Prospective manipulation of the gut microbiome with microbial ecosystem therapeutic 4 (MET4) in HPV-related locoregionally-advanced oropharyngeal cancer squamous cell carcinoma (LA-OPSCC) undergoing primary chemoradiation: ROMA2 study.

BACKGROUND: Gut microbiome modulation to boost antitumor immune responses is under investigation. METHODS: ROMA-2 evaluated the microbial ecosystem therapeutic (MET)-4 oral consortia, a mixture of cultured human stool-derived immune-responsiveness associated bacteria, given with chemoradiation (CRT) in HPV-related oropharyngeal cancer patients. Co-primary endpoints were safety and changes in stool cumulative MET-4 taxa relative abundance (RA) by 16SRNA sequencing. Stools and plasma were collected pre/post-MET-4 intervention for microbiome and metabolome analysis. RESULTS: Twenty-nine patients received ≥1 dose of MET-4 and were evaluable for safety: drug-related adverse events (AEs) occurred in 13/29 patients: all grade 1-2 except one grade 3 (diarrhea). MET-4 was discontinued early in 7/29 patients due to CRT-induced toxicity, and in 1/29 due to MET-4 AEs. Twenty patients were evaluable for ecological endpoints: there was no increase in stool MET-4 RA post-intervention but trended to increase in stage III patients (p = 0.06). MET-4 RA was higher in stage III vs I-II patients at week 4 (p = 0.03) and 2-month follow-up (p = 0.01), which correlated with changes in plasma and stool targeted metabolomics. CONCLUSIONS: ROMA-2 did not meet its primary ecologic endpoint, as no engraftment was observed in the overall cohort. Exploratory findings of engraftment in stage III patients warrants further investigation of microbiome interventions in this subgroup.

Transitions in oral and gut microbiome of HPV+ oropharyngeal squamous cell carcinoma following definitive chemoradiotherapy (ROMA LA-OPSCC study).

BACKGROUND: Oral and gut microbiomes have emerged as potential biomarkers in cancer. We characterised the oral and gut microbiomes in a prospective observational cohort of HPV+ oropharyngeal squamous cell carcinoma (OPSCC) patients and evaluated the impact of chemoradiotherapy (CRT). METHODS: Saliva, oropharyngeal swabs over the tumour site and stool were collected at baseline and post-CRT. 16S RNA and shotgun metagenomic sequencing were used to generate taxonomic profiles, including relative abundance (RA), bacterial density, α-diversity and ß-diversity. RESULTS: A total of 132 samples from 22 patients were analysed. Baseline saliva and swabs had similar taxonomic composition (R2 = 0.006; p = 0.827). Oropharyngeal swabs and stool taxonomic composition varied significantly by stage, with increased oral RA of Fusobacterium nucleatum observed in stage III disease (p < 0.05). CRT significantly reduced the species richness and increased the RA of gut-associated taxa in oropharyngeal swabs (p < 0.05), while it had no effect in stool samples. These findings remained significant when adjusted by stage, smoking status and antibiotic use. CONCLUSIONS: Baseline oral and gut microbiomes differ by stage in this HPV+ cohort. CRT caused a shift towards a gut-like microbiome composition in oropharyngeal swabs. Stage-specific features and the transitions in oral microbiome might have prognostic and therapeutic implications.

Tumor-Associated Microbiome: Where Do We Stand?

The study of the human microbiome in oncology is a growing and rapidly evolving field. In the past few years, there has been an exponential increase in the number of studies investigating associations of microbiome and cancer, from oncogenesis and cancer progression to resistance or sensitivity to specific anticancer therapies. The gut microbiome is now known to play a significant role in antitumor immune responses and in predicting the efficacy of immune-checkpoint inhibitors in cancer patients. Beyond the gut, the tumor-associated microbiome-microbe communities located either in the tumor or within its body compartment-seems to interact with the local microenvironment and the tumor immune contexture, ultimately impacting cancer progression and treatment outcome. However, pre-clinical research focusing on causality and mechanistic pathways as well as proof-of-concept studies are still needed to fully understand the potential clinical utility of microbiome in cancer patients. Moreover, there is a need for the standardization of methodology and the implementation of quality control across microbiome studies to allow for a better interpretation and greater comparability of the results reported between them. This review summarizes the accumulating evidence in the field and discusses the current and upcoming challenges of microbiome studies.

