Immunogram defines four cancer-immunity cycle phenotypes with distinct clonal selection patterns across solid tumors.

BACKGROUND: The cancer-immunity cycle (CI cycle) provides a theoretical framework to illustrate the process of the anticancer immune response. Recently, the update of the CI cycle theory emphasizes the importance of tumor's immunological phenotype. However, there is lack of immunological phenotype of pan-cancer based on CI cycle theory. METHODS: Here, we applied a visualizing method termed 'cancer immunogram' to visualize the state of CI cycle of 8460 solid tumors from TCGA cohort. Unsupervised clustering of the cancer immunogram was performed using the nonnegative matrix factorization (NMF) analysis. We applied an evolutionary genomics approach (dN/dS ratio) to evaluate the clonal selection patterns of tumors with distinct immunogram subtypes. RESULTS: We defined four major CI cycle patterns across 32 cancer types using a cancer immunogram approach. Immunogram-I was characterized by 'hot' and 'exhausted' features, indicating a favorable prognosis. Strikingly, immunogram-II, immunogram-III, and immunogram-IV represented distinct immunosuppressive patterns of 'cold' tumor. Immunogram-II was characterized by 'cold' and 'radical' features, which represented increased expression of immune inhibitor molecules and high levels of positive selection, indicating the worst prognosis. Immunogram-III was characterized by 'cold' and 'recognizable' features and upregulated expression of MHC I molecules. Immunogram-IV was characterized by 'cold' and 'inert' features, which represented overall immunosuppression, lower levels of immunoediting and positive selection, and accumulation of more tumor neoantigens. In particular, favorable overall survival was observed in metastatic urothelial cancer patients with immunogram-I and immunogram-IV after immune checkpoint inhibitor (ICI) therapy. Meanwhile, a higher response rate to ICI therapy was observed in metastatic gastric cancer patients with immunogram-I phenotype. CONCLUSIONS: Our findings provide new insight into the interaction between immunity and cancer evolution, which may contribute to optimizing immunotherapy strategies.

[Long-term follow-up study of post-COVID-19 patients].

AIM: To evaluate dynamic changes in the lungs, hemostasis system, immune system in different terms after coronavirus pneumonia. MATERIALS AND METHODS: Ventilation-perfusion single-photon emission computed tomography/computed tomography (CT), functional methods of lung investigation, evaluation of hemostasis system, immune status and specific humoral immune response were performed and evaluated in different terms after coronavirus pneumonia. A total of 71 patients were examined according to this protocol. We examined patients with the lesion volume not less than 50% according to chest CT. All patients were divided into 2 groups depending on the distance from the acute stage of coronavirus pneumonia. Group 1 included patients who were examined early (3060 days after hospital discharge), group 2 included patients who were examined later (61180 days after hospital discharge). RESULTS: We obtained gradual regression of pathologically-modified tissue from 67.3% during the inpatient phase to 30.9% during the early period and to 19.7% during the late period of examination, according to CT scan of the chest organs. The same tendency was demonstrated by diffusion capacity of the lungs. Perfusion scintigraphy data showed a decrease in perfusion deficit from 26.012.8% during the early period of examination to 19.46.2% during the late period of examination. On the contrary, ventilatory scintigraphy demonstrates the increase of isotope passage time through the alveolar-capillary membrane over time (from 48.231.3 minutes in the early period to 83.637.2 minutes in the late period). An increase in D-dimer was detected in 24% of patients in the early group. The levels of inflammatory markers, indices of immune status, and specific humoral immune response did not differ in the two described groups. CONCLUSION: The results demonstrate gradual regression of pathological changes caused by coronavirus infection.

A novel scoring method based on RNA-Seq immunograms describing individual cancer-immunity interactions.

