UVB-Induced Tumor Heterogeneity Diminishes Immune Response in Melanoma.

Although clonal neo-antigen burden is associated with improved response to immune therapy, the functional basis for this remains unclear. Here we study this question in a novel controlled mouse melanoma model that enables us to explore the effects of intra-tumor heterogeneity (ITH) on tumor aggressiveness and immunity independent of tumor mutational burden. Induction of UVB-derived mutations yields highly aggressive tumors with decreased anti-tumor activity. However, single-cell-derived tumors with reduced ITH are swiftly rejected. Their rejection is accompanied by increased T cell reactivity and a less suppressive microenvironment. Using phylogenetic analyses and mixing experiments of single-cell clones, we dissect two characteristics of ITH: the number of clones forming the tumor and their clonal diversity. Our analysis of melanoma patient tumor data recapitulates our results in terms of overall survival and response to immune checkpoint therapy. These findings highlight the importance of clonal mutations in robust immune surveillance and the need to quantify patient ITH to determine the response to checkpoint blockade.

Measuring the Efficiency of Purging by non-random Mating in Human Populations.

Human populations harbor a high concentration of deleterious genetic variants. Here, we tested the hypothesis that non-random mating practices affect the distribution of these variants, through exposure in the homozygous state, leading to their purging from the population gene pool. To do so, we produced whole-genome sequencing data for two pairs of Asian populations exhibiting different alliance rules and rates of inbreeding, but with similar effective population sizes. The results show that populations with higher rates of inbred matings do not purge deleterious variants more efficiently. Purging therefore has a low efficiency in human populations, and different mating practices lead to a similar mutational load.

The evolutionary dynamics of local adaptations under genetic rescue is determined by mutational load and polygenicity.

Inbred populations often suffer from increased mutational load and reduced fitness due to lower efficacy of purifying selection in groups with small effective population sizes. Genetic rescue (GR) is a conservation tool that is studied and deployed with the aim of increasing the fitness of such inbred populations by assisted migration of individuals from closely related outbred populations. The success of GR depends on several factors-such as their demographic history and distribution of dominance effects of mutations-that may vary across populations. While we understand the impact of these factors on the dynamics of GR, their impact on local adaptations remains unclear. To this end, we conduct a population genetics simulation study to evaluate the impact of trait complexity (Mendelian vs. polygenic), dominance effects, and demographic history on the efficacy of GR. We find that the impact on local adaptations depends highly on the mutational load at the time of GR, which is in turn shaped dynamically by interactions between demographic history and dominance effects of deleterious variation. Over time local adaptations are generally restored post-GR, though in the short term they are often compromised in the process of purging deleterious variation. We also show that while local adaptations are almost always fully restored, the degree to which ancestral genetic variation affecting the trait is replaced by donor variation can vary drastically and is especially high for complex traits. Our results provide insights on the impact of GR on trait evolution and considerations for the practical implementation of GR.

Genomic Impact of Whaling in North Atlantic Fin Whales.

It is generally recognized that large-scale whaling in the 19th and 20th century led to a substantial reduction of the size of many cetacean populations, particularly those of the baleen whales (Mysticeti). The impact of these operations on genomic diversity of one of the most hunted whales, the fin whale (Balaenoptera physalus), has remained largely unaddressed because of the paucity of adequate samples and the limitation of applicable techniques. Here, we have examined the effect of whaling on the North Atlantic fin whale based on genomes of 51 individuals from Icelandic waters, representing three temporally separated intervals, 1989, 2009 and 2018 and provide a reference genome for the species. Demographic models suggest a noticeable drop of the effective population size of the North Atlantic fin whale around a century ago. The present results suggest that the genome-wide heterozygosity is not markedly reduced and has remained comparable with other baleen whale species. Similarly, there are no signs of apparent inbreeding, as measured by the proportion of long runs of homozygosity, or of a distinctively increased mutational load, as measured by the amount of putative deleterious mutations. Compared with other baleen whales, the North Atlantic fin whale appears to be less affected by anthropogenic influences than other whales such as the North Atlantic right whale, consistent with the presence of long runs of homozygosity and higher levels of mutational load in an otherwise more heterozygous genome. Thus, genome-wide assessments of other species and populations are essential for future, more specific, conservation efforts.

