The demands of curtailing the impact of the coronavirus disease 2019 (COVID-19) global pandemic have disrupted the world’s ability to care for patients with thoracic pathologies. Those who undergo thoracic surgical therapeutic procedures are a high-risk category, likely to have impaired lung function but also high risk for exposing clinical teams to aerosolised viral loads. In light of this global pandemic, thorough pre-procedural planning, adequate personal protective equipment (PPE), experienced personnel and judicious anaesthetic and intra-operative measures will serve to be instrumental in ensuring positive patient outcomes whilst still protecting the safety of healthcare workers. The novel coronavirus, now termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a significant global impact in the space of 4 months. This has had resounding effects on the ability to deliver adequate healthcare to all those who require it. The demands of curtailing the impact of the pandemic have disrupted the world’s ability to care for patients with thoracic pathologies. The ever increasing burden of the coronavirus disease 2019 (COVID-19) pandemic coupled with the finite amount of hospital resources has forced surgeons to prioritise cases and protect patients. At the same time, however, careful consideration must also be given to the hospital policies and procedures that need to be implemented in order to protect the healthcare teams from viral transmission. Numerous bodies from multiple cancer, surgical and research organisations have come together to form consensus statements on how we can mitigate the risk of transmission through optimisation of pre-operative, intra-operative and post-operative procedures [Therapeutic procedures on the lung during the era of COVID-19 automatically place clinical teams to a high risk of exposure from aerosolised viral loads. Procedures such as bronchoscopy, tracheostomy, double-lumen endotracheal tube placement, airway surgery, laparoscopy and lung surgery with post-operative parenchymal air leaks all pose a risk. Multi-disciplinary strategies should initially be put in place to triage patients to treatment pathways that are often alternative to surgery. These should be balanced against the COVID-19 status of the hospital, the level of hospital resources available and the COVID-19 trajectory of the hospital  [2 of 1.0 and positive end-expiratory pressure of 5 cm H2O, in patients who are supine before tracheostomy to show physiological readiness to tolerate the procedure, with strategies to mitigate aerosolisation is also advised [Tracheostomy and bronchoscopy both have a continuing role in managing weaning from extended periods of mechanical ventilation during this pandemic. Working groups have determined that for tracheostomy, patient selection and timing of procedure are paramount . The pro advised  reported advised  reviewedDuring the conduct of thoracic surgery and elective lung resection, multiple guidance documents have been produced. Rakovich and colleagues  assimila2 insufflation during these procedures remains unclear; however, precautions should be undertaken to decrease exposure to surgical smoke, decrease production of surgical plume and filter any gaseous products (e.g. CO2) through pre-approved filters. Van den Eynde and colleagues [2 insufflation as well as high-energy electrocautery and ultrasonic devices.In the world of Minimally Invasive and Robotic Surgery, similar working groups have been formed. Porter and colleagues  conductelleagues  describeThe COVID-19 pandemic has dramatically affected the conduct of healthcare practices globally. This has sparked the development of various guidelines to reduce the risk of viral transmission during thoracic surgery and allied specialties. In light of this global pandemic, there is a growing body of evidence, which is largely guideline, expert opinion and consensus driven, yet still represents the current best platform from which we can inform our precautions in the management of this uncertain threat. Thorough pre-procedural planning, adequate PPE, experienced personnel and judicious anaesthetic and intra-operative measures will serve to be instrumental in ensuring positive patient outcomes whilst still protecting the safety of healthcare workers.


Significance: Surgical simulators, both virtual and physical, are increasingly used as training tools for teaching and assessing surgical technical skills. However, the metrics used for assessment in these simulation environments are often subjective and inconsistent.Aim: We propose functional activation metrics, derived from brain imaging measurements, to objectively assess the correspondence between brain activation with surgical motor skills for subjects with varying degrees of surgical skill.Approach: Cortical activation based on changes in the oxygenated hemoglobin (HbO) of 36 subjects was measured using functional near-infrared spectroscopy at the prefrontal cortex (PFC), primary motor cortex, and supplementary motor area (SMA) due to their association with motor skill learning. Inter-regional functional connectivity metrics, namely, wavelet coherence (WCO) and wavelet phase coherence were derived from HbO changes to correlate brain activity to surgical motor skill levels objectively.Results: One-way multivariate analysis of variance found a statistically significant difference in the inter-regional WCO metrics for physical simulator based on Wilk’s Λ for expert versus novice, Conclusion: We show that brain functional connectivity WCO metric corresponds to surgical motor skills in the laparoscopic physical simulators. Functional connectivity between the CPFC and the SMA is lower for subjects that exhibit expert surgical motor skills than untrained subjects in laparoscopic physical simulators. Current studies have shown the potential of non-invasive brain imaging to quantify cortical activation differences for subjects with varying degrees of surgical motor skills.Herein, we report the inter-regional functional connectivity between the three above-mentioned cortical regions, namely, the PFC, the M1, and the SMA. Beyond the difference in activation levels of each of these cortical regions as demonstrated in Ref. 22.1a priori power analysis, based on two-sample Thirty-six right-handed subjects were recruited in this Institutional Review Board-approved study conducted at the Massachusetts General Hospital and the University at Buffalo. The subjects were split into two cohorts. The first cohort included novice and expert surgeons and the second cohort included training medical students. The second cohort was further divided into three distinct groups: FLS training group, virtual basic laparoscopic skills trainer (VBLaST) training group, and control group. An All the participants were instructed on how to perform the task with standardized verbal instruction indicating the goal of the task and rules for the task completion. The optical probe was positioned on the participants with great care to avoid any hair between source/detector and scalp, and robust coupling with the skin. Each participant’s experimental protocol consisted of a block design of rest and stimulus period . Each surgeon performed five trials, whereas the control group performed three trials, as shown in the table