Because of the complexity of cancer-immune system interactions, combinations of biomarkers will be required for predicting individual patient responses to treatment and for monitoring combination strategies to overcome treatment resistance. To this end, the "immunogram" has been proposed as a comprehensive framework to capture all relevant immunological variables. Here, we developed a method to convert transcriptomic data into immunogram scores (IGS). This immunogram includes 10 molecular profiles, consisting of innate immunity, priming and activation, T cell response, interferon γ (IFNG) response, inhibitory molecules, regulatory T cells, myeloid-derived suppressor cells (MDSCs), recognition of tumor cells, proliferation, and glycolysis. Using genes related to these 10 parameters, we applied single-sample gene set enrichment analysis (ssGSEA) to 9417 bulk RNA-Seq data from 9362 cancer patients with 29 different solid cancers in The Cancer Genome Atlas (TCGA). Enrichment scores were z-score normalized (Z) for each cancer type or the entire TCGA cohort. The IGS was defined by the formula IGS = 3 + 1.5 × Z so that patients would be well distributed over a range of scores from 1 to 5. The immunograms constructed in this way for all individual patients in the entire TCGA cohort can be accessed at "The RNA-Seq based Cancer Immunogram Web" (https://yamashige33.shinyapps.io/immunogram/).

In-Vitro Assessment of Hapten-Induced Immune Regulation in Children with Asthenoneurotic Syndrome.

The study employed in vitro assay to examine the peculiarities of immune status in children with functional disorders of the autonomic nervous system diagnosticated as asthenoneurotic syndrome. In contrast to control children without asthenoneurotic syndrome, the examined group was characterized by significantly (p<0.05) elevated hapten-specific immunological sensitization (indicated by anti-Al IgG), induction of inflammatory reactions (IL-1), activation of apoptosis (CD3+CD95+ and р53) observed against the background inhibition of adaptive immune response (CD3+, CD3+CD4+, CD3+CD8+, CD16+56+, and CD19+), as well as hyperexpression of glutamic acid, NO, and VEGF combined with deficiency of serotonin. In cultured immunocompetent cells derived from children with hapten-modified immune status, the combined application of cytokine stimulator IL-1 with hapten sensitizer aluminum or with endocrine stimulator cortisol significantly (p<0.05) up-regulated expression of IL-8 and IL-10, but down-regulated production of IL-17 in a dose-dependent manner.

Analytical validation of an Enzyme Immunoassay for the measurement of urinary oxytocin in dogs and wolves.

Assessing changes in oxytocin (OT) levels in response to a variety of social stimuli has become of major interest in the field of behavioral endocrinology. OT is involved in the regulation of various aspects of social behavior such as tolerance, and the formation and maintenance of social bonds but also the regulation of stress. All of these aspects have been identified as potential targets of selection during the domestication process. Therefore, comparing the role of the oxytocinergic system in various aspects of dog and wolf social behavior, might help to understand whether this system was involved in the domestication process. Studies assessing OT levels in dogs and wolves have used invasively collected plasma and serum samples and non-invasively collected urine samples. However, when using an assay system on a new species a careful and complete validation of the method is of crucial importance, and to date no proper validation, to assess urinary OT levels in dogs and wolves, has been reported. We therefore conducted an analytical validation of an Enzyme Immunoassay (EIA) for the measurement of OT in urine of dogs and wolves, using a commercially available EIA. Stability tests revealed that OT levels degrade over time when stored at 4 °C, but are little affected by repeated thawing. In addition, our results indicate that the variance in OT levels is slightly lower when phosphoric acid is added following collection to prevent OT degradation. Long term storage tests revealed that urinary OT levels are least variable when stored as extracts in ethanol at -20 °C, rather than as unextracted urine samples. Validation results were acceptable with regard to parallelism, but values for accuracy and extraction efficiency were not meeting the standard criteria usually applied to steroid EIAs, especially when assessed for the lower range of the assay. The results of this study highlight the importance of an analytical assay validation, since even if validation parameters are not optimal, if published, they allow readers to estimate the relevance of studies using the validated method.

Validation of an enzyme-immunoassay for the non-invasive monitoring of faecal testosterone metabolites in male cheetahs (Acinonyx jubatus).