Signaling Pathways in the Pathogenesis of Barrett's Esophagus and Esophageal Adenocarcinoma.

Barrett's esophagus (BE) is a premalignant lesion that can develop into esophageal adenocarcinoma (EAC). The development of Barrett's esophagus is caused by biliary reflux, which causes extensive mutagenesis in the stem cells of the epithelium in the distal esophagus and gastro-esophageal junction. Other possible cellular origins of BE include the stem cells of the mucosal esophageal glands and their ducts, the stem cells of the stomach, residual embryonic cells and circulating bone marrow stem cells. The classical concept of healing a caustic lesion has been replaced by the concept of a cytokine storm, which forms an inflammatory microenvironment eliciting a phenotypic shift toward intestinal metaplasia of the distal esophagus. This review describes the roles of the NOTCH, hedgehog, NF-κB and IL6/STAT3 molecular pathways in the pathogenesis of BE and EAC.

Comprehensive Genetic Study of Malignant Cervical Paraganglioma.

Malignant middle ear paraganglioma (MEPGL) is an exceedingly rare tumor of the neuroendocrine system. In general, MEPGLs represent as slow growing and hypervascularized benign neoplasms. The genetic basis of MEPGL tumorigenesis has been poorly investigated. We report a case of malignant MEPGL accompanied by the comprehensive genetic analysis of the primary tumor and metastasis. Based on whole-exome sequencing data, the germline pathogenic mutation p.R230H in the SDHB gene, encoding for subunit B of mitochondrial complex II, was found in a patient. Analysis of somatic mutation spectra revealed five novel variants in different genes, including a potentially deleterious variant in UNC13C that was common for the tumor and metastasis. Identified somatic variants clustered into SBS1 and SBS5 mutational signatures. Of note, the primary tumor was characterized by Ki-67 4% and had an elevated mutational load (1.4/Mb); the metastasis' mutational load was about 4.5 times higher (6.4/Mb). In addition, we revealed somatic loss of the wild-type SDHB allele, as well as loss of heterozygosity (LOH) at the 11p locus. Thus, germline mutation in SDHB combined with somatic LOH seem to be drivers that lead to the tumor's initiation and progression. Other somatic changes identified can be additional disease-causing factors. Obtained results expand our understanding of molecular genetic mechanisms associated with the development of this rare tumor.

Genomic trajectories of a near-extinction event in the Chatham Island black robin.

BACKGROUND: Understanding the micro--evolutionary response of populations to demographic declines is a major goal in evolutionary and conservation biology. In small populations, genetic drift can lead to an accumulation of deleterious mutations, which will increase the risk of extinction. However, demographic recovery can still occur after extreme declines, suggesting that natural selection may purge deleterious mutations, even in extremely small populations. The Chatham Island black robin (Petroica traversi) is arguably the most inbred bird species in the world. It avoided imminent extinction in the early 1980s and after a remarkable recovery from a single pair, a second population was established and the two extant populations have evolved in complete isolation since then. Here, we analysed 52 modern and historical genomes to examine the genomic consequences of this extreme bottleneck and the subsequent translocation. RESULTS: We found evidence for two-fold decline in heterozygosity and three- to four-fold increase in inbreeding in modern genomes. Moreover, there was partial support for temporal reduction in total load for detrimental variation. In contrast, compared to historical genomes, modern genomes showed a significantly higher realised load, reflecting the temporal increase in inbreeding. Furthermore, the translocation induced only small changes in the frequency of deleterious alleles, with the majority of detrimental variation being shared between the two populations. CONCLUSION: Our results highlight the dynamics of mutational load in a species that recovered from the brink of extinction, and show rather limited temporal changes in mutational load. We hypothesise that ancestral purging may have been facilitated by population fragmentation and isolation on several islands for thousands of generations and may have already reduced much of the highly deleterious load well before human arrival and introduction of pests to the archipelago. The majority of fixed deleterious variation was shared between the modern populations, but translocation of individuals with low mutational load could possibly mitigate further fixation of high-frequency deleterious variation.

The Counteracting Effects of Demography on Functional Genomic Variation: The Roma Paradigm.