below. The surgical cutting task was performed until the completion or stopped after 5 min. Then a rest period of one minute was given. This cycle of the rest period and task continued for the number of trials indicated above. Subject demographics are summarized in 2.2,,,–We utilized two different surgical training simulators that employ the pattern cutting task. As a physical surgical trainer, we used the official FLS box trainer used in Board certification.2.3All study participants were asked to perform the pattern cutting task. The objective was to use laparoscopic tools to cut a marked circle on a piece of gauze as accurately and quickly as possible. Each subject was instructed on how to perform the task using a standardized verbal dictation indicating the pattern cutting task’s rules and goals. Each session consisted of each subject performing the pattern cutting task with rest periods between each subsequent trial. Further information regarding study design can be found in Nemani et al.2.4–,,–All fNIRS data processing were completed using HOMER2, a validated and published open-source software suite implemented in Matlab , which provides a set of Matlab scripts used for analyzing fNIRS data.2.5,,,,,,,To objectively quantify functional connectivity between time series from different cortical regions, we utilize the WCO and WPCO metrics. WCO as a function of frequency is defined below:2.6The inter-regional functional connectivity metrics (WCO and WPCO) from the first cohort of novice and expert surgeons were used to conduct a one-way multivariate analysis of variance (one-way MANOVA) in SPSS version 27 (IBM) to determine whether there is any significant difference in the inter-regional  functional connectivity metrics between novice and expert surgeons using Wilks’ Lambda. The Shapiro-Wilk test was used to test normality for each of the dependent variables  for the independent variable, novice, and expert. Also, the Levene test was used to test homogeneity of variance. All the significance levels were set at 33.1Supplementary Materials.To investigate significant inter-regional functional connectivity differences between expert and novice surgeons on physical (FLS) or virtual (VBLaST) simulators, we report in 3.2Since functional connectivity changes between the CPFC and SMA, CPFC-SMA was responsive to increased surgical motor skill proficiency in expert versus novice during physical (FLS) simulator task, so we calculated CPFC-SMA WCO and WPCO metrics for FLS practice in medical student trainees. 4While surgical simulators are significantly gaining ground for surgical skill training and assessments,Inter-regional functional connectivity within the neurovascular coupling frequency range (0.02 to 0.05 Hz) is postulated to be related to neuronal communication. Neurovascular coupling is the interaction between neural activity and vascular response in terms of regional cerebral blood supply and HbO during brain activity. The interaction between HbO time-series between two brain regions can be assessed using various methods, including WCO, a measure of the correlation between two time-series, and WPCO based on the degree of coincidence of instantaneous phase over the entire time-series.,During surgical tasks in physical and virtual simulators, our neuroimaging approach utilized the most recent advances in portable functional brain imaging using fNIRS with increased specificity to cortical tissue due to short separation regression.5This study showed that functional connectivity changes based on WCO and WPCO metrics corresponded to the surgical motor skill proficiency, and these connectivity changes were in the neurovascular coupling frequency range in the cortical regions. Our study showed that surgical experts and surgically trained subjects exhibited functional activation correlations and the instantaneous phase’s coincidence over the CPFC and SMA time-series. These results further our understanding of neural correlates of the interplay between motor and frontal regions related to fine motor learning associated with surgical training and can be used for future assessment paradigms.Click here for additional data file.


The correlations between microbiota dysbiosis and cancer have gained extensive attention and been widely explored. As a leading cancer diagnosis worldwide, lung cancer poses a great threat to human health. The healthy human lungs are consistently exposed to external environment and harbor a specific pattern of microbiota, sharing many key pathological and physiological characteristics with the intestinal tract. Although previous findings uncovered the critical roles of microbiota in tumorigenesis and response to anticancer therapy, most of them were focused on the intestinal microbiota rather than lung microbiota. Notably, the considerable functions of microbiota in maintaining lung homeostasis should not be neglected as the microbiome dysbiosis may promote tumor development and progression through production of cytokines and toxins and multiple other pathways. Despite the fact that increasing studies have revealed the effect of microbiome on the induction of lung cancer and different disease status, the underlying mechanisms and potential therapeutic strategies remained unclear. Herein, we summarized the recent progresses about microbiome in lung cancer and further discussed the role of microbial communities in promoting lung cancer progression and the current status of therapeutic approaches targeting microbiome to alleviate and even cure lung cancer. Currently the human microbiome has received extensive attention and been demonstrated to play a critical role in various aspects of human health and disease status via immunity, metabolism and inflammation2.The human body coexists with a complex array of commensal microbiome that colonizes the host microenvironment forming a dynamic micro-ecological system developed during evolution. The commensal microbiome shares a symbiotic relationship with the host during the long-term coexistence development which eventually forms a dynamic microecosystem4. It is generally divided into two histological-pathological types including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Treatments for lung cancer include surgery, chemotherapy, radiotherapy, targeted therapy and emerging immunotherapy. And the best recommendations depend on patient’s TNM stage and unique health situations5. However, most patients are diagnosed at an advanced stage, with high mortality and poor benefit from limited treatment options6. On the other hand, multiorgan metastatic and relapse in pre-treatment and post-treatment are critical causes of death without effective therapy. There are growing emergency and social demand in exploring the carcinogenesis and new therapeutics for this deadly disease. Lung cancer has been widely considered to be a complicated disease caused by interactions between host and environmental factors7. Among diverse environmental risk factors, microbes present a vital part in maintaining microecological balance and regulating host immune responses to multi-treatments. Although the healthy lung tissues were long considered as a sterile environment, it was found recently that there were certain microbial species existed in the lung tissues impacting the balance between