopsies, an increased amount of phospho-STAT3 positive cells was discovered. Plasma cytokine profiling showed that ten of the 92 assayed cytokines were elevated both in Felty syndrome and large granular lymphocyte leukemia, and three of these cytokines were also increased in patients with uncomplicated rheumatoid arthritis. In conclusion, somatic STAT3 mutations and STAT3 activation are as frequent in Felty syndrome as they are in large granular lymphocyte leukemia. Considering that the symptoms and treatment modalities are also similar, a unified reclassification of these two syndromes is warranted.

Systematic drug screening reveals specific vulnerabilities and co-resistance patterns in endocrine-resistant breast cancer.

BACKGROUND: The estrogen receptor (ER) inhibitor tamoxifen reduces breast cancer mortality by 31 % and has served as the standard treatment for ER-positive breast cancers for decades. However, 50 % of advanced ER-positive cancers display de novo resistance to tamoxifen, and acquired resistance evolves in 40 % of patients who initially respond. Mechanisms underlying resistance development remain poorly understood and new therapeutic opportunities are urgently needed. Here, we report the generation and characterization of seven tamoxifen-resistant breast cancer cell lines from four parental strains. METHODS: Using high throughput drug sensitivity and resistance testing (DSRT) with 279 approved and investigational oncology drugs, exome-sequencing and network analysis, we for the first time, systematically determine the drug response profiles specific to tamoxifen resistance. RESULTS: We discovered emerging vulnerabilities towards specific drugs, such as ERK1/2-, proteasome- and BCL-family inhibitors as the cells became tamoxifen-resistant. Co-resistance to other drugs such as the survivin inhibitor YM155 and the chemotherapeutic agent paclitaxel also occurred. CONCLUSION: This study indicates that multiple molecular mechanisms dictate endocrine resistance, resulting in unexpected vulnerabilities to initially ineffective drugs, as well as in emerging co-resistances. Thus, combatting drug-resistant tumors will require patient-tailored strategies in order to identify new drug vulnerabilities, and to understand the associated co-resistance patterns.

T cells and cancer - why do the killers become exhausted?

Nascent tumors are mostly eliminated by the immune system. During carcinogenesis mutated cells find a way to escape from the immune system´s surveillance. As the tumor microenvironment evolves it becomes increasingly difficult for T cells to recognize and kill cancer cells. Recently, novel immunological targets have been recognized and potent immunomodulatory drugs discovered in clinical trials. This has resulted in the emergence of immunotherapy as a novel potent therapy for cancer in addition to chemotherapy, targeted therapy, operative therapy and radiotherapy.

A bibliometric analysis of the gender gap in the authorship of leading medical journals.

BACKGROUND: Monitoring gender representation is critical to achieve diversity and equity in academia. One way to evaluate gender representation in academia is to examine the authorship of research publications. This study sought to determine the gender of first and senior authors of articles in leading medical journals and assess trends in the gender gap over time. METHODS: We gather bibliometric data on original research articles (n = 10,558) published in 2010-2019 in five leading medical journals to audit publication and citation frequency by gender. We explored their association with scientific fields, geographical regions, journals, and collaboration scope. RESULTS: We show that there are fewer women as senior (24.8%) than leading authors (34.5%, p < 0.001). The proportion of women varied by country with 9.1% last authors from Austria, 0.9% from Japan, and 0.0% from South Korea. The gender gap decreased longitudinally and faster for the last (-24.0 articles/year, p < 0.001) than first authors (-14.5 articles/year, p = 0.024) with pronounced country-specific variability. We also demonstrate that usage of research keywords varied by gender, partly accounting for the difference in citation counts. CONCLUSIONS: In summary, gender representation has increased, although with country-specific variability. The study frame can be easily applied to any journal and time period to monitor changes in gender representation in science.