Demographic history plays a major role in shaping the distribution of genomic variation. Yet the interaction between different demographic forces and their effects in the genomes is not fully resolved in human populations. Here, we focus on the Roma population, the largest transnational ethnic minority in Europe. They have a South Asian origin and their demographic history is characterized by recent dispersals, multiple founder events, and extensive gene flow from non-Roma groups. Through the analyses of new high-coverage whole exome sequences and genome-wide array data for 89 Iberian Roma individuals together with forward simulations, we show that founder effects have reduced their genetic diversity and proportion of rare variants, gene flow has counteracted the increase in mutational load, runs of homozygosity show ancestry-specific patterns of accumulation of deleterious homozygotes, and selection signals primarily derive from preadmixture adaptation in the Roma population sources. The present study shows how two demographic forces, bottlenecks and admixture, act in opposite directions and have long-term balancing effects on the Roma genomes. Understanding how demography and gene flow shape the genome of an admixed population provides an opportunity to elucidate how genomic variation is modeled in human populations.

Environment dependence of the expression of mutational load and species' range limits.

Theoretical and empirical research on the causes of species' range limits suggest the contribution of several intrinsic and extrinsic factors, with potentially complex interactions among them. An intrinsic factor proposed by recent theory is mutational load increasing towards range edges because of genetic drift. Furthermore, environmental quality may decline towards range edges and enhance the expression of load. Here, we tested whether the expression of mutational load associated with range limits in the North American plant Arabidopsis lyrata was enhanced under stressful environmental conditions by comparing the performance of within- versus between-population crosses at common garden sites across the species' distribution and beyond. Heterosis, reflecting the expression of load, increased with heightened estimates of genomic load and with environmental stress caused by warming, but the interaction was not significant. We conclude that range-edge populations suffer from a twofold genetic Allee effect caused by increased mutational load and stress-dependent load linked to general heterozygote deficiency, but there is no synergistic effect between them.

Interacting Genetic Lesions of Melanoma in the Tumor Microenvironment: Defining a Viable Therapy.

Melanoma is the most aggressive form of skin cancer with an estimated 106,110 newly diagnosed cases in the United States of America in 2021 leading to an approximated 7180 melanoma-induced deaths. Cancer typically arises from an accumulation of somatic mutations and can be associated with mutagenic or carcinogenic exposure. A key characteristic of melanoma is the extensive somatic mutation rate of 16.8 mutations/Mb, which is largely attributed to UV exposure. Bearing the highest mutational load, many of them occur in key driver pathways, most commonly the BRAFV600E in the mitogen-activated protein kinase (MAPK) pathway. This driver mutation is targeted clinically with FDA-approved therapies using small molecule inhibitors of oncogenic BRAFV600E and MEK, which has greatly expanded therapeutic intervention following a melanoma diagnosis. Up until 2011, therapeutic options for metastatic melanoma were limited, and treatment typically fell under the spectrum of surgery, radiotherapy, and chemotherapy.Attributed to the extensive mutation rate, as well as having the highest number of neoepitopes, melanoma is deemed to be extremely immunogenic. However, despite this highly immunogenic nature, melanoma is notorious for inducing an immunosuppressive microenvironment which can be relieved by checkpoint inhibitor therapy. The two molecules currently approved clinically are ipilimumab and nivolumab, which target the molecules CTLA-4 and PD-1, respectively.A plethora of immunomodulatory molecules exist, many with redundant functions. Additionally, these molecules are expressed not only by immune cells but also by tumor cells within the tumor microenvironment. Tumor profiling of these cell surface checkpoint molecules is necessary to optimize a clinical response. The presence of immunomodulatory molecules in melanoma, using data from The Cancer Genome Atlas and validation of expression in two model systems, human melanoma tissues and patient-derived melanoma cells, revealed that the expression levels of B and T lymphocyte attenuator (BTLA), TIM1, and CD226, concurrently with the BRAFV600E mutation status, significantly dictated overall survival in melanoma patients. These molecules, along with herpesvirus entry mediator (HVEM) and CD160, two molecules that are a part of the HVEM/BTLA/CD160 axis, had a higher expression in human melanoma tissues when compared to normal skin melanocytes and have unique roles to play in T cell activation. New links are being uncovered between the expression of immunomodulatory molecules and the BRAFV600E genetic lesion in melanoma. Small molecule inhibitors of the MAPK pathway regulate the surface expression of this multifaceted molecule, making BTLA a promising target for immuno-oncology to be targeted in combination with small molecule inhibitors, potentially alleviating T regulatory cell activation and improving patient prognosis.