health and pathogenesis in the lung microenvironment with the advancements of high-throughput next generation sequencing (NGS) technologies9. Increasing studies have profiled the microbiome in respiratory samples from healthy adult lungs11. It has discovered the most common phyla including Bacteroides, Firmicutes, and Proteobacteria and genera like Streptococcus, Pseudomonas, Veillonella, and Prevotella12. Nowadays researches in the lung microbiome and important discoveries in the microbiome’s association with lung diseases are growing rapidly. It is believed that improved understanding of this association will provide novel insights into the pathogenesis of lung diseases. In this review, we summarized and evaluated the current development of the interplay and the underlying mechanisms between microbiota and lung cancer. Furthermore, we also discussed the prospects about the carcinogenesis and therapeutic applications of microbiome on lung cancer.Lung cancer poses a great threat to global public health and is ranked as the most common cancer  with over 2.09 million diagnosis and 1.7 million deaths worldwide in 201813. At present, more and more efforts have been focused on the most compelling commensal microbial ecosystem interacting with the human body—especially the gut microbiome which is considered as a forgotten organ mediating host homeostasis via complex mechanisms14. Emerging gut microbiota research are fully facilitated by the rapid development and application of high-throughput molecular technologies15, bioinformatics and metagenomics17. It was reported that gut microbiome was closely correlated with various chronic diseases such as gastroenteric cancers within which carcinogenic H. pylori was colonized frequently, as well as multiple pathological processes of remote organs in the host19. Conversely, lungs were regarded as a pair of sterile organs until new studies assisted with high-throughput sequencing technologies challenged the old false dogma20. It is now generally recognized that microbial disturbances influence a variety of lung diseases. The lung microbiota is composed of bacteria, fungi, and virus, which derived from the inhalation of mucosal secretions, nasopharynx, oropharynx, and environmental air exchange23. Under healthy conditions, bacteria of the genus Propionibacterium, Streptococcus, Haemophilus, and Veillonella coexist with fungi such as Aspergillus, Penicillium, and Candida, but they do not cause infection of human lungs24. It is not surprising when considering multiple unknown interactions in other tissues. All these interactions among microbiome, immunity and metabolism in these microbial niches affect multiple lung pathogenesis of COPD, asthma, cystic fibrosis and lung cancer28. Recently, increasing interests have been raised about the field including commensal lung microbiome communities and the possible mechanisms maintaining microecological effects on human respiratory system. Based on previous investigations, several controversial hypotheses have been proposed including “Microbiota-Brain-Gut axis”13, “Microbiota-Gut-Liver axis”29, and “Microbiota-Gut-Skin axis”30.Microbiome was defined by the bacteria, fungi, virus, protozoa and their related genes and genomes, as well as metabolites31. Some researchers have brought forward that oral microbiome may be the primary sources of lung microbiome from a long-term and validated observation 31. The respiratory tract and gut could communicate with each other via biological processes including micro-aspiration and inhalation32. Bacterial metabolites from the intestinal tract can regulate the differentiation tendency of naive T cells, effector T cells, Tregs, or Th17 release, which further induces systematic inflammation and immunity response33. Furthermore, they could be transported into host bloodstream to regulate the systemic immune activity and alter the microbial communities located in respiratory tract35. Although some theoretical models had been put forward, no specific studies were conducted to approach the relationship and communicating manners in terms of the species diversity and the relative abundance of microbiota at different sites within respiratory and gastrointestinal tract.In fact, the human body is a dynamically balanced integrity and microorganisms in various body sites can interact with each other directly including mucosal dispersion, respiratory and digestive activities, or indirectly via inflammatory substances, cytokine, and metabolites in systematic circulation as shown in Fig. 36. On the other hand, Lozupone et al. reported that the progressive maturation pattern of diversity, stability, and resilience of the lung microbiota from birth to adult mice was consistent with both human lung and gut microbiota during the first 3 years37. Previous studies revealed that early-life formation of microbiota and immunologic environment in human airways and gastrointestinal tract may be derived from the skin and external environment40. Despite distinct differences in micro-anatomic features, composition and population dynamics in gut and lung microbiota, these two organs share a similar homeostasis and certain physiological characteristics such as microbiota maturation process, mucosal immune system, co-evolution and communication with immune cells and continuous exposure to outside environment.The temperature and pH environment in gastrointestinal tract are relatively constant and migration of microbes is unidirectional and always altered by complicated physical and chemical conditions. By contrast, the lung is frequently exchanging gas with outside environment to maintain abundant reserves of oxygen and microbiota. Furthermore, there is no physical barrier and gradient diversity of pressure and temperature in upper respiratory tract which provide bidirectional conditions for lung-resident microbial migration and dynamic changes42. These significant discoveries lead us to reconsider whether the microbiome interaction network really exists and modulates host susceptibility to either internal or external pathogenic factors. Gut microbiome have been confirmed contributing to the chronic obstructive pulmonary diseases43, the progression of asthma44, and the worsening acute lung injury45. More and more studies have also implicated the connections and modulatory effects of specific microbial metabolites in gut and lung via circulation47. For example, a significant reduction of the microbial metabolites including the fatty acids, acetate, butyrate and propionate, as well as isoacids in the feces from patients with bronchial asthma was observed when compared with healthy controls48. And Faecalibacterium prausnitzii and Akkermansia muciniphila have been reported to suppress the inflammatory responses in pediatric allergic asthma through inducing anti-inflammatory cytokine IL-10 and inhibiting the secretion of pro-inflammatory cytokines like IL-1247. Previous studies also found that alterations in colonic luminal, serum, and hippocampal metabolomic profiles in mice treated with ketogenic diet were significantly correlated with seizure protection, supporting the distant modulation of intestinal microbiome in other organs49. Bingula et al. reported that the gut bacteria and their fragments were taken up into DCs (dendritic cells) and