The publishing of medical research papers has traditionally been dominated by men. To better understand whether gender diversity in the authorship of research papers has changed recently, we analyzed over 10,000 articles published between 2010 and 2019 in five top medical journals. Usually, the first author is recognized as the leading contributor, whereas the last author supervises the study. We found that there were fewer women in senior positions compared to first author positions. The percentage of women as authors varied across countries. Over time, the gender gap decreased, but at different rates depending on the author's position and country. Keywords selected by researchers to describe their work varied between genders. Our findings show progress in gender representation, but with country-specific differences. This study can be used as a model to track gender representation in other journals and time periods.

A bibliometric analysis of geographic disparities in the authorship of leading medical journals.

BACKGROUND: It has previously been reported that authors from developing countries are underrepresented in medical journals. Here, we aimed to build a comprehensive landscape of the geographical representation in medical research publications. METHODS: We collected bibliometric data of original research articles (n = 10,558) published between 2010 and 2019 in five leading medical journals and geolocated these by the institute of the corresponding authors. We introduced two simple metrics, the International Research Impact and the Domestic Self-Citation Index, to assess publishing and citing patterns by cities and countries. RESULTS: We show that only 32 countries published more than 10 publications in 10 years equaling 98.9% of all publications. English-speaking countries USA (48.2%), UK (15.9%), Canada (5.3%), and Australia (3.2%) are most represented, but with a declining trend in recent years. When normalized to citation count, 9/32 countries published ≥ 10% more than expected. In total, 85.7% of the publication excess originate from the USA and UK. We demonstrate similar geographical bias at the municipal level. Finally, we discover that journals more commonly publish studies from the country in which the journal is based and authors are more likely to cite work from their own country. CONCLUSIONS: The study reveals Anglocentric dominance, domestic preference, but increased geographical representation in recent years in medical publishing. Similar audits could mitigate possible national and regional disparities in any academic field.

Geographical representation in authorships of research articles is insufficiently understood. We analyzed data from over 10,000 research articles published between 2010­2019 in top medical journals. Anglocentric countries (USA, UK, Canada, and Australia) accounted for most publications, but their proportion has recently declined. When considering citations, i.e. formal references connecting new findings to observations from previously published articles, 1/3 of the studied countries published ≥10% more articles than expected. When publishing and citing articles, journals and researchers tended to favor publications from their own countries. While some improvement in geographical representation has occurred, our findings expose an Anglocentric bias and national preference, which might bias medical publishing. The approach used in this study may be used in future efforts to monitor geographical representation in publication authorships.

Real-world experience of novel multiple myeloma treatments in a large, single-center cohort in Finland.

In this single-center study, we aimed to describe the characteristics, treatment patterns, and outcomes of patients with multiple myeloma (MM) following treatment with bortezomib, carfilzomib, daratumumab, ixazomib, lenalidomide or pomalidomide-based regimens. Data were collected retrospectively from a study cohort of patients receiving a MM treatment in the Hospital District of Helsinki and Uusimaa (HUS) in Finland between 2016-2020. In total, 472 patients were included in the study. Median age was 68.2 years and nearly 25% had a high cytogenetic risk according to the International Myeloma Working Group categorization. In 2018-2020, the spectrum of regimens used as third- or later-line therapy was notably broader than in 2016-2017. The overall response rates for patients who received the most novel regimens (available ≤ 5 years) in second or third line of therapy (n = 67/430) and fourth line or later (n = 78/151) were 53.3% and 25.0%, respectively. In this real-world MM patient cohort, the response rates for these novel agents were lower compared to those reported in clinical trials. Given the higher cytogenetic risk profile and more advanced disease stage at the time when treated with novel agents, patients could have benefited from effective novel therapies earlier in their treatment pathway. What is the NEW aspect of your work? (ONE sentence) This study characterized the treatment of Finnish multiple myeloma patients during the era of most novel therapies (after 2016) and also included information on the cytogenetic risk profile of this real-world population.What is the CENTRAL finding of your work? (ONE sentence) There are clear differences between real-world populations treated with most novel combinations and those of randomized controlled trials (RCTs), which is reflected by the poorer treatment outcomes in the real-world setting.What is (or could be) the SPECIFIC clinical relevance of your work? (ONE sentence) Given the high cytogenetic risk profile and advanced disease stage at the time when treated with novel agents, patients could have benefited from effective novel therapies earlier in their treatment pathway.