In mammals, the sex hormone testosterone is the major endocrine variable to objectify testicular activity and thus reproductive function in males. Testosterone is involved in the development and function of male reproductive physiology and sex-related behaviour. The development of a reliable androgen enzyme-immunoassay (EIA) to monitor faecal testosterone metabolites (fTM) is a powerful tool to non-invasively assess the gonadal status of males. We validated an epiandrosterone EIA for male cheetahs by performing a testosterone radiometabolism study followed by high-performance liquid chromatography (HPLC) analyses and excluding possible cross-reactivities with androgenic metabolites not derived from testosterone metabolism. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations within one day in response to a testosterone injection, (2) a significant increase in fTM concentrations within one day in response to a gonadotropin-releasing hormone (GnRH) injection, which failed following a placebo injection, and (3) significant differences in fTM concentrations between adult male and adult female cheetahs and between adult and juvenile male cheetahs of a free-ranging population. Finally, we demonstrated stability of fTM concentrations measured in faecal samples exposed to ambient temperatures up to 72h. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor testicular activity in male cheetahs.

Validation of an enzyme immunoassay for the measurement of faecal glucocorticoid metabolites in Eurasian (Lynx lynx) and Iberian lynx (Lynx pardinus).

Stress hormone levels are important indicator of an animal's well-being, as stress has harmful effects on reproduction, growth and immune function. The development of enzyme immunoassays (EIA) to monitor faecal glucocorticoid metabolites (fGM) contributes a powerful tool to assess an animal's adrenal status non-invasively. We aimed to identify a suitable EIA for monitoring fGM by assessing the suitability of six different EIAs for detecting quantitative changes in fGM concentrations in response to an ACTH challenge test in Eurasian lynx. FGM were characterised in a male Eurasian lynx that received an injection of (3)H-cortisol. Using HPLC analyses radiolabeled metabolites were compared with immunoreactive metabolites. The second aim was to biologically validate the established EIA for monitoring adrenocortical activity of captive Iberian lynxes after a translocation to new enclosures in relation to behaviour. Additionally faecal samples of ten pregnant Iberian lynxes from the peripartal period were analysed. The ACTH challenge revealed an 11ß-hydroxyetiocholanolone EIA as the most sensitive assay to reflect acute fGM elevations in the Eurasian lynx. HPLC immunograms demonstrated that the 11ß-hydroxyetiocholanolone EIA measured significant amounts of immunoreactivities corresponding to radiolabeled metabolites with strong similarities across both lynx species. Additionally, HPLC and GC-MS analyses confirmed the presence of 11ß-hydroxyetiocholanolone in faeces of both, the Eurasian and the Iberian lynx. Longitudinal fGM profiles of Iberian lynx revealed increases in concentrations associated with management events. During the peripartal period, however, fGM concentrations were not significantly elevated. Our results show that the 11ß-hydroxyetiocholanolone EIA is a reliable tool to assess fGM in both lynx species.

The immunogram of inflammatory breast cancer.

Inflammatory breast cancer (IBC) is the most aggressive and fatal clinical presentation of breast cancer. Despite the term "inflammatory", based on the clinical presentation, IBC is biologically driven by an immunosuppressive tumor microenvironment (TME). Whether IBC can be switched into an immune-inflamed TME by immune-checkpoint inhibitors (ICIs) is a matter of debate. Presently, measurable biomarkers of IBC-TME have never been synthetized into a comprehensive portray of the immune-milieu (i.e., an immunogram), describing the immune-vulnerability of IBC and potentially predicting the response to ICIs. We propose an immunogram for IBC, based on preclinical and clinical studies, including six parameters: the presence of immune-effector cells, of immune-suppressive cells and of immune checkpoints, the general immune status, the activation of immune-suppressive pathways, the tumor foreignness. The IBC immunogram suggests the existence of a preexisting immune TME that is suppressed by mechanisms of immune-escape but might be restored by ICIs. The combination of chemotherapy and ICIs in patients with IBC is based on a strong biological rationale. However, the design and the development of clinical trials assessing the incorporation of ICIs raise many methodological and practical issues. In parallel with the further comprehension of IBC biology, the prospective validation and integration of biomarkers predictive of response to ICIs are warranted.

Understanding resistance to immune checkpoint inhibitors in advanced breast cancer.