Modelling ground thermal regime in bordering (dis)continuous permafrost environments.

Permafrost controls geomorphological dynamics in maritime Antarctic ecosystems. Here, we analyze and model ground thermal regime in bordering conditions between continuous and discontinuous permafrost to better understand its relationship with the timing of glacial retreat. In February 2017, a transect including 10 sites for monitoring ground temperatures was installed in the eastern fringe of Byers Peninsula (Livingston Island, northern Antarctic Peninsula), together with one station recording air temperatures and snow thickness. The sites were selected following the Mid-Late Holocene deglaciation of the area at a distance ranging from 0.30 to 3.15 km from the current Rotch Dome glacier front. The transect provided data on the effects of topography, snow cover and the timing of ice-free exposure, on the ground thermal regime. From February 2017 to February 2019, the mean annual air temperature was -2.0 °C, which was >0.5 °C higher than 1986-2015 average in the Western Antarctic Peninsula region. Mean annual ground temperature at 10 cm depth varied between 0.3 and -1.1 °C, similar to the modelled Temperatures on the Top of the Permafrost (TTOP) that ranged from 0.06 ±â€¯0.08 °C to -1.33 ±â€¯0.07 °C. The positive average temperatures at the warmest site were related to the long-lasting presence of snow which favoured warmer ground temperatures and may trigger permafrost degradation. The role of other factors (topography, and timing of the deglaciation) explained intersite differences, but the overall effect was not as strong as snow cover.

Southward migration of the zero-degree isotherm latitude over the Southern Ocean and the Antarctic Peninsula: Cryospheric, biotic and societal implications.

The seasonal movement of the zero-degree isotherm across the Southern Ocean and Antarctic Peninsula drives major changes in the physical and biological processes around maritime Antarctica. These include spatial and temporal shifts in precipitation phase, snow accumulation and melt, thawing and freezing of the active layer of the permafrost, glacier mass balance variations, sea ice mass balance and changes in physiological processes of biodiversity. Here, we characterize the historical seasonal southward movement of the monthly near-surface zero-degree isotherm latitude (ZIL), and quantify the velocity of migration in the context of climate change using climate reanalyses and projections. From 1957 to 2020, the ZIL exhibited a significant southward shift of 16.8 km decade-1 around Antarctica and of 23.8 km decade-1 in the Antarctic Peninsula, substantially faster than the global mean velocity of temperature change of 4.2 km decade-1, with only a small fraction being attributed to the Southern Annular Mode (SAM). CMIP6 models reproduce the trends observed from 1957 to 2014 and predict a further southward migration around Antarctica of 24 ± 12 km decade-1 and 50 ± 19 km decade-1 under the SSP2-4.5 and SSP5-8.5 scenarios, respectively. The southward migration of the ZIL is expected to have major impacts on the cryosphere, especially on the precipitation phase, snow accumulation and in peripheral glaciers of the Antarctic Peninsula, with more uncertain changes on permafrost, ice sheets and shelves, and sea ice. Longer periods of temperatures above 0 °C threshold will extend active biological periods in terrestrial ecosystems and will reduce the extent of oceanic ice cover, changing phenologies as well as areas of productivity in marine ecosystems, especially those located on the sea ice edge.

Impact of climate change on snowpack dynamics in coastal Central-Western Greenland.