Immunotherapy for Neuro-oncology.

Immunotherapy has changed the landscape of treatment of many solid and hematological malignancies and is at the forefront of cancer breakthroughs. Several circumstances unique to the central nervous system (CNS) such as limited space for an inflammatory response, difficulties with repeated sampling, corticosteroid use for management of cerebral edema, and immunosuppressive mechanisms within the tumor and brain parenchyma have posed challenges in clinical development of immunotherapy for intracranial tumors. Nonetheless, the success of immunotherapy in brain metastases (BMs) from solid cancers such as melanoma and non-small cell lung cancer (NSCLC) proves that the CNS is not an immune-privileged organ and is capable of initiating and regulating immune responses that lead to tumor control. However, the development of immunotherapeutics for the most malignant primary brain tumor, glioblastoma (GBM), has been challenging due to systemic and profound tumor-mediated immunosuppression unique to GBM, intratumoral and intertumoral heterogeneity, and lack of stably expressed clonal antigens. Here, we review recent advances in the field of immunotherapy for neuro-oncology with a focus on BM, GBM, and rare CNS cancers.

Criticality in tumor evolution and clinical outcome.

How mutation and selection determine the fitness landscape of tumors and hence clinical outcome is an open fundamental question in cancer biology, crucial for the assessment of therapeutic strategies and resistance to treatment. Here we explore the mutation-selection phase diagram of 6,721 tumors representing 23 cancer types by quantifying the overall somatic point mutation load (ML) and selection (dN/dS) in the entire proteome of each tumor. We show that ML strongly correlates with patient survival, revealing two opposing regimes around a critical point. In low-ML cancers, a high number of mutations indicates poor prognosis, whereas high-ML cancers show the opposite trend, presumably due to mutational meltdown. Although the majority of cancers evolve near neutrality, deviations are observed at extreme MLs. Melanoma, with the highest ML, evolves under purifying selection, whereas in low-ML cancers, signatures of positive selection are observed, demonstrating how selection affects tumor fitness. Moreover, different cancers occupy specific positions on the ML-dN/dS plane, revealing a diversity of evolutionary trajectories. These results support and expand the theory of tumor evolution and its nonlinear effects on survival.

Immune recognition of irradiated cancer cells.

Ionizing irradiation has been extensively employed for the clinical management of solid tumors, with therapeutic or palliative intents, for decades. Until recently, radiation therapy (RT) was believed to mediate antineoplastic activity mostly (if not only) as a consequence of cancer cell-intrinsic effects. Indeed, the macromolecular damage imposed to malignant cells by RT initiates one or multiple signal transduction cascades that drive a permanent proliferative arrest (cellular senescence) or regulated cell death. Both these phenomena show a rather linear dose-response correlation. However, RT also mediates consistent immunological activity, not only as an "on-target effect" originating within irradiated cancer cells, but also as an "off-target effect" depending on the interaction between RT and stromal, endothelial, and immune components of the tumor microenvironment. Interestingly, the immunological activity of RT does not exhibit linear dose-response correlation. Here, we discuss the mechanisms whereby RT alters the capacity of the immune system to recognize and eliminate irradiated cancer cells, either as an "on-target" or as on "off-target" effect. In particular, we discuss the antagonism between the immunostimulatory and immunosuppressive effects of RT as we delineate combinatorial strategies to boost the former at the expenses of the latter.

In vivo vaccination with cell line-derived whole tumor lysates: neoantigen quality, not quantity matters.