INTRODUCTION: The addition of immune checkpoint inhibitors (ICIs) to frontline chemotherapy has improved survival for patients with advanced triple-negative breast cancer (TNBC) expressing programmed death-ligand 1 (PD-L1). Nonetheless, most patients develop resistance, with outcomes remaining poor for this population. Moreover, unsatisfactory activity has been observed with ICIs in PD-L1-negative TNBC and in other breast cancer (BC) subtypes, warranting a deeper understanding of resistance to ICIs in BC. AREAS COVERED: We discuss the immune landscape of distinct BC subtypes, review the clinical activity of immunotherapy in BC, and highlight strategies under development to overcome resistance to ICIs. EXPERT OPINION: Activity and resistance to ICIs in BC are strongly related to the intrinsic immunophenotype of the tumor tissue. Several promising biomarkers reflecting the immunological state of BC are emerging, with only PD-L1 expression currently adopted into clinical practice. However, limitations make of PD-L1 a sub-optimal biomarker for patient selection, which require efforts to integrate this marker with other immunological features. Concomitantly, a wide variety of drug combinations designed to overcome immune-resistance are being evaluated, with some encouraging signals observed in early-phase trials. Combination strategies tailored to patient and tumor immunophenotype may allow to overcome resistance and fully exploit the potential of ICIs.

A 12-chemokine gene signature is associated with the enhanced immunogram scores and is relevant for precision immunotherapy.

Advances in the understanding of checkpoint blockade immunotherapy have suggested that boosting the cancer-immunity cycle (CIC) can help induce regression of tumors. However, good efficacy only occurs in a subset of patients. Predictive biomarkers that can reflect the tumor microenvironment (TME) and CIC may have great potential. More recently, the presence of intratumoral tertiary lymphoid structures (TLSs) has also been correlated with clinical benefit in patients. In this study, we comprehensively measured the immunogram scores (IGSs) for the CIC and explored the associations between immunological and mutational features and a 12-chemokine metagene TLS signature in data from The Cancer Genome Atlas (TCGA). Three immunotherapy datasets were further applied for validation. In the TCGA dataset, we observed that the 12-chemokine TLS signature score was positively associated with the enhanced IGSs as represented by increased tumor mutational burden (TMB) and neoantigen burden (TNB), enriched immune cell (IC) infiltration, and elevated cytolytic activity and checkpoint expression. Specifically, in bladder cancer and melanoma, the high 12-chemokine TLS signature score was found to potentially reflect an expanded cancer-immunity status characterized by high TNB and an immune-inflamed feature. The predictive and prognostic value of the 12-chemokine TLS signature was further validated in several immunotherapy datasets. The score of the 12-chemokine TLS signature may serve as a pancancer marker of the immune-active phenotype. The 12-chemokine TLS signature showed promise as a predictive and prognostic biomarker for ICB efficacy, especially in melanoma and bladder cancer.

Validation of a commercial enzyme immunoassay to assess urinary oxytocin in humans.

Within the last decade, oxytocin (OT) has attracted a lot of attention in the context of various human social behaviors. Besides its importance in regulating physiological processes in females related to giving birth and lactation, OT is involved in the establishment and maintenance of social relationships, trust and emotion recognition. However, results are not always consistent across studies, which may partly be due to the incomplete validation of methods used to assess OT levels. Carefully validating a method before its use is of crucial importance to ensure that it can be used to accurately, reliably and repeatedly assess OT levels. With this study we evaluated a commercially available Enzyme Immunoassay to assess OT in human urine samples by conducting a careful analytical validation. Results indicate that, with regard to parallelism and immunoreactivity, human urinary OT can be assessed reliably. However, extraction methods need further improvement to optimize measures of accuracy and extraction efficiency, especially in the lower range of the assay system. Tests on OT stability indicate that OT is affected by degradation when stored at 4°C or room temperature. Storing urine samples over longer periods revealed that OT levels are most stable when stored as ethanol extracts at -20°C compared to being stored as samples at -20°C or -80°C. Although some of the validated parameters did not reach the intended quality criteria, this study highlights the importance of such in depth validation procedures and reporting results to make them available to researchers embarking on projects utilizing such methods.

Hair cortisol analyses in different mammal species: choosing the wrong assay may lead to erroneous results.