Snow patterns in ice-free areas of Greenland play important roles in ecosystems. Within a changing climate, a comprehensive understanding of the snow responses to climate change is of interest to anticipate forthcoming dynamics in these areas. In this study, we analyze the future snowpack evolution of a polar maritime Arctic location, Qeqertarsuaq (Disko Island, Central-Western Greenland). A physically-based snow model (FSM2) is validated and forced with CMIP6 projections for SSP2-4.5 and SSP5-8.5 greenhouse gasses emission scenarios, using two models: CanESM5 and MIROC6. The future snowpack evolution is assessed through four key seasonal (October to May) snow climate indicators: snow depth, snow days, snowfall fraction and ablation rate. Comparison against the observed air temperature for the reference climate period demonstrates superior accuracies for MIROC6 SSP2.4-5, with anomalies at 19 %, compared to CanESM5 SSP5.8-5 (25 %) and CanESM5 SSP2.4-5 (78 %). In terms of precipitation, CanESM5 SSP2.4-5 and SSP2.4-5 exhibit smaller anomalies against the observed data (5 %) in contrast to MIROC6 SSP2.4-5 (15 %) and MIROC6 SSP2.8-5 (17 %). Results demonstrate distinct snowpack responses to climate change depending on the model and emission scenario. For CanESM5, seasonal snow depth anomalies with respect to the reference period range from - 38 % (SSP2-4.5, 2040-2050 period) to - 74 % (SSP5-8.5, 2090-2100 period). MIROC6 projects lower snowpack reductions, with a decrease ranging from - 38 % (SSP2-4.5, 2040-2050 period) to - 57 % (SSP5-8.5, 2090-2100 period). Similar reductions are anticipated for snowfall and snow days. Changes in the snowpack evolution are primarily driven by positive trends in downwelling longwave radiation and air temperature. The projected increase in precipitation by the mid to late 21st century will lead to more frequent rain-on-snow events, intensifying snowpack melting. These findings help enhance the comprehension of future snow dynamics in the ice-free zones of Greenland, as well as the associated hydrological and ecological changes.

Immunotherapy in elderly head and neck cancer patients: a systematic review and meta-analysis.

Introduction: Over the past years, there has been a growing interest in the role of immunotherapy in locally advanced (LA) and recurrent and metastatic head and neck squamous cell carcinoma (R/M HNSCC). High-quality data from prospective trials are lacking for the elderly subpopulation. This systematic review and meta-analysis aims to review the efficacy and safety of immunotherapy in older patients. Methods: A systematic literature search was conducted. Randomized clinical trials providing outcome data on a subgroup of elderly (>65 years old) were available for meta-analysis. Primary outcomes of interest were OS and PFS for efficacy analysis. Results: Seven studies were included in the systematic review and four in the efficacy analysis. The pooled analysis of OS and PFS showed a consistent benefit (HR 0.78 and 0.91, respectively). Conclusions: Immunotherapy may be an effective and well-tolerated treatment option in the elderly population, but more prospective and randomized data are needed. Systematic Review Registration: PROSPERO (CRD42022333891).

Study of late toxicity biomarkers of locally advanced head and neck cancer patients treated with radiotherapy plus cisplatin or cetuximab points to the relevance of skin macrophages (TOX-TTCC-2015-01).

PURPOSE: Radiotherapy (RT) with concomitant cisplatin (CRT) or cetuximab (ERT) are accepted treatment options for locally advanced squamous cell carcinoma of the head and neck (LA-SCCHN). Long-term adverse events (AEs) have a vast impact on patients' quality of life. This study explored tissue biomarkers which could help predict late toxicity. METHODS/PATIENTS: Single-institution prospective study including patients aged ≥ 18 with histologically confirmed newly diagnosed LA-SCCHN treated with RT and either concomitant cisplatin q3w or weekly cetuximab, according to institutional protocols. All patients underwent pre- and post-treatment skin biopsies of neck regions included in the clinical target volume. Angiogenesis, macrophages, and extracellular matrix (ECM) markers were evaluated by immunohistochemistry (IHC). RESULTS: From April 15, 2016, to December 11, 2017; 31 patients were evaluated [CRT = 12 (38.7%) and ERT = 19 (61.3%)]. 27 patients (87%) had received induction chemotherapy. All patients finished RT as planned. IHC expression of vasculature (CD34) and collagen (Masson's Trichrome) did not differ significantly between and within CRT and ERT arms. Conversely, an increased CD68 and CD163 macrophage infiltration expression was observed after treatment, without significant impact of treatment modality. Patients with higher late toxicity showed lower expression of macrophage markers in pre-treatment samples compared with those with lower late toxicity, with statistically significant differences for CD68. CONCLUSIONS: Angiogenesis and ECM biomarkers did not differ significantly between CRT and ERT. Macrophage markers increased after both treatments and deserve further investigation as predictors of late toxicity in LA-SCCHN patients. [Protocol code: TOX-TTCC-2015-01/Spanish registry of clinical studies (REec): 2015-003012-21/Date of registration: 27/01/2016].