BACKGROUND: Cancer vaccines provide a complex source of neoantigens. Still, increasing evidence reveals that the neoantigen quality rather than the quantity is predictive for treatment outcome. METHODS: Using the preclinical Mlh1-/- tumor model, we performed a side-by side comparison of two autologous cell-line derived tumor lysates (namely 328 and A7450 T1 M1) harboring different tumor mutational burden (TMB; i.e. ultra-high: 328; moderate-high: A7450 T1 M1). Mice received repetitive prophylactic or therapeutic applications of the vaccine. Tumor incidence, immune responses and tumor microenvironment was examined. RESULTS: Both tumor cell lysates delayed tumor formation in the prophylactic setting, with the A7450 T1 M1 lysate being more effective in decelerating tumor growth than the 328 lysate (median overall survival: 37 vs. 25 weeks). Comparable results were achieved in therapeutic setting and could be traced back to antigen-driven immune stimulation. Reactive T cells isolated from A7450 T1 M1-treated mice recognized autologous Mlh1-/- tumor cells in IFNγ ELISpot, but likewise YAC-1 cells, indicative for stimulation of both arms of the immune system. By deciphering local effects, vaccines shaped the tumor microenvironment differently. While A7450 T1 M1 prophylactically vaccinated tumors harbored low numbers of myeloid-derived suppressor cells (MDSC) and elevated CD8-T cell infiltrates, vaccination with the 328 lysate evoked MDSC infiltration. Similar effects were seen in the therapeutic setting with stable disease induction only upon A7450 T1 M1 vaccination. Untangling individual response profiles revealed strong infiltration with LAG3+ and PD-L1+ immune cells when treatments failed, but almost complete exclusion of checkpoint-expressing lymphocytes in long-term survivors. CONCLUSIONS: By applying two tumor cell lysates we demonstrate that neoantigen quality outranks quantity. This should be considered prior to designing cancer vaccine-based combination approaches.

The Multifaceted Nature of Tumor Microenvironment in Breast Carcinomas.

Heterogeneity in breast carcinomas can be appreciated at various levels, from morphology to molecular alterations, and there are well-known genotypic-phenotypic correlations. Clinical decision-making is strictly focused on the evaluation of tumor cells and is based on the assessment of hormone receptors and of the HER2 status, by means of a combination of immunohistochemical and in situ hybridization techniques. The tumor microenvironment (TME) also shows a multifaceted nature stemming from the different actors populating the intratumoral and the peritumoral stroma of breast carcinomas. Of note, we have now evidence that tumor-infiltrating lymphocytes (TILs) are clinically meaningful as their quantification in the intratumoral stroma strongly correlates with good prognosis, in particular in triple-negative and HER2-positive breast cancer patients. Nevertheless, TILs are just one of the many actors orchestrating the complexity of the TME, which is populated by immune and non-immune cells (cancer-associated fibroblasts, cancer-associated adipocytes), as well as non-cellular components such as chemical inflammation mediators. In this review article we will overview the main features of the distinct cell compartments by discussing (i) the potential impact the TME may have on the prognostic stratification of breast cancers and (ii) the possible predictive value of some markers in the context of immunotherapy in light of the recent results of phase III studies in advanced and early triple-negative breast cancer patients.

Immunotherapy for Neuro-Oncology.

Immunotherapy has changed the landscape of treatment of many solid and hematological malignancies and is at the forefront of cancer breakthroughs. Several circumstances unique to the central nervous system (CNS) such as limited space for an inflammatory response, difficulties with repeated sampling, corticosteroid use for management of cerebral edema, and immunosuppressive mechanisms within the tumor and brain parenchyma have posed challenges in clinical development of immunotherapy for intracranial tumors. Nonetheless, the success of immunotherapy in brain metastases (BMs) from solid cancers such as melanoma and non-small cell lung cancer (NSCLC) proves that the CNS is not an immune-privileged organ and is capable of initiating and regulating immune responses that lead to tumor control. However, the development of immunotherapeutics for the most malignant primary brain tumor, glioblastoma (GBM), has been challenging due to systemic and profound tumor-mediated immunosuppression unique to GBM, intratumoral and intertumoral heterogeneity, low mutation burden, and lack of stably expressed clonal antigens. Here, we review recent advances in the field of immunotherapy for neuro-oncology with a focus on BM and GBM.

Emerging biomarkers for the combination of radiotherapy and immune checkpoint blockers.

Over the past few years, multiple immune checkpoint blockers (ICBs) have achieved unprecedented clinical success and have been approved by regulatory agencies for the treatment of an increasing number of malignancies. However, only a limited fraction of patients responds to ICBs employed as a standalone intervention, calling for the development of combinatorial regimens. Radiation therapy (RT) stands out as a very promising candidate for this purpose. Indeed, RT mediates antineoplastic effects not only by cytotoxic and cytostatic mechanisms, but also by modulating immunological functions, both locally (within the irradiated field) and systemically. As combinatorial regimens involving RT and ICBs are being developed and clinically tested at an accelerating pace, it is paramount to identify biomarkers that reliably predict the likelihood of individual patients to respond. Here, we discuss emerging biomarkers that may potentially predict the response of cancer patients to RT plus ICBs.

