HLA-I diversity and tumor mutational burden by comprehensive next-generation sequencing as predictive biomarkers for the treatment of non-small cell lung cancer with PD-(L)1 inhibitors.

OBJECTIVES: Immune checkpoint inhibitors (ICIs) improved outcomes in non-small cell lung cancer (NSCLC) patients. We report the predictive utility of human leukocyte antigen class I (HLA-I) diversity and tumor mutational burden (TMB) by comprehensive next-generation sequencing. METHODS: 126 patients were included. TMB high was defined as ≥ 10 nonsynonymous mutations/Mb. Patients exhibit high HLA-I diversity if at least one locus was in the upper 15th percentile for DNA alignment scores. RESULTS: No difference in response rate (RR; 44.4% versus 30.9%; p = 0.1741) or 6-month survival rate (SR; 75.6% versus 77.8%; p = 0.7765) was noted between HLA-I high diversity and low diversity patients. HLA-I high diversity patients did significantly more often exhibit durable clinical benefit (DCB), defined as response or stable disease lasting minimally 6 months (64.4% [29/45] versus 43.2% [35/81]; p = 0.0223). TMB high patients exhibited higher RR (49.1% versus 25.4%; p = 0.0084) and SR 6 months after start ICI (85.5% versus 70.4%; p = 0.0468) than TMB low patients. The proportion of patients with DCB, did not differ significantly between TMB high and low subgroups (60.0% [33/55] versus 42.3% [30/71]; p = 0.0755). Patients with combined dual high TMB and HLA-I diversity had higher RR (63.2% versus 22.2%; p = 0.0033), but SR at 6 months did not differ significantly (84.2% versus 64,4%; p = 0.1536). A significantly higher rate of patients experienced DCB in dual high compared to the dual low group (73.7% [14/19] versus 35.6% [16/45]; p = 0.0052). Triple positive patients (high TMB and HLA-I diversity and PD-L1 positive) had higher RR (63.6% versus 0.0%; p = 0.0047) and SR at 6 months (100% versus 66.7%; p = 0.0378) compared to triple-negative patients. CONCLUSION: HLA-I diversity was able to predict durable clinical benefit in ICI treated NSCLC patients, but failed to confirm as a predictor of response or survival. TMB confirmed as a predictive biomarker.

Diagnostic Validation of a Comprehensive Targeted Panel for Broad Mutational and Biomarker Analysis in Solid Tumors.

The use of targeted Next Generation Sequencing (NGS) for the diagnostic screening of somatic variants in solid tumor samples has proven its high clinical value. Because of the large number of ongoing clinical trials for a multitude of variants in a growing number of genes, as well as the detection of proven and emerging pan-cancer biomarkers including microsatellite instability (MSI) and tumor mutation burden (TMB), the currently employed diagnostic gene panels will become vastly insufficient in the near future. Here, we describe the validation and implementation of the hybrid capture-based comprehensive TruSight Oncology (TSO500) assay that is able to detect single-nucleotide variants (SNVs) and subtle deletions and insertions (indels) in 523 tumor-associated genes, copy-number variants (CNVs) of 69 genes, fusions with 55 cancer driver genes, and MSI and TMB. Extensive validation of the TSO500 assay was performed on DNA or RNA from 170 clinical samples with neoplastic content down to 10%, using multiple tissue and specimen types. Starting with 80 ng DNA and 40 ng RNA extracted from formalin-fixed and paraffine-embedded (FFPE) samples revealed a precision and accuracy >99% for all variant types. The analytical sensitivity and specificity were at least 99% for SNVs, indels, CNVs, MSI, and gene rearrangements. For TMB, only values around the threshold could yield a deviating outcome. The limit-of-detection for SNVs and indels was well below the set threshold of 5% variant allele frequency (VAF). This validated comprehensive genomic profiling assay was then used to screen 624 diagnostic samples, and its success rate for adoption in a clinical diagnostic setting of broad solid tumor screening was assessed on this cohort.

The Dynamics of Nucleotide Variants in the Progression from Low-Intermediate Myeloma Precursor Conditions to Multiple Myeloma: Studying Serial Samples with a Targeted Sequencing Approach.

Multiple myeloma (MM), or Kahler's disease, is an incurable plasma cell (PC) cancer in the bone marrow (BM). This malignancy is preceded by one or more asymptomatic precursor conditions, monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering multiple myeloma (SMM). The molecular mechanisms and exact cause of this progression are still not completely understood. In this study, the mutational profile underlying the progression from low-intermediate risk myeloma precursor conditions to MM was studied in serial BM smears. A custom capture-based sequencing platform was developed, including 81 myeloma-related genes. The clonal evolution of single nucleotide variants and short insertions and deletions was studied in serial BM smears from 21 progressed precursor patients with a median time of progression of six years. From the 21 patients, four patients had no variation in one of the 81 studied genes. Interestingly, in 16 of the 17 other patients, at least one variant present in MM was also detected in its precursor BM, even years before progression. Here, the variants were present in the pre-stage at a median of 62 months before progression to MM. Studying these paired BM samples contributes to the knowledge of the evolutionary genetic landscape and provides additional insight into the mutational behavior of mutant clones over time throughout progression.