Cancer cell plasticity defines response to immunotherapy in cutaneous squamous cell carcinoma.

Immune checkpoint blockade (ICB) approaches have changed the therapeutic landscape for many tumor types. However, half of cutaneous squamous cell carcinoma (cSCC) patients remain unresponsive or develop resistance. Here, we show that, during cSCC progression in male mice, cancer cells acquire epithelial/mesenchymal plasticity and change their immune checkpoint (IC) ligand profile according to their features, dictating the IC pathways involved in immune evasion. Epithelial cancer cells, through the PD-1/PD-L1 pathway, and mesenchymal cancer cells, through the CTLA-4/CD80 and TIGIT/CD155 pathways, differentially block antitumor immune responses and determine the response to ICB therapies. Accordingly, the anti-PD-L1/TIGIT combination is the most effective strategy for blocking the growth of cSCCs that contain both epithelial and mesenchymal cancer cells. The expression of E-cadherin/Vimentin/CD80/CD155 proteins in cSCC, HNSCC and melanoma patient samples predicts response to anti-PD-1/PD-L1 therapy. Collectively, our findings indicate that the selection of ICB therapies should take into account the epithelial/mesenchymal features of cancer cells.

Circulating tumour DNA kinetics in recurrent/metastatic head and neck squamous cell cancer patients.

PURPOSE: Immune checkpoint blockade (ICB) has become a standard of care in the treatment of recurrent/metastatic head and neck squamous cell cancer (R/M HNSCC). However, only a subset of patients benefit from treatment. Quantification of plasma circulating tumour DNA (ctDNA) levels and on-treatment kinetics may permit real-time assessment of disease burden under selective pressures of treatment. PATIENTS AND METHODS: R/M HNSCC patients treated with systemic therapy, platinum-based chemotherapy (CT) or ICB, underwent serial liquid biopsy sampling. Biomarkers tested included ctDNA measured by CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) and markers of host inflammation measured by neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR). RESULTS: Among 53 eligible patients, 16 (30%) received CT, 30 (57%) ICB [anti-PD1/L1] monotherapy and 7 (13%) combination immunotherapy (IO). Median progression-free survival (PFS) and overall survival (OS) were 2.8 months (95% CI, 1.3-4.3) and 8.2 months (95% CI, 5.6-10.8), respectively. Seven (13%) patients experienced a partial response and 21 (40%) derived clinical benefit. At baseline, median ctDNA variant allele frequency (VAF) was 4.3%. Baseline ctDNA abundance was not associated with OS (p = 0.56) nor PFS (p = 0.54). However, a change in ctDNA VAF after one cycle of treatment (ΔVAF (T1-2)) was predictive of both PFS (p< 0.01) and OS (p< 0.01). Additionally, decrease in ΔVAF identified patients with longer OS despite early radiological progression, 8.2 vs 4.6 months, hazard ratio 0.44 (95% CI, 0.19-0.87) p = 0.03. After incorporating NLR and PLR into multivariable Cox models, ctDNA ∆VAF retained an association with OS. CONCLUSIONS: Early dynamic changes in ctDNA abundance, after one cycle of treatment, compared to baseline predicted both OS and PFS in R/M HNSCC patients on systemic therapy.

When Less May Be Enough: Dose Selection Strategies for Immune Checkpoint Inhibitors Focusing on AntiPD-(L)1 Agents.