Size matters: Dissecting key parameters for panel-based tumor mutational burden analysis.

Tumor mutational burden (TMB) represents a new determinant of clinical benefit from immune checkpoint blockade that identifies responders independent of PD-L1 expression levels and is currently being explored in clinical trials. Although TMB can be measured directly by comprehensive genomic approaches such as whole-genome and exome sequencing, broad availability, short turnaround times, costs and amenability to formalin-fixed and paraffin-embedded tissue support the use of gene panel sequencing for approximating TMB in routine diagnostics. However, data on the parameters influencing panel-based TMB estimation are limited. Here, we report an extensive in silico analysis of the TCGA data set that simulates various panel sizes and compositions. We demonstrate that panel size is a critical parameter that influences confidence intervals (CIs) and cutoff values as well as important test parameters including sensitivity, specificity, and positive predictive value. Moreover, we evaluate the Illumina TSO500 panel, which will be made available for TMB estimation, and propose dynamic, entity-specific cutoff values based on current clinical trial data. Optimizing the cost-benefit ratio, our data suggest that panels between 1.5 and 3 Mbp are ideally suited to estimate TMB with small CIs, whereas smaller panels tend to deliver imprecise TMB estimates for low to moderate TMB (0-30 muts/Mbp), connected with insufficient separation of hypermutated tumors from non-hypermutated tumors.

Measurement of tumor mutational burden (TMB) in routine molecular diagnostics: in silico and real-life analysis of three larger gene panels.

Assessment of Tumor Mutational Burden (TMB) for response stratification of cancer patients treated with immune checkpoint inhibitors is emerging as a new biomarker. Commonly defined as the total number of exonic somatic mutations, TMB approximates the amount of neoantigens that potentially are recognized by the immune system. While whole exome sequencing (WES) is an unbiased approach to quantify TMB, implementation in diagnostics is hampered by tissue availability as well as time and cost constrains. Conversely, panel-based targeted sequencing is nowadays widely used in routine molecular diagnostics, but only very limited data are available on its performance for TMB estimation. Here, we evaluated three commercially available larger gene panels with covered genomic regions of 0.39 Megabase pairs (Mbp), 0.53 Mbp and 1.7 Mbp using i) in silico analysis of TCGA (The Cancer Genome Atlas) data and ii) wet-lab sequencing of a total of 92 formalin-fixed and paraffin-embedded (FFPE) cancer samples grouped in three independent cohorts (non-small cell lung cancer, NSCLC; colorectal cancer, CRC; and mixed cancer types) for which matching WES data were available. We observed a strong correlation of the panel data with WES mutation counts especially for the gene panel >1Mbp. Sensitivity and specificity related to TMB cutpoints for checkpoint inhibitor response in NSCLC determined by wet-lab experiments well reflected the in silico data. Additionally, we highlight potential pitfalls in bioinformatics pipelines and provide recommendations for variant filtering. In summary, our study is a valuable data source for researchers working in the field of immuno-oncology as well as for diagnostic laboratories planning TMB testing.

Germline Stem Cell Competition, Mutation Hot Spots, Genetic Disorders, and Older Fathers.

Some de novo human mutations arise at frequencies far exceeding the genome average mutation rate. Examples include the common mutations at one or a few sites in the genes that cause achondroplasia, Apert syndrome, multiple endocrine neoplasia type 2B, and Noonan syndrome. These mutations are recurrent, provide a gain of function, are paternally derived, and are more likely to be transmitted as the father ages. Recent experiments have tested whether the high mutation frequencies are due to an elevated mutation rate per cell division, as expected, or to an advantage of the mutant spermatogonial stem cells over wild-type stem cells. The evidence, which includes the surprising discovery of testis mutation clusters, rules out the former model but not the latter. We propose how the mutations might alter spermatogonial stem cell function and discuss how germline selection contributes to the paternal age effect, the human mutational load, and adaptive evolution.