Fibroblast subsets in non-small cell lung cancer: Associations with survival, mutations, and immune features.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are molecularly heterogeneous mesenchymal cells that interact with malignant cells and immune cells and confer anti- and protumorigenic functions. Prior in situ profiling studies of human CAFs have largely relied on scoring single markers, thus presenting a limited view of their molecular complexity. Our objective was to study the complex spatial tumor microenvironment of non-small cell lung cancer (NSCLC) with multiple CAF biomarkers, identify novel CAF subsets, and explore their associations with patient outcome. METHODS: Multiplex fluorescence immunohistochemistry was employed to spatially profile the CAF landscape in 2 population-based NSCLC cohorts (n = 636) using antibodies against 4 fibroblast markers: platelet-derived growth factor receptor-alpha (PDGFRA) and -beta (PDGFRB), fibroblast activation protein (FAP), and alpha-smooth muscle actin (αSMA). The CAF subsets were analyzed for their correlations with mutations, immune characteristics, and clinical variables as well as overall survival. RESULTS: Two CAF subsets, CAF7 (PDGFRA-/PDGFRB+/FAP+/αSMA+) and CAF13 (PDGFRA+/PDGFRB+/FAP-/αSMA+), showed statistically significant but opposite associations with tumor histology, driver mutations (tumor protein p53 [TP53] and epidermal growth factor receptor [EGFR]), immune features (programmed death-ligand 1 and CD163), and prognosis. In patients with early stage tumors (pathological tumor-node-metastasis IA-IB), CAF7 and CAF13 acted as independent prognostic factors. CONCLUSIONS: Multimarker-defined CAF subsets were identified through high-content spatial profiling. The robust associations of CAFs with driver mutations, immune features, and outcome suggest CAFs as essential factors in NSCLC progression and warrant further studies to explore their potential as biomarkers or therapeutic targets. This study also highlights multiplex fluorescence immunohistochemistry-based CAF profiling as a powerful tool for the discovery of clinically relevant CAF subsets.

Single-cell characterization of anti-LAG-3 and anti-PD-1 combination treatment in patients with melanoma.

BackgroundRelatlimab plus nivolumab (anti-lymphocyte-activation gene 3 plus anti-programmed death 1 [anti-LAG-3+anti-PD-1]) has been approved by the FDA as a first-line therapy for stage III/IV melanoma, but its detailed effect on the immune system is unknown.MethodsWe evaluated blood samples from 40 immunotherapy-naive or prior immunotherapy-refractory patients with metastatic melanoma treated with anti-LAG-3+anti-PD-1 in a phase I trial using single-cell RNA and T cell receptor sequencing (scRNA+TCRαß-Seq) combined with other multiomics profiling.ResultsThe highest LAG3 expression was noted in NK cells, Tregs, and CD8+ T cells, and these cell populations underwent the most significant changes during the treatment. Adaptive NK cells were enriched in responders and underwent profound transcriptomic changes during the therapy, resulting in an active phenotype. LAG3+ Tregs expanded, but based on the transcriptome profile, became metabolically silent during the treatment. Last, higher baseline TCR clonality was observed in responding patients, and their expanding CD8+ T cell clones gained a more cytotoxic and NK-like phenotype.ConclusionAnti-LAG-3+anti-PD-1 therapy has profound effects on NK cells and Tregs in addition to CD8+ T cells.Trial registrationClinicalTrials.gov (NCT01968109)FundingCancer Foundation Finland, Sigrid Juselius Foundation, Signe and Ane Gyllenberg Foundation, Relander Foundation, State funding for university-level health research in Finland, a Helsinki Institute of Life Sciences Fellow grant, Academy of Finland (grant numbers 314442, 311081, 335432, and 335436), and an investigator-initiated research grant from BMS.