Early clinical trials investigating antiPD(L)-1 agents rarely reached a maximum tolerated dose (MTD), and efficacy signals were observed even at the lowest dose levels. Most extended treatment intervals investigated indicated that these drugs do not follow a direct dose-toxicity or dose-efficacy relationship. Within this context and considering the high cost of antiPD(L)-1 agents, there is a significant debate on whether lower doses or the administration of such agents at an extended interval should be prospectively evaluated in already-approved agents, or at least be considered in novel combination trials involving antiPD(L)-1 drugs. Herein, we review the dosing, overall response rates, and incidence of treatment-related adverse events of antiPD(L)-1 agents in early dose-escalation trials and discuss the appropriateness of recommended Phase 2 dose selection as well as the final regulatory approved doses of such agents. Efficacy and safety data from randomized dose-range Phase 2 trials and real-world data (RWD) on the usage of lower doses and/or non-standard extended treatment intervals are also examined. As the accumulating evidence suggests lower doses or extended dosing intervals of antiPD(L)-1 may achieve a similar clinical benefit in comparison to the currently approved doses, we address the clinical and financial toxicity implications of using potentially higher doses than necessary. Last, we discuss ways to resolve the current dosing conundrum of antiPD-(L)1 agents such as performing near-equivalence studies and propose a framework for future development of immunotherapeutics to find the lowest efficacious dose instead of MTD.

Impact of pharmacodynamic biomarkers in immuno-oncology phase 1 clinical trials.

BACKGROUND: Phase 1 immuno-oncology (IO) trials frequently involve pharmacodynamic (PD) biomarker assessments involving tumour biopsies and/or blood collection, with increasing use of molecular imaging. PD biomarkers are set to play a fundamental role in early drug development of immuno-oncology (IO) agents. In the IO era, the impact of PD biomarkers for confirmation of biologic activity and their role in subsequent drug development have not been investigated. METHODS: Phase 1 studies published between January 2014 and December 2020 were reviewed. Studies that reported on-treatment PD biomarkers [tissue-derived (tissue-PD), blood-based (blood-PD) and imaging-based (imaging-PD)] were analysed. PD biomarker results and their correlation with clinical activity endpoints were evaluated. Authors' statements on the influence of PD biomarkers on further drug development decisions, and subsequent citations of PD biomarker study results were recorded. RESULTS: Among 386 trials, the most frequent IO agent classes evaluated were vaccines (32%) and PD-(L)1 inhibitors (25%). No PD biomarker assessments were reported in 100 trials (26%). Of the remaining 286, blood-PD, tissue-PD, and imaging-PD data were reported in 270 (94%), 94 (33%), and 12 (4%) trials, respectively. Assessments of more than one PD biomarker type were reported in 82 studies (29%). Similar proportions of blood-PD (9%), tissue-PD (7%), and imaging-PD studies (8%) had positive results that correlated with clinical activity. Results of 22 PD biomarker studies (8%) were referenced in subsequent clinical trials. CONCLUSIONS: Most phase 1 IO studies performed PD biomarker assessments. Overall, positive PD biomarker results were infrequently correlated with clinical activity or cited in subsequent trials, suggesting a limited impact on subsequent drug development. With emerging health regulatory emphasis on optimal dose selection based on PD activity, more informative and integrative multiplexed assays that capture the complexity of tumour-host immunity interactions are warranted to improve phase 1 IO trial methodology.

A risk stratification model for toxicities in phase 1 immunotherapy trials.

INTRODUCTION: Despite the increased number of novel immunotherapy (IO) agents under current development, their toxicity profile remains to be fully elucidated. METHODS: An IO risk stratification model was developed based on 5 different variables: treatment-related deaths; rate of grade ≥3 treatment-related adverse events or treatment-emergent adverse events; grade ≥2 encephalopathy or central nervous system toxicity; grade ≥2 cytokine release syndrome; and the number and type of dose-limiting toxicity. Phase 1 IO trials published from January 2014 to December 2020 were reviewed and categorised based on our risk stratification model into three categories: low-, intermediate- and high-risk. Clinical trial variables were associated with the high-risk category. To review the quality of reporting across phase 1 IO trials, a subset of studies was further examined by the use of the ASCO/SITC Trial Reporting in Immuno-Oncology (TRIO) standards. RESULTS: Different IO compounds demonstrated diverse risk profiles. In multivariable analysis, combination versus IO single agent treatment, and testing IO agents different from anti-programmed death-1/programmed death ligand-1 (anti-PD1/L1), anti-cytotoxic t-lymphocyte antigen-4 (anti-CTLA4) antibodies and anti-cancer vaccines were associated with a higher toxicity risk. None of the studies examined in our dataset reported all the items included in the TRIO standards. CONCLUSIONS: Our results have important implications for future clinical trial design. Additionally, standards for reporting are urgently needed.