macrophages through phagocytosis and then migrate into gut or lungs to regulate immune response50. Tsay et al. demonstrated that gut flora can induce lung inflammatory reaction against bacterial pneumonia and enhance neutrophils infiltration through TLR4 in mice51. Taken together, the complex and interventional ecosystems regulate various pathological processes and maintain the physiological equilibrium of gut and lung. Thus, a new hypothesis was put forward as “Microbiota-Gut-Lung axis” based on the diverse and complex gut-lung microbiome networks established based on numerous long-tern epidemiological observations. However, the mechanisms underlie the “Microbiota-Gut-Lung axis” remains elusive and more robust evidences are still required to kindle the lamp.Intriguingly, increasing clinical studies indicated that multiple lung diseases were more likely to develop in patients with gastrointestinal disorders54. The carcinogen acetaldehyde and deoxycholic acid produced by microbiome were reported to be involved in esophagus and liver carcinogenesis56. Visconti et al. reported that the blood and fecal metabolites were analyzed together with the characterization of gut microbiome by metagenomic shotgun sequencing. Their findings supported a correlation between active pathways of gut bacteria and metabolites found in feces and blood57. Metabolism is essential for maintaining human body homeostasis through numerous pathologic and physiologic processes58. There are also emerging studies approaching lung microbiome associated with host metabolism. Cribbs et al. discovered that specific metabolic profiles correlated with bacterial organisms were associated with glycerophospholipid and lineolate pathways, which play an important role in the pathogenesis of pneumonia in HIV-infected individuals through 16s rRNA sequencing in the bronchoalveolar lavage fluid59. Bei et al. also found that primary metabolites secreted by Pseudomonas aeruginosa using substrates produced by Rothia mucilaginosa might contribute to its pathogenesis in the progression of cystic fibrosis60. One of the high-profile metabolites is short-chain fatty acids (SCFA) which is produced by large amounts of commensal microbes and acts as a crucial signaling molecule in host cells. And many studies have focused on the role of SCFA in host gut and immunity, while the functions of SCFA in respiratory system, epithelium and immunity remain unclear. It has been shown by Gauguet et al. that mice lacking SCFA in the gut are prone to suffer from more bacterial load like Staphylococcus aureus which could be modulated by pulmonary Th17 immunity61. Previous study by Cait et al. demonstrated that dietary supplementation with short-chain fatty acids (SCFAs) can ameliorate this enhanced asthma susceptibility by modulating the activity of T cells and DCs in mice62. Besides, some researches have remonstrated that modulation of gut microbiome in preclinical model can alter host immune response and susceptibility to pulmonary infection factors64. Furthermore, SCFAs has been reported to regulate differentiation of bone marrow cell and maintain host immunological homeostasis66. Under certain circumstances, SCFAs can modulate the composition of gut microbiome and induce myelopoiesis resulting in an anti-inflammatory milieu in the airways68. Therefore, the concept of “gut-bone marrow-lung axis” has been brought forward, which represents a mechanistic explanation on how the gut microbiota derived SCFAs modulate the host immune system against exogenous pathogenic factors. However, currently most of the studies are focused more on the possible association between the intestinal-pulmonary axis and lung inflammatory diseases with few studies involving lung cancer. Although recent investigations in microbial metabolism associated with cancer occurrence and progression are shedding novel insight, limited evidences are available to establish solid connections concerning distal metabolic regulations between gut and lung and the mechanism still awaits further investigation.Dysregulation of host metabolism by microbiome alterations have been intensively studied in the intestinal tract69. The special subgroup CD4 + Th17 cells play an important role in microbial interactions, mucosal immunity functions and host response to inflammatory diseases of intestinal tract, lungs and skin70. Mantis et al. reported that IgA mainly regulated bacterial virulence in the gut by blocking bacterial adherence to mucosal epithelial cells71. The microecology with low-density and less-stable microbiome was susceptible to lose balance of commensal bacteria and be invaded by exogenous pathogens72. A recent study revealed that high density of commensal microbiota might promote the clinical outcome of vaccine for infants73. Gut microbiota can stimulate Th17 response and modulate the generation of IL-17, which is involved in the elimination of certain pathogens75. In addition, IL-17 pathway is also involved in the pathogenesis of several pulmonary pathologies including asthma, sarcoidosis, obliterative bronchiolitis, and bone marrow transplant–related pneumonitis79. Gollwitzer et al. reported that bacteria resident in the lung regulated the expression of certain innate immunity genes including IL-5, IL-10, and IFN, and the expression level of PD-L1 on CD11bC DCs and FoxP3+CD25+ Treg cells were higher in the lungs of SPF (specific pathogen-free) neonates than GF (germ free) mice80. Steed et al. reported that a microbially associated metabolite, desaminotyrosine (DAT), protects host from influenza via enhancing type I IFN stimulation and reducing lung cancer immunopathology81. Similarly, Takeshi Ichinohe et al. found that commensal microbiota could regulate immunity in respiratory mucosa through inflammasomes and provided immune activation signals at steady state after influenza virus infection63. The most recent study revealed that the fermentable fiber inulin could alter gut microbiota structure and the associated metabolites like short-chain fatty acids which eventually improve the response of mice to influenza virus infection by dampening damage induced by neutrophils and enhancing anti-viral CD8 + T cell responses67. And enrichment of lung microbiome with oral taxa was found to be associated with Th17 inflammation, in which TLR4 responses were impacted by lung microbiome composition25. Moreover, commensal microbiota was shown to drive proliferation and activation of Vg6 + Vd1 + T cells in lung cancer82. Nevertheless, there is no consistent definition of a healthy or beneficial lung microbiota, partly due to limited understanding in approaching the association between lung-resident microbiome and host immunity.The microbiome regulates host immune activity directly or indirectly by mediating host susceptibility to various pathogenic factors and therapeutic outcomes. The dynamic interaction between microbiome and immune system enables host to recognize and prevent bacterial or fungal invasions and infections. In preclinical studies germ-free (GF) mice lacking intestinal microbiome displayed severe immune dysplasia with incomplete mucous layer, immunoglobulin secretion disorder, and decreased size and number of lymph nodes83. Here, we made a summary of previous studies in microbiome correlated with lung cancers have