Wild animals are faced with a broad range of environmental stressors and research is needed to better understand their effect on populations. Hormone analysis based on enzyme immunoassays (EIAs) can provide valuable information on adrenocortical activity (stress), and assessment of cortisol in hair may allow the quantification of cortisol production. To validate hair hormone analysis, we compared two EIAs based on antibodies against cortisol-3-CMO-BSA and cortisol-21-HS-BSA for hair glucocorticoid (hGC) measurements in Egyptian mongoose, Iberian lynx, Alpine marmot, Asiatic black bear, spotted hyena and cheetah, with results obtained by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) measurements. Both EIAs were also characterized by HPLC immunograms. Our results revealed that the cortisol-21-HS EIA measured 2.3- to 12-fold higher hGC concentrations than the cortisol-3-CMO assay. In dependence of the species, high-performance liquid chromatography (HPLC) immunograms showed that up to 70% of immunoreactivities determined by the cortisol-21-HS constituted of unknown unpolar compounds leading to an overestimation of hGC. The cortisol-3-CMO EIA expressed a better specificity, with 32.1-67.4% of immunoreactivity represented by cortisol and cortisone. The LC-MS/MS analyses (gold standard) revealed that the cortisol-3-CMO EIA also resulted in an (up to 3-fold) overestimation of hGC, but EIA results were correlated with LC-MS/MS in the mongoose, the lynx, the spotted hyena and the marmot. No correlation was obtained for Asiatic black bears. As a result of our study, we strongly recommend to test any cortisol EIA for its specificity towards extracted hair components. In all analyzed species, except the Asiatic black bear, cortisone and cortisol were simultaneously present in hair extracts; consequently, an appropriate EIA should cross-react to these two glucocorticoid hormones and express negligible affinity towards substances with less polarity than corticosterone. Choosing the wrong EIA for hGC analyses may lead to overestimations of hGC or-in the worst case-to results that do not mirror real adrenocortical activity.

Integrative immunogenomic analysis of gastric cancer dictates novel immunological classification and the functional status of tumor-infiltrating cells.

OBJECTIVES: A better understanding of antitumor immunity will help predict the prognosis of gastric cancer patients and tailor the appropriate therapies in each patient. Therefore, we propose a novel immunological classification of gastric cancer. METHODS: We performed whole-exome sequencing (WES), RNA-Seq and flow cytometry in 29 gastric cancer patients who received surgery. The TCGA data set of 323 gastric cancer patients and RNA-Seq data of 45 patients who received pembrolizumab (Kim et al. Nat Med 2018; 24: 1449-1458) were also analysed. RESULTS: Immunogram analysis of cancer-immunity interaction of gastric cancer revealed immune signatures of four main types, designated Hot1, Hot2, Intermediate and Cold. Immunologically hot tumors displayed a dysfunctional T-cell signature, while cold tumors had an exclusion signature. Ex vivo tumor-infiltrating lymphocyte analysis documented T-cell dysfunction with the expression of checkpoint molecules and impaired cytokine production. The T-cell function was more profoundly damaged in Hot1 than Hot2 tumors. Patients in Hot2 subtypes had better survival in our cohort and TCGA cohort. Although these immunological subtypes overlapped to some degree with the molecular subtypes in the TCGA, intratumoral immune responses cannot be predicted solely based on histological or molecular subtyping of gastric cancer. Molecular and immunological classifications complement each other to predict the responses to anti-PD-1 therapy and have the potential to be a biomarker for the treatment of gastric cancer. CONCLUSION: The immunological classification of gastric cancer resulted in four subtypes. Hot tumors were further divided into two subtypes, between which the functional status of T cells was different.

An Immunogram for an Individualized Assessment of the Antitumor Immune Response in Patients With Hepatocellular Carcinoma.