Paclitaxel Plus Cetuximab as Induction Chemotherapy for Patients With Locoregionally Advanced Head and Neck Squamous Cell Carcinoma Unfit for Cisplatin-Based Chemotherapy.

Objectives: Induction chemotherapy (ICT) followed by definitive treatment is an accepted non-surgical approach for locoregionally advanced head and neck squamous cell carcinoma (LA-HNSCC). However, ICT remains a challenge for cisplatin-unfit patients. We evaluated paclitaxel and cetuximab (P-C) as ICT in a cohort of LA-HNSCC patients unfit for cisplatin. Materials and Methods: This is a retrospective analysis of patients with newly diagnosed LA-HNSCC considered unfit for cisplatin-based chemotherapy (age >70 and/or ECOG≥2 and/or comorbidities) treated with weekly P-C followed by definitive radiotherapy and cetuximab (RT-C) between 2010 and 2017. Toxicity and objective response rate (ORR) to ICT and RT-C were collected. Median overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method. Cox regression analysis was performed to determine baseline predictors of OS and PFS. Results: A total of 57 patients were included. Grade 3-4 toxicity rate to ICT was 54.4%, and there was a death deemed treatment-related (G5). P-C achieved an ORR of 66.7%, including 12.3% of complete responses (CR). After P-C, 45 patients (78.9%) continued with concomitant RT-C. Twenty-six patients (45.6%) achieved a CR after definitive treatment. With a median follow-up of 21.7 months (range 1.2-94.6), median OS and PFS were 22.9 months and 10.7 months, respectively. The estimated 2-year OS and PFS rates were 48.9% and 33.7%, respectively. Disease stage had a negative impact on OS (stage IVb vs. III-IVa: HR = 2.55 [1.08-6.04], p = 0.03), with a trend towards worse PFS (HR = 1.92 [0.91-4.05], p = 0.09). Primary tumor in the larynx was associated with improved PFS but not OS (HR = 0.45 [0.22-0.92], p = 0.03, and HR = 0.69 [0.32-1.54], p = 0.37, respectively). Conclusion: P-C was a well-tolerated and active ICT regimen in this cohort of LA-HNSCC patients unfit for cisplatin-based chemotherapy. P-C might represent a valid ICT option for unfit patients and may aid patient selection for definitive treatment.

Contemporary dose-escalation methods for early phase studies in the immunotherapeutics era.

Phase 1 dose-escalation trials are crucial to drug development by providing a framework to assess the toxicity of novel agents in a stepwise and monitored fashion. Despite widely adopted, rule-based dose-escalation methods (such as 3 + 3) are limited in finding the maximum tolerated dose (MTD) and tend to treat a significant number of patients at subtherapeutic doses. Newer methods of dose escalation, such as model-based and model-assisted designs, have emerged and are more accurate in finding MTD. However, these designs have not yet been broadly embraced by investigators. In this review, we summarise the advantages and disadvantages of contemporary dose-escalation methods, with emphasis on model-assisted designs, including time-to-event designs and hybrid methods involving optimal biological dose (OBD). The methods reviewed include mTPI, keyboard, BOIN, and their variations. In addition, the challenges of drug development (and dose-escalation) in the era of immunotherapeutics are discussed, where many of these agents typically have a wide therapeutic window. Fictional examples of how the dose-escalation method chosen can alter the outcomes of a phase 1 study are described, including the number of patients enrolled, the trial's timeframe, and the dose level chosen as MTD. Finally, the recent trends in dose-escalation methods applied in phase 1 trials in the immunotherapeutics era are reviewed. Among 856 phase I trials from 2014 to 2019, a trend towards the increased use of model-based and model-assisted designs over time (OR = 1.24) was detected. However, only 8% of the studies used non-rule-based dose-escalation methods. Increasing familiarity with such dose-escalation methods will likely facilitate their uptake in clinical trials.

Bugs as drugs: The role of microbiome in cancer focusing on immunotherapeutics.