been listed in the Table 194. The results demonstrated the close relationship between microbial communities and respiratory tract. The initiations of surface boundary tumors are often associated with the host mucosal immune barrier destruction. When the mucosal surface is damaged, the microenvironment of the original tissue and commensal microbiome will be reconstructed if the injury cannot be repaired in time. Otherwise, this damage will continue to intensify and lead to recurring inflammation which may induce cancer at the end. It is possible that microbiome located in the surface-bound tumor or intratumor utilize tumor-derived carbon sources and other nutrients to interact with the tumor immune microenvironment in the long-term coexistence96. Of note, the most significant microbial carcinogenetic effect is the chronic infections by HBV and HCV induced liver cancer development97. Interestingly, a recent study found that the fungal microbiome was abundantly enriched in pancreas and triggered pancreatic carcinogenesis via MBL activation98. In aggregate, increasing evidences have supported that dysbiosis of commensal microbial communities can alter the host susceptibility to carcinogenic factors. These findings reflecting the carcinogenesis development can provide a novel insight to help us understand the process from normal tissue to precancerous lesions and to advanced lung cancer.Cancer is generally thought to be a multifactorial pathological process, where normal cells begin to proliferate in an unprogrammed manner resulting in inhibition of apoptosis, autophagy, inflammations and DNA damage. There are increasing commensal and pathogenic microorganisms defined in the human body with reported carcinogenic properties and most of them are significantly correlated with carcinogenesis epidemiologically99. Until now, more and more attention has been paid to intestinal microbiota associated with colorectal cancer development, which has also provided us a better understanding about contributions of microbiome to cancer100. A most recent study by Manzano et al. revealed a distinct mutation signature from organoids injected with genotoxic pks+ E. coli, which indicated a direct role of bacteria in the occurrence of oncogenic mutations101. Multiple signaling pathways are involved in the process of carcinogenesis associated with microbiome interventions104. In addition, destruction of boundary surface between host and microbes could also activate pattern recognition receptors and their signaling cascades which leads to imbalance in the symbiotic microenvironment. The existence of gut microbes was also involved in other aspects of cancer initiation and progression such as angiogenesis, invasion, tumor immune microenvironment and apoptosis which can be promising research hotspots for in-depth investigation105.Genotoxic toxins or metabolites produced by bacteria can directly damage host DNA and induce genomic instability via reactive oxygen or nitrogen species or natural killer immune receptors which cause cancer-like characteristic alterations when cumulative damage effect overrides the host self-repair ability125. Some researchers proposed that a disordered microbial dysbiosis might provoke dysregulation in host physiology and contribute to exacerbations in chronic lung diseases. Several observations reported that respiratory viruses were identified in the respiratory specimens of 39–56% of chronic obstructive pulmonary disease (COPD) patients compared to 6–19% at clinical baseline127. It was also revealed that pathogenic bacteria existed in 51–70% of patients during disease exacerbations compared to 25–48% in the initial stable clinical baseline128. Another large cohort survey reported that CXCL8/IL-8 was significantly associated with lung microbiome diversity and community structure, which can mediate host inflammatory responses during COPD exacerbations in some subjects129. Another slowly progressive lung disease is idiopathic fibrosis (IPF) has been confirmed to harbor a distinct microbiome from that of healthy lung status130, and a randomized trial reported that antibiotic therapy could be beneficial to survival of IPF patients131. Robert et al. revealed that radiographic honeycombing altered the lung microbiota of patients with IPF, which might further exacerbate the anatomic disruption of IPF in a bidirectional interaction125. Despite growing evidence of the associations, the causal significance of an altered lung microbiota in COPD and IPF remains elusive. Furthermore, lung microbiome including bacteria or virus infection could potentially invade epithelial cells of the airways inducing host immune response or triggering the wound healing cascade in chronic pathogenic stimuli132. Overall, these hypothesis driven theories or epidemiological observations or cohort studies, especially whether the microbiome correlated with chronic lung diseases participate in the exacerbation or initiation of lung cancer, still awaits further investigations. Emerging studies have also raised interests about correlations between lung cancer and microbiome by high-throughput sequencing and epidemiological analysis. It was found that significantly high abundance of Granulicatella, Thermus, Legionella, and Streptococcus were observed in lung tumor tissues compared with control groups93. Of note, Tsay et al. demonstrated that the enrichment of the lower airway microbiota with oral taxa (Streptococcus and Veillonella) was significantly correlated with the upregulation of lung carcinogenic ERK and PI3K signaling pathways. In vitro exposure of airway epithelial cells to Veillonella, Prevotella, and Streptococcus also led to upregulation of these same signaling pathways91. Gomes et al. reported that lung cancer microbiota was enriched in Proteobacteria and more diverse in squamous carcinoma than adenocarcinoma, particularly in males and heavier smokers11. Another study by Greathouse et al. revealed that mutations in TP53 were correlated with the presence of Acidovorax in the lung microenvironment84. The emerging technologies revealed that the niche effects of lung-resident microbiome on lung cancer should not be neglected. From a global perspective, Pseudomonas, Streptococcus, Staphylococus, Veillonella, and Moraxella were frequently reported as the most relevant lung cancer-related microbiome133. Notably, some lung commensal microbiota including lung cancer-related microbiome including Proteobacteria, Streptococcus, Bacteroidetes, Veillonella, and Moraxella have also been identified significantly correlated with lung inflammatory diseases125. Yet, limited by technology development and ethics, the nasal secretions, oral saliva, sputum, and BAL fluid are often used indirectly for lung microbiome research. Most studies based on indirect samples and evidences were problematic and failed to illustrate the molecular mechanisms in this field. Despite the inevitable limitations in preclinical animal model and cell lines, emerging advances in organoid technology has allowed for innovative and meaningful investigations of 3D human lung tissues. There are increasing discussions about the feasibility of utilizing lung organoids to approach lung tissue cell–cell interaction mechanism and the potentiality of IL-17 signaling pathway during lung infection135. Future