In clinical practice, the cancer-immunity cycle of an individual patient with hepatocellular carcinoma (HCC) must be described to support the clinical management of cancer. The present study explored the immunograms of patients with liver cancer based on liver RNA sequencing data to visually display the individualized cancer-immunity cycles. Two independent HCC cohorts [The Cancer Genome Atlas (TCGA) and Liver Cancer-RIKEN, Japan (LIRI-JP) HCC cohorts] with whole exome sequencing (WES) data, RNA sequencing data, and clinical data from TCGA and International Cancer Genome Consortium (ICGC) were enrolled in this study. This study constructed HCC immunograms of cancer immune cells to visually explore the anticancer immune responses of patients with HCC. The patterns of the HCC immunograms were categorized into two clusters: hot and cold HCC immunograms. Favorable overall survival (OS) and disease-free survival (DFS) were observed in the hot immunogram cluster in the TCGA cohort. The results for LIRI-JP cohort were similar to the TCGA cohort. The OS of patients with HCC presenting the hot immunogram was longer than patients with the cold immunogram in the LIRI-JP HCC cohort. Compared with cold immunograms, hot immunograms were characterized by higher levels of immune cell infiltration and stronger immune signatures, including cytolytic activity, IFN-γ signature, immunocostimulator, immunoinhibitor, chemokine, adhesion molecule, MHC I, MHC II, and non-class MHC levels. The main difference in molecular features between hot and cold immunograms was reflected in WNT-CTNNB1 alterations and copy number variant (CNV) and loss of heterozygosity (LOH) scores, which are the molecular features associated with resistance to immunotherapy and tumor escape. The immunogram patterns were distinct in terms of the different molecular features of HCC tumors. The HCC immunogram for the cancer-immune cycle was able to visualize the personalized antitumor immune response of patients with HCC, and the patterns of the HCC immunograms contributed to the clinical outcomes of patients, which may facilitate an individualized assessment of the antitumor immune response for optimal personalized immunotherapy.

It Is a Capital Mistake to Theorize Who to Treat with Checkpoint Inhibitors before One Has Data.

Immunotherapy results in remarkable clinical benefit in a subset of cancer patients by activating the patient's own immune system. The factors determining which cancer patients will benefit are diverse. Success in realizing precision immunotherapy needs collaboration to bring together multiple diverse data sets. Defining multi-factorial biomarker algorithms for immunotherapy requires new approaches and methodologies that use deep molecular and cellular profiling of the tumor microenvironment, systemic immunity with clinical metadata from clinical trials, and other databases.

Defining the immunogram of breast cancer: a focus on clinical trials.

In phase III ImPassion130 trial, the addition of immunotherapy to chemotherapy improved overall survival in metastatic triple-negative breast cancer patients. This benefit was significant only in patients harboring PD-L1-positive tumors, suggesting that stratification according to response biomarkers is needed to achieve consistent responses. Besides PD-L1 expression, a variety of potential biomarkers are under investigation for predicting immunotherapy efficacy in breast cancer, such as tumor-infiltrating lymphocytes, gene signatures, tumor mutational burden, microsatellite instability, and gut microbiome. Enriching future trials through these biomarkers could help identifying the population of responders, realizing what has been called precision immunotherapy.

Oncograms Visualize Factors Influencing Long-Term Survival of Cancer Patients Treated with Adenoviral Oncolytic Immunotherapy.

The first US Food and Drug Administration (FDA)- and EMA-approved oncolytic virus has been available since 2015. However, there are no markers available that would predict benefit for the individual patient. During 2007-2012, we treated 290 patients with advanced chemotherapy-refractory cancers, using 10 different oncolytic adenoviruses. Treatments were given in a Finnish Medicines Agency (FIMEA)-regulated individualized patient treatment program (the Advanced Therapy Access Program [ATAP]), which required long-term follow-up of patients, which is presented here. Focusing on the longest surviving patients, some key clinical and biological features are presented as "oncograms." Some key attributes that could be captured in the oncogram are suggested to predict treatment response and survival after oncolytic adenovirus treatment. The oncogram includes immunological laboratory parameters assessed in peripheral blood (leukocytes, neutrophil-to-lymphocyte ratio, interleukin-8 [IL-8], HMGB1, anti-viral neutralizing antibody status), features of the patient (gender, performance status), tumor features (histological tumor type, tumor load, region of metastases), and oncolytic virus-specific features (arming of the virus). The retrospective approach used here facilitates verification in a prospective controlled trial setting. To our knowledge, the oncogram is the first holistic attempt to identify the patients most likely to benefit from adenoviral oncolytic virotherapy.

Changes in the TCRß Repertoire and Tumor Immune Signature From a Cutaneous Melanoma Patient Immunized With the CSF-470 Vaccine: A Case Report.