The human microbiome comprising microorganisms, their collective genomes and metabolic products has gained tremendous research interest in oncology, as multiple cohorts and case studies have demonstrated discernible interpatient differences in this ecosystem based on clinical variables including disease type, stage, diet, antibiotic usage, cancer treatments, therapeutic responses and toxicities. The modulation of the gut microbiome is the subject of many ongoing preclinical and clinical investigations, through the manipulation of diet, as well as the use of prebiotics, probiotics, specific antibiotics, fecal microbial transplantation, microbial consortia and stool substitutes. Standardization and quality control are needed to maximize the information being generated in this growing field, ranging from technical assays to measure microbiome composition, to methodological aspects in the analysis and reporting of results. Proof-of-mechanism and proof-of-concept clinical trials with appropriate controls are needed to confirm or refute the feasibility, safety and ultimately the clinical utility of human microbiome modulation in cancer patients.

Two-Target Quantitative PCR To Predict Library Composition for Shallow Shotgun Sequencing.

When determining human microbiota composition, shotgun sequencing is a powerful tool that can generate high-resolution taxonomic and functional information at once. However, the technique is limited by missing information about host-to-microbe ratios observed in different body compartments. This limitation makes it difficult to plan shotgun sequencing assays, especially in the context of high sample multiplexing and limited sequencing output and is of particular importance for studies employing the recently described shallow shotgun sequencing technique. In this study, we evaluated the use of a quantitative PCR (qPCR)-based assay to predict host-to-microbe ratio prior to sequencing. Combining a two-target assay involving the bacterial 16S rRNA gene and the human beta-actin gene, we derived a model to predict human-to-microbe ratios from two sample types, including stool samples and oropharyngeal swabs. We then validated it on two independently collected sample types, including rectal swabs and vaginal secretion samples. This assay enabled accurate prediction in the validation set in a range of sample compositions between 4% and 98% nonhuman reads and observed proportions varied between -18.8% and +19.2% from the expected values. We hope that this easy-to-use assay will help researchers to plan their shotgun sequencing experiments in a more efficient way. IMPORTANCE When determining human microbiota composition, shotgun sequencing is a powerful tool that can generate large amounts of data. However, in sample compositions with low or variable microbial density, shallowing sequencing can negatively affect microbial community metrics. Here, we show that variable sequencing depth decreases measured alpha diversity at differing rates based on community composition. We then derived a model that can determine sample composition prior to sequencing using quantitative PCR (qPCR) data and validated the model using a separate sample set. We have included a tool that uses this model to be available for researchers to use when gauging shallow sequencing viability of samples.

Predictive Value of Skeletal Muscle Mass in Recurrent/Metastatic Head and Neck Squamous Cell Carcinoma Patients Treated With Immune Checkpoint Inhibitors.

BACKGROUND: Reduced muscle mass has been associated with increased treatment complications in several tumor types. We evaluated the impact of skeletal muscle index (SMI) on prognosis and immune-related adverse events (IrAEs) in a cohort of recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) treated with immune checkpoints inhibitors (ICI). METHODS: A single-institutional, retrospective study was performed including 61 consecutive patients of R/M HNSCC diagnosed between July 2015 and December 2018. SMI was quantified using a CT scan at L3 to evaluate body composition. Median baseline SMI was used to dichotomize patients in low and high SMI. Kaplan-Meier estimations were used to detect overall survival (OS) and progression-free survival (PFS). Toxicity was recorded using Common Terminology Criteria for Adverse Event v4.3. RESULTS: Patients were 52 men (85.2%) with mean of age 57.7 years (SD 9.62), mainly oral cavity (n = 21; 34.4%). Low SMI was an independent factor for OS in the univariate (HR, 2.06; 95% CI, 1.14-3.73, p = 0.017) and multivariate Cox analyses (HR, 2.99; 95% CI, 1.29-6.94; p = 0.011). PFS was also reduced in patients with low SMI (PFS HR, 1.84; 95% CI, 1.08-3.12; p = 0.025). IrAEs occurred in 29 (47.5%) patients. There was no association between low SMI and IrAEs at any grade (OR, 0.56; 95% CI, 0.20-1.54; p = 0.261). However, grades 3 to 4 IrAEs were developed in seven patients of whom three had low SMI. CONCLUSIONS: Low SMI before ICI treatment in R/M HNSCC patients had a negative impact on OS and PFS. Further prospective research is needed to confirm the role of body composition as a predictive biomarker in ICI treatment.