research should try to use organoids to better simulate and explore the roles of microorganisms in lung cancer and the possible molecular mechanisms due to its success in investigation of microbiota associated with colorectal cancer101.Prior studies have uncovered some correlations between microbiome and lung inflammatory and organizational structure in several respiratory diseases including COPD (chronic pulmonary disease), IPF (idiopathic fibrosis), asthma, CF (cystic fibrosis) and non-CF Bronchiectasis, as shown in the Table 2136. And alpha-diversity in tumor microbiome and an intra-tumoral microbiome signature were highly correlated with long-tern survival in patients. They also proved that human-into-mice fecal microbiota transplantation (FMT) experiments differentially affected tumor growth, as well as tumor immune infiltration through modulating the tumor microbiome137. To identify the characterization of the tumor microbiome, researchers from Israel conducted a comprehensive analysis of 1526 tumor tissues with adjacent normal tissues from seven cancer types  which revealed that the intratumor bacteria were mostly intracellular in both cancer and immune cells and supported significant correlations between intratumor bacteria or their predicted functions with tumor types and subtypes, patients’ smoking status, and the response to immunotherapy96. In addition to bacteria, a pan-cancer comprehensive analysis based on the International Cancer Genomic Consortium (ICGC) and the Cancer Genome Atlas (TCGA) has drawn a landscape of viral associations in human cancers, which detected a high prevalence of known tumor-associated viruses138. Yu et al. profiled the lung tissue microbiota and linked its composition and diversity to human lifestyle and clinical outcomes93. Also, Liu et al. reported that profile of lung cancer tissue microbiota is distinct from emphysema89. Consistently, Peters et al. demonstrated the existence of associations in lung tumor or normal tissue microbiome diversity and composition with patient recurrence-free (RFS) and disease-free survival (DFS)90. Above all, there is still an urgent need for much more explorations approaching the potentials of intra-tumor microbiome in cancer development and therapies.Increasing studies have also approached to the role of intratumor tissue microbiome in cancer development and therapies. T. Geller et al. found that intratumor bacteria might contribute to gemcitabine resistance of pancreatic ductal adenocarcinoma (PDAC), in which 76% of 133 human PDACs were positive for bacteria139. It was shown that bacteria in the lung may create a proinflammatory environment that promote lung cancer progression in the murine mouse model82. The GF or antibiotic treated mice were significantly protected from lung cancer development induced by Kras mutation and p53 loss when compared with SPF mice. And the commensal bacteria stimulated production of Myd88-dependent IL-1β and IL-2β from myeloid cells, inducing proliferation and activation of Vγ6+Vγ1+ γδ T cells that produced IL-17 and other effector molecules to promote inflammation and tumor cell proliferation. A similar result was also discovered by K.L. Greathouse et al. that a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue and a separate group of taxa are identified, in which Acidovorax is enriched in smokers with squamous cell carcinoma84. A reasonable hypothesis is that lung microbiota homeostasis may promote the pathological processes of lung cancer. Interestingly, it was observed in spontaneous colon tumor models that the morbidity of solid tumors was significantly lower in germ-free mice compared to mice raised conventionally140. Furthermore, high relative microbial abundance and diversity were found to be positively correlated with patient’s better responses to PD-1-based immunotherapy treatment141. On the other hand, the process of colonization and maturation of lung-resident microbiome community also participates in the maturation of lung and promotes host homeostasis and tolerance, as well as confers susceptibility to lung disorders during exposure to complex external environment142. Gollwitzer et al. allergic airway inflammation was significantly attenuated in adult mice, due to an increase in expression of the surface ligands PD-L1, PD-L2, and CD40 after HDM (house dust mites) treatment80. Nevertheless, current knowledge cannot elaborate the causal relationships of lung microbiome alterations accompanied with disease progression, since most researches are based on long-term observations and cohort studies. It is more likely that lung microbiome may play a dual role in maintaining body stability and promoting cancer as shown in Fig. Furthermore, some pioneer preclinical studies have tried to utilize GF mice  and SPF mice  model to elucidate the real and precise correlations between lung microbiome and lung cancer closely143. Patients with advanced lung cancer always suffer from poor prognosis and limited treatment options, despite substantial advances in the oncological management of lung cancer achieved during the past three decades. Therefore, early detection and improved treatments for lung cancer are becoming increasingly urgent. The current explorations of the clinical applications of microorganisms remain at its early stages including probiotics, diet interventions and FMT  in the preclinical model. Understanding the relationship between human microbiota, especially gut microbiota and lung cancer, may open a new window for the diagnosis and treatment of lung cancer.Currently, the traditional treatments of lung cancer can be categorized into surgical resection, radiotherapy, chemotherapy, and immunotherapy. Even now, nearly 75% of the lung cancer patients have developed into advanced stage at the time of diagnosis (stage III/IV)85, melanoma144 and pancreatic ductal adenocarcinoma145. As shown in Table 94. Previous studies have provided novel insights into the microbiota alteration during the development and exacerbation of various lung diseases, which help establish a non-invasive detection method118. Zheng et al. identified and established the specific gut microbial signatures for the prediction of early-stage lung cancer94. Yan et al. found that Neisseria, Steptococcus, and Porphyromonas were significantly higher in the saliva from lung cancer patients, which may serve as potential biomarkers for the disease detection/classification92. A pilot study using 16S rRNA sequencing revealed that greater abundance of Bacteroidaceae, Lachnospiraceae, and Ruminococcaceae in lung tissue were significantly associated with a decreased risk of recurrence-free (RFS) and disease-free survival (DFS)90. There is no doubt that further clinical studies are necessary to establish microbial markers for predicting lung cancer in the future. Moreover, the precise role of salivary, sputum and feces microbiome in lung cancer initiation and progression is largely unknown and the potential molecular mechanisms awaits further investigation.Nowadays, the widely used and effective diagnostic tools for lung cancer are chest X-ray and CT in clinic. However, the examination by low-dose spiral CT is still unable to fully popularized due to its high cost and inconvenience. The best option for lung cancer screening is to examine