The allogeneic therapeutic vaccine CSF-470 has demonstrated a significant benefit over medium-dose IFNα2b in the distant metastasis-free survival for stages IIB-IIC-III cutaneous melanoma patients in a randomized phase II/III clinical trial (CASVAC-0401, NCT01729663). At the end of the 2-year CSF-470 immunization protocol, patient #006 developed several lung and one subcutaneous melanoma metastases; this later was excised. In this report, we analyzed the changes throughout vaccination of immune populations in blood and in the tumor tissue, with special focus on the T-cell repertoire. Immunohistochemistry revealed a marked increase in CD8+, CD4+, and CD20+ lymphocytes infiltrating the metastasis relative to the primary tumor. Lymphocytes were firmly attached to dying-tumor cells containing Granzyme-B granules. Whole-exon sequencing assessment indicated a moderate-to-high tumor mutational burden, with BRAFV600E as the main oncogenic driver. Mutational signature presented large numbers of mutations at dipyrimidines, typical of melanoma. Relevant tumor and immune-related genes from the subcutaneous metastasis were addressed by RNA-Seq analysis, revealing expression of typical melanoma antigens and proliferative tumor-related genes. Stimulatory and inhibitory immune transcripts were detected as well as evidence of active T-cell effector function. Peripheral blood monitoring revealed an increase in CD4+ and CD8+ cells by the end of the immunization protocol. By CDR3-T-cell receptor ß (TCRß) sequencing, generation of new clones and an increase in oligoclonality was observed in the peripheral T-cells immune repertoire throughout immunization. A shift, with the expansion of selected preexisting and newly arising clones with reduction of others, was detected in blood. In tumor-infiltrating lymphocytes, prevalent clones (50%) were both new and preexisting that were expanded in blood following CSF-470 immunization. These clones persisted in time, since 2 years after completing the immunization, 51% of the clones present in the metastasis were still detected in blood. This is the first report of the modulation of the TCRß repertoire from a melanoma patient immunized with the CSF-470 vaccine. After immunization, the changes observed in peripheral immune populations as well as in the tumor compartment suggest that the vaccine can induce an antitumor adaptive immune repertoire that can reach tumor lesions and persists in blood for at least 2 years.

An Immunogram for the Cancer-Immunity Cycle: Towards Personalized Immunotherapy of Lung Cancer.

INTRODUCTION: The interaction of immune cells and cancer cells shapes the immunosuppressive tumor microenvironment. For successful cancer immunotherapy, comprehensive knowledge of antitumor immunity as a dynamic spatiotemporal process is required for each individual patient. To this end, we developed an immunogram for the cancer-immunity cycle by using next-generation sequencing. METHODS: Whole exome sequencing and RNA sequencing were performed in 20 patients with NSCLC (12 with adenocarcinoma, seven with squamous cell carcinoma, and one with large cell neuroendocrine carcinoma). Mutated neoantigens and cancer germline antigens expressed in the tumor were assessed for predicted binding to patients' human leukocyte antigen molecules. The expression of genes related to cancer immunity was assessed and normalized to construct a radar chart composed of eight axes reflecting seven steps in the cancer-immunity cycle. RESULTS: Three immunogram patterns were observed in patients with lung cancer: T-cell-rich, T-cell-poor, and intermediate. The T-cell-rich pattern was characterized by gene signatures of abundant T cells, regulatory T cells, myeloid-derived suppressor cells, checkpoint molecules, and immune-inhibitory molecules in the tumor, suggesting the presence of antitumor immunity dampened by an immunosuppressive microenvironment. The T-cell-poor phenotype reflected lack of antitumor immunity, inadequate dendritic cell activation, and insufficient antigen presentation in the tumor. Immunograms for both the patients with adenocarcinoma and the patients with nonadenocarcinoma tumors included both T-cell-rich and T-cell-poor phenotypes, suggesting that histologic type does not necessarily reflect the cancer immunity status of the tumor. CONCLUSIONS: The patient-specific landscape of the tumor microenvironment can be appreciated by using immunograms as integrated biomarkers, which may thus become a valuable resource for optimal personalized immunotherapy.