Inhibition of the Glycolytic Activator PFKFB3 in Endothelium Induces Tumor Vessel Normalization, Impairs Metastasis, and Improves Chemotherapy.

Abnormal tumor vessels promote metastasis and impair chemotherapy. Hence, tumor vessel normalization (TVN) is emerging as an anti-cancer treatment. Here, we show that tumor endothelial cells (ECs) have a hyper-glycolytic metabolism, shunting intermediates to nucleotide synthesis. EC haplo-deficiency or blockade of the glycolytic activator PFKFB3 did not affect tumor growth, but reduced cancer cell invasion, intravasation, and metastasis by normalizing tumor vessels, which improved vessel maturation and perfusion. Mechanistically, PFKFB3 inhibition tightened the vascular barrier by reducing VE-cadherin endocytosis in ECs, and rendering pericytes more quiescent and adhesive (via upregulation of N-cadherin) through glycolysis reduction; it also lowered the expression of cancer cell adhesion molecules in ECs by decreasing NF-κB signaling. PFKFB3-blockade treatment also improved chemotherapy of primary and metastatic tumors.

Glycolytic regulation of cell rearrangement in angiogenesis.

During vessel sprouting, endothelial cells (ECs) dynamically rearrange positions in the sprout to compete for the tip position. We recently identified a key role for the glycolytic activator PFKFB3 in vessel sprouting by regulating cytoskeleton remodelling, migration and tip cell competitiveness. It is, however, unknown how glycolysis regulates EC rearrangement during vessel sprouting. Here we report that computational simulations, validated by experimentation, predict that glycolytic production of ATP drives EC rearrangement by promoting filopodia formation and reducing intercellular adhesion. Notably, the simulations correctly predicted that blocking PFKFB3 normalizes the disturbed EC rearrangement in high VEGF conditions, as occurs during pathological angiogenesis. This interdisciplinary study integrates EC metabolism in vessel sprouting, yielding mechanistic insight in the control of vessel sprouting by glycolysis, and suggesting anti-glycolytic therapy for vessel normalization in cancer and non-malignant diseases.

The Cancer Cell Oxygen Sensor PHD2 Promotes Metastasis via Activation of Cancer-Associated Fibroblasts.

Several questions about the role of the oxygen sensor prolyl-hydroxylase 2 (PHD2) in cancer have not been addressed. First, the role of PHD2 in metastasis has not been studied in a spontaneous tumor model. Here, we show that global PHD2 haplodeficiency reduced metastasis without affecting tumor growth. Second, it is unknown whether PHD2 regulates cancer by affecting cancer-associated fibroblasts (CAFs). We show that PHD2 haplodeficiency reduced metastasis via two mechanisms: (1) by decreasing CAF activation, matrix production, and contraction by CAFs, an effect that surprisingly relied on PHD2 deletion in cancer cells, but not in CAFs; and (2) by improving tumor vessel normalization. Third, the effect of concomitant PHD2 inhibition in malignant and stromal cells (mimicking PHD2 inhibitor treatment) is unknown. We show that global PHD2 haplodeficiency, induced not only before but also after tumor onset, impaired metastasis. These findings warrant investigation of PHD2's therapeutic potential.

Role of PFKFB3-driven glycolysis in vessel sprouting.

Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.

Predicting the future: towards symbiotic computational and experimental angiogenesis research.

Understanding the fundamental organisational principles underlying the complex and multilayered process of angiogenesis is the mutual aim of both the experimental and theoretical angiogenesis communities. Surprisingly, these two fields have in the past developed in near total segregation, with neither fully benefiting from the other. However, times are changing and here we report on the new direction that angiogenesis research is taking, where from well-integrated collaborations spring new surprises, experimental predictions and research avenues. We show that several successful ongoing collaborations exist in the angiogenesis field and analyse what aspects of their approaches led them to achieve novel and impactful biological insight. We conclude that there are common elements we can learn from for the future, and provide a list of guidelines to building a successful collaborative venture. Specifically, we find that a near symbiosis of computation with experimentation reaps the most impactful results by close cyclical feedback and communication between the two disciplines resulting in continual refinement of models, experimental directions and our understanding. We discuss high impact examples of predictive modelling from the wider, more established integrated scientific domains and conclude that the angiogenesis community can do nothing but benefit from joining this brave new, integrated world.