groups with high-risk disease characteristics including age, gender, long-term smoking, and occupational exposure. Different from the previous methods, it will be better to explore the interaction between gut microbiota and lung cancer, and try to find the microbial alterations and specific microbes that are closely associated with lung cancer, which can provide better targets to pick out the high-risk groups for chest X-ray and CT. With development and popularization of deep sequencing, the associations between microbiota in different human body sites and various diseases have held interests from both researchers and clinicians. It has been reported that there is a significant correlation between microbiota landscape alterations and development of various cancer including lung cancer146. Furthermore, a unique microbial signature with enhanced IL-1β, IL-6, and TNF-α expression compared with naïve microbiota, was observed in post-radiation mouse model tissue147. It is promising to identify microbes hypersensitive to radiation as predictive biotargets for improvement of curative effects. Microbiota might serve as a therapeutic strategy to reduce radiation-induced toxicity and improve the prognosis of lung cancer patients after radiotherapy148. Recent studies have implicated that gut microbiome plays a crucial role in drug metabolism, chemotherapy-induced toxicity and host response sensitivity149. The gut microbiota can directly modulate drug absorption and metabolism via microorganisms and microbial enzymes151. In addition, gut microbiota can also indirectly affect the rate of metabolism in oral and systemic administration via regulating gene expression, local mucosal barrier response, and the physiology of distant organs153. Experiments in vivo and in vitro have indicated a complex and multi-level intervention relationships between chemotherapeutic agents and human microbiota. Some special microbial species and metabolites have been confirmed to inhibit the antineoplastic drug gemcitabine and promote the prodrug CB1954 in blood circulation154. Another subset of mainstream antineoplastic drugs is genotoxic platinum drugs, exhibiting anti-cancer effect via inhibition of DNA replication and by targeting plasma membranes and mitochondria, which leads to adverse drug reactions including intestinal toxicity, nephrotoxicity, blood–brain barrier integrity disorder and deafness156. A possible explanation is that destruction of the intestinal mucosal barrier enables microorganisms or pathogens invade into the mesenteric lymph nodes and blood circulation157. Furthermore, accumulating studies prove that effects of antineoplastic drugs with broad spectrum can be reduced, and antibiotics abuse may not only aggravate the side effects of anti-tumor drugs but also cause severe additional systemic side-effects159. At present, most studies of microbiome and chemotherapeutic drugs remains in the stage of animal experiments, and there is only little research directly exploring the gut microbiota alterations and functions in post-chemotherapy patients with lung cancer. Additional mechanistic studies and clinical trials are still required to investigate whether modulation model of gut microbiota could become an effective clinical approach to assist treatment of lung cancer with chemotherapeutic drugs and minimize drug-induced toxicity.Radiotherapy for advanced lung cancer has become a routine treatment in clinical practice, although it brings some unexpected side effects such as immune damage and radiation-induced toxicity. However, the relationship between gut microbiome and ionizing radiotherapy of cancer remains unclear as there was not too much progresses in this field. A recent study revealed that mice fecal microbiota transplantation could reduce radiation-induced damage without promoting cancer cell proliferation and migration in vivo161. For example, the microbiome of 249 cancer patients who underwent PD-1 immunotherapy was examined by a French research group. Among them, 69 patients were given antibiotics due to other diseases at the beginning of treatment which will disrupt the intestinal flora. Surprisingly, patients treated with antibiotics have shorter cancer-recurrence time and survival time than those who did not receive antibiotics, indicating that antibiotics consumption could greatly reduce the effectiveness of immunotherapy141. A follow-up study compared the gut microbiota of the two groups of patients and isolated the probiotics Akkermansia muciniphila from the stool of the recovered patients. And this probiotic has been proved to be effective in the prevention of obesity and diabetes in previous studies. Interestingly, this study also demonstrates its contributions to cancer immunotherapy. Moreover, researchers implanted the feces of the recovered patients into germ free mice and those who received “effective” feces was responding quickly to PD-1 inhibitors. In addition, the oral probiotic Akkermansia muciniphila can also restore the same effect of immunotherapy162. One possible reason is that a higher diversity of microbiome communities might be positively correlated with T cell activity, which in turn causes cancer cells to be killed more thoroughly. Inversely, patients with “bad bacteria” have more regulatory T cells which could suppress the host immune response. A recent study of Chinese patients with advanced non-small cell lung cancer who treated with the immunological checkpoint inhibitor PD-1 showed that patients with higher gut microbiota diversity presented better response to anti-PD-1 immuno-checkpoint inhibitors. Patients with favorable gut microbiome (such as those with high diversity) exhibit enhanced memory T cell and natural killer cell signatures in the periphery blood163. Shi et al. revealed that systemic administration of Bifidobacterium potently stimulated STING signaling and increased cross-priming of dendritic cells after anti-CD47 treatment, which converts the non-responder mice into responder164. Paulo et al. brought forward a hypothesis that gut microbiome may help prime an immune response through TLR4-signaling165. Tumor-associated myeloid cells might be activated by commensal gut bacteria  to produce TNF and other inflammatory cytokines that mediate the tumor microenvironment and anti-tumor effect of immunotherapy159. After treatment with cyclophosphamide, the translocation of specific commensal bacteria into mesenteric lymph nodes enhanced Th17 responses in the spleen and the induction of memory Th1 responses, which proved the important role of commensal microorganisms in MyD88 and TLR signaling158. Intriguingly, a new research demonstrated that the commensal lung bacteria stimulated the production of Myd88-dependent IL-1β, IL-2β, and IL-17 and other effector molecules to promote inflammation and tumor cell proliferation82. These studies revealed a close relationship between intestinal flora and cancer immunotherapy which provided new insights for improving the effectiveness of tumor immunotherapy. Nevertheless, further understanding of the effects and mechanisms of microbiome and immunotherapy needs more explorations.It was previously reported that the disorder of the intestinal microbiota may affect the immunotherapeutic effect for cancer166. One of the major effects of administration