Antitumor immune effects of preoperative sitravatinib and nivolumab in oral cavity cancer: SNOW window-of-opportunity study.

BACKGROUND: Sitravatinib, a tyrosine kinase inhibitor that targets TYRO3, AXL, MERTK and the VEGF receptor family, is predicted to increase the M1 to M2-polarized tumor-associated macrophages ratio in the tumor microenvironment and have synergistic antitumor activity in combination with anti-programmed death-1/ligand-1 agents. SNOW is a window-of-opportunity study designed to evaluate the immune and molecular effects of preoperative sitravatinib and nivolumab in patients with oral cavity squamous cell carcinoma. METHODS: Patients with newly-diagnosed untreated T2-4a, N0-2 or T1 >1 cm-N2 oral cavity carcinomas were eligible. All patients received sitravatinib 120 mg daily from day 1 up to 48 hours pre-surgery and one dose of nivolumab 240 mg on day 15. Surgery was planned between day 23 and 30. Standard of care adjuvant radiotherapy was given based on clinical stage. Tumor photographs, fresh tumor biopsies and blood samples were collected at baseline, at day 15 after sitravatinib alone, and at surgery after sitravatinib-nivolumab combination. Tumor flow cytometry, multiplex immunofluorescence staining and single-cell RNA sequencing (scRNAseq) were performed on tumor biopsies to study changes in immune-cell populations. Tumor whole-exome sequencing and circulating tumor DNA and cell-free DNA were evaluated at each time point. RESULTS: Ten patients were included. Grade 3 toxicity occurred in one patient (hypertension); one patient required sitravatinib dose reduction, and one patient required discontinuation and surgery delay due to G2 thrombocytopenia. Nine patients had clinical-to-pathological downstaging, with one complete response. Independent pathological treatment response (PTR) assessment confirmed a complete PTR and two major PTRs. With a median follow-up of 21 months, all patients are alive with no recurrence. Circulating tumor DNA and cell-free DNA dynamics correlated with clinical and pathological response and distinguished two patient groups with different tumor biological behavior after sitravatinib alone (1A) versus sitravatinib-nivolumab (1B). Tumor immunophenotyping and scRNAseq analyses revealed differential changes in the expression of immune cell populations and sitravatinib-targeted and hypoxia-related genes in group 1A vs 1B patients. CONCLUSIONS: The SNOW study shows sitravatinib plus nivolumab is safe and leads to deep clinical and pathological responses in oral cavity carcinomas. Multi-omic biomarker analyses dissect the differential molecular effects of sitravatinib versus the sitravatinib-nivolumab and revealed patients with distinct tumor biology behavior. TRIAL REGISTRATION NUMBER: NCT03575598.

Pan-cancer analysis of longitudinal metastatic tumors reveals genomic alterations and immune landscape dynamics associated with pembrolizumab sensitivity.

Serial circulating tumor DNA (ctDNA) monitoring is emerging as a non-invasive strategy to predict and monitor immune checkpoint blockade (ICB) therapeutic efficacy across cancer types. Yet, limited data exist to show the relationship between ctDNA dynamics and tumor genome and immune microenvironment in patients receiving ICB. Here, we present an in-depth analysis of clinical, whole-exome, transcriptome, and ctDNA profiles of 73 patients with advanced solid tumors, across 30 cancer types, from a phase II basket clinical trial of pembrolizumab (NCT02644369) and report changes in genomic and immune landscapes (primary outcomes). Patients stratified by ctDNA and tumor burden dynamics correspond with survival and clinical benefit. High mutation burden, high expression of immune signatures, and mutations in BRCA2 are associated with pembrolizumab molecular sensitivity, while abundant copy-number alterations and B2M loss-of-heterozygosity corresponded with resistance. Upon treatment, induction of genes expressed by T cell, B cell, and myeloid cell populations are consistent with sensitivity and resistance. We identified the upregulated expression of PLA2G2D, an immune-regulating phospholipase, as a potential biomarker of adaptive resistance to ICB. Together, these findings provide insights into the diversity of immunogenomic mechanisms that underpin pembrolizumab outcomes.