SITC cancer immunotherapy resource document: a compass in the land of biomarker discovery.

Since the publication of the Society for Immunotherapy of Cancer\u27s (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune escape mechanisms which can develop in each patient. Therefore, the development of robust biomarkers for each immunotherapy strategy, enabling rational patient selection and the design of precise combination therapies, is key for the continued success and improvement of immunotherapy. In this document, we summarize and update established biomarkers, guidelines, and regulatory considerations for clinical immune biomarker development, discuss well-known and novel technologies for biomarker discovery and validation, and provide tools and resources that can be used by the biomarker research community to facilitate the continued development of immuno-oncology and aid in the goal of durable responses in all patients

Tissue Proteomes: Quantitative Mass Spectrometry of Murine Liver and Ovarian Endometrioma

A human genome contains more than 20 000 protein-encoding genes. A human proteome, instead, has been estimated to be much more complex and dynamic. The most powerful tool to study proteins today is mass spectrometry (MS). MS based proteomics is based on the measurement of the masses of charged peptide ions in a gas-phase. The peptide amino acid sequence can be deduced, and matching proteins can be found, using software to correlate MS-data with sequence database information. Quantitative proteomics allow the estimation of the absolute or relative abundance of a certain protein in a sample. The label-free quantification methods use the intrinsic MS-peptide signals in the calculation of the quantitative values enabling the comparison of peptide signals from numerous patient samples. In this work, a quantitative MS methodology was established to study aromatase overexpressing (AROM+) male mouse liver and ovarian endometriosis tissue samples. The workflow of label-free quantitative proteomics was optimized in terms of sensitivity and robustness, allowing the quantification of 1500 proteins with a low coefficient of variance in both sample types. Additionally, five statistical methods were evaluated for the use with label-free quantitative proteomics data. The proteome data was integrated with other omics datasets, such as mRNA microarray and metabolite data sets. As a result, an altered lipid metabolism in liver was discovered in male AROM+ mice. The results suggest a reduced beta oxidation of long chain phospholipids in the liver and increased levels of pro-inflammatory fatty acids in the circulation in these mice. Conversely, in the endometriosis tissues, a set of proteins highly specific for ovarian endometrioma were discovered, many of which were under the regulation of the growth factor TGF-β1. This finding supports subsequent biomarker verification in a larger number of endometriosis patient samples.Siirretty Doriast

The gut microbial metabolite formate exacerbates colorectal cancer progression

The gut microbiome is a key player in the immunomodulatory and protumorigenic microenvironment during colorectal cancer (CRC), as different gut-derived bacteria can induce tumour growth. However, the crosstalk between the gut microbiome and the host in relation to tumour cell metabolism remains largely unexplored. Here we show that formate, a metabolite produced by the CRC-associated bacterium Fusobacterium nucleatum, promotes CRC development. We describe molecular signatures linking CRC phenotypes with Fusobacterium abundance. Cocultures of F. nucleatum with patient-derived CRC cells display protumorigenic effects, along with a metabolic shift towards increased formate secretion and cancer glutamine metabolism. We further show that microbiome-derived formate drives CRC tumour invasion by triggering AhR signalling, while increasing cancer stemness. Finally, F. nucleatum or formate treatment in mice leads to increased tumour incidence or size, and Th17 cell expansion, which can favour proinflammatory profiles. Moving beyond observational studies, we identify formate as a gut-derived oncometabolite that is relevant for CRC progression

Proteomics in thrombosis research

A State of the Art lecture titled “Proteomics in Thrombosis Research” was presented at the ISTH Congress in 2021. In clinical practice, there is a need for improved plasma biomarker-based tools for diagnosis and risk prediction of venous thromboembolism (VTE). Analysis of blood, to identify plasma proteins with potential utility for such tools, could enable an individualized approach to treatment and prevention. Technological advances to study the plasma proteome on a large scale allows broad screening for the identification of novel plasma biomarkers, both by targeted and nontargeted proteomics methods. However, assay limitations need to be considered when interpreting results, with orthogonal validation required before conclusions are drawn. Here, we review and provide perspectives on the application of affinityand mass spectrometry-based methods for the identification and analysis of plasma protein biomarkers, with potential application in the field of VTE. We also provide a future perspective on discovery strategies and emerging technologies for targeted proteomics in thrombosis research. Finally, we summarize relevant new data on this topic, presented during the 2021 ISTH Congress

Biomedical analysis of formalin-fixed, paraffin-embedded tissue samples: The Holy Grail for molecular diagnostics

More than a century ago in 1893, a revolutionary idea about fixing biological tissue specimens was introduced by Ferdinand Blum, a German physician. Since then, a plethora of fixation methods have been investigated and used. Formalin fixation with paraffin embedment became the most widely used types of fixation and preservation method, due to its proper architectural conservation of tissue structures and cellular shape. The huge collection of formalin-fixed, paraffin-embedded (FFPE) sample archives worldwide holds a large amount of unearthed information about diseases that could be the Holy Grail in contemporary biomarker research utilizing analytical omics based molecular diagnostics. The aim of this review is to critically evaluate the omics options for FFPE tissue sample analysis in the molecular diagnostics field

Isolation and analysis of T cell receptors against a drug-selected secondary mutation in BCR-ABL for adoptive T cell therapy

Adoptive T cell therapy with T cell receptor (TCR) engineered T cells is a promising approach for cancer treatment. Target antigens are categorized into tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). TAAs are overexpressed on cancer cells and often shared between different cancers, however, targeting TAAs has the risk of inducing autoimmune responses. In contrast to TAAs, TSAs are expressed exclusively on cancer cells, but only some of them are shared between patients. Since drug-selected secondary mutations in chronic myeloid leukemia (CML) are both cancer-specific and shared between patients, they become attractive targets. CML is a myeloproliferative disease. Over 95% of CML cases are associated with Philadelphia (Ph) chromosome t(9;22). Such abnormal chromosome results in the BCR-ABL hybrid gene that encodes a constantly active tyrosine kinase. Currently, tyrosine kinase inhibitors (TKIs) are used as the first-line treatment, as they effectively avoid uncontrollable cell division by blocking the ATP binding site of the kinase. Nevertheless, drug resistance is often observed due to drug-selected secondary mutations that prevent the TKIs from binding to the BCR-ABL protein. As relapsed CML patients with compound mutations are resistant to most or all TKIs, TCR gene therapy could benefit these patients. In this project, we aimed to discover neoepitopes from drug-selected secondary mutations in CML and to generate neoepitope-specific TCRs for clinical use. ABabDII mice expressing a human TCR repertoire and HLA-A2 molecules were immunized with two selected mutant peptides and analyzed. Although the peptide comprising mutation T315I did not elicit immune responses, two specific TCRs against the peptide comprising mutation E255V (ABL-E255V) were isolated from ABabDII mice. The T9141 TCR exhibiting a superior avidity was further analyzed for off-target toxicity. As no allo- and cross-reactivities were detected, T9141 was constructed to express minimal murinized human TCR constant region (T9141-mmc) as in the clinic. The expression and the functionality of T9141-mmc were comparable to T9141. Furthermore, ABL-E255V was demonstrated to be endogenously processed and presented by the ABL-minigene-E255V-transduced cancer cell lines. Taking a step forward, we introduced the point mutation E255V into Ph+ CML cell lines to simulate the antigen expression of blast cells in CML patients. Although no T cell responses against the genome-edited BV173 cells were found, detectable T cell responses against the genome-edited K562-A2 cells were observed, suggesting that the induced T cell responses varied with the BCR-ABL expression level in CML cell lines. Accordingly, the potency of T9141 could differ among CML patients. Therefore, it is recommended to analyze the BCR-ABL expression level in corresponding CML patients, which should allow us to evaluate the efficacy of TCR gene therapy with T9141. Taken together, T9141 is optimal for TCR gene therapy; ABL-E255V could be a potential target