of prebiotics and probiotics is to achieve optimal host immune homeostasis through maintaining the diversity and relative numbers of Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria and so on168. On the other hand, the pharmaceutical and biotechnology companies worldwide are looking for microbial drug targets for developing and promoting chemotherapy and targeted therapy for cancer. Drug targeting microbiome have potentials to lessen the side effects resulting from chemotherapy, in which a clinical trial suggested a clinical benefit from administering neomycin concurrent with irinotecan to diminish the side effects169. In another preclinical study, mice treated with the novel small-molecule inhibitors targeting bacterial β-glucuronidase were protected from irinotecan (an anticancer drug) induced diarrhea171. However, the current limited investigation and knowledge about the beneficial microbiota and molecular mechanisms cannot provide the best method to dissect the host microbiome. Whether the microbial changes will cause unexpected local homeostasis disorders, inflammatory response or even precancerous lesions remains elusive. Recently, FDA issued a safety alert on the application of FMT for the risk of serious adverse events due to transmission of pathogenic organisms172. Even though emerging achievements have indicated a promising application of microbiome in anti-cancer action, future studies should focus more on the causal effects of lung microbiome alterations on lung diseases and identify the healthy and beneficial lung microbiome.At present, mature products targeting microbiome that have entered the commercial market include probiotics, prebiotics, and synbiotics, having showed generally safety in different clinical practice. The general effects revealed by increasing clinical data include promoting gastrointestinal homeostasis and integrity, regulating metabolism via productions of SCFAs (short-chain fatty acids) and vitamin or second bile salts, participating digestive activities and neutralization of inflammation and carcinogens3. As shown in Fig. 175. It will be very interesting to dissect the lung-resident microbiome but there are technical challenges regarding to the characterization of the low-biomass lung microbiome by the next generation sequencing technologies177. Second, the role of microbial components such as fungi and virus other than bacteria, is largely unexplored in lung cancer, partly due to their relatively lower abundance and lack of well-characterized reference genomes. Third, currently most studies are conducted in laboratory mice. The difference in microbiome structure between human and mice can result in limited translational potential from murine studies to human diseases. Therefore, more effective and human relevant preclinical models approaching the causal relationship between microbiome and tumor is particularly important. For example, the development of patient derived organoid will be a good option180. Fourth, the role of microbiota in the progression of lung cancer has attracted more attention to the interaction between tumor immune microenvironment and microbiota82, but lung tumor microenvironment derived microbiota was also reported to acted directly on the tumor cells91. Finally, there’s an associated effect between the gut microbiota and immunotherapy in the advanced NSCLC163. And the microbial biomarkers in early-stage lung cancer, or probiotics for treatment of lung cancer still need to be explored. Although the huge potential of microbiome has drawn a great landscape for prevention and treatment of lung cancer, it was generally recognized that development of this field requires more multidisciplinary and in-depth exploration. A better understanding of microbiome in the process of cancer initiation and different response to multiple treatments may provide great opportunities for promoting diagnosis and prognosis of lung cancers patients.Lung cancer has been the leading cause of cancer-related deaths worldwide mainly due to its initially asymptomatic and typically diagnosis at an advanced stage


The conventional pedicle-screw-based dynamic stabilization process involves dissection of the Wiltse plane to cannulate the pedicles, which cannot be undertaken with minimal surgical invasion. Despite some reports having demonstrated satisfactory outcomes of dynamic stabilization in the management of low-grade spondylolisthesis, the extensive soft tissue dissection involved during pedicle screw insertion substantially compromises the designed rationale of motion (muscular) preservation. The authors report on a novel method for minimally invasive insertion of dynamic screws and a mini case series.The authors describe innovations for inserting dynamic screws via the cortical bone trajectory (CBT) under spinal navigation. All the detailed surgical procedures and clinical data are demonstrated.A total of four  patients (mean age 64.75 years) with spinal stenosis at L4–5 were included. By a combination of microscopic decompression and image-guided CBT screw insertion, laminectomy and dynamic screw stabilization were achieved via one small skin incision (less than 3 cm). These patients’ back and leg pain improved significantly after the surgery.This innovative dynamic screw stabilization via the CBT involved no discectomy , no interbody fusion, and little muscle dissection (not even of the Wiltse plane). As a minimally invasive surgery, CBT appeared to be a viable alternative to the conventional pedicle-screw-based dynamic stabilization approach. Various fusion techniques have been accepted as the surgical management for disc degenerative disease or spondylolisthesis, including anterior, posterior, transforaminal, and extreme lateral lumbar interbody fusion  –4. In thPedicle-screw based dynamic stabilization systems use elastic materials, for example, a synthetic cord-and-spacer or metallic spring, instead of a rigid metallic rod, to connect between individual pedicle screws . PlacemeThe authors report in this technical note an innovative method to insert four dynamic screws via a 3-cm skin incision to neutralize a degenerative spine or to stabilize low-grade spondylolisthesis. Under image guidance, the screws were placed along the cortical bone trajectory (CBT) and assembled with dynamic cords. The surgery involved microscopic decompression of the bilateral neuroforamen and dynamic screw insertion, but no discectomy  or interbody fusion procedures. Compared to the standard minimally invasive TLIF, the novel technique preserved the intervertebral disc and required less muscle dissection. Although the theoretical benefit of reducing ASD requires longer-term follow up to confirm, this technique spared arthrodesis and was done with minimal soft tissue or facet destruction. This is the first report to describe dynamic screw placement via the CBT.Four consecutive patients , with an average age of 64.75 years, who underwent minimally invasive dynamic screw stabilization with the Dynesys Dynamic Stabilization  system were retrospectively analyzed.After general anesthesia, the patient who had a L4–5 herniated disc with bilateral foraminostenosis Fig.  was put After decompression, the O-arm  was brought into the room to acquire images for navigated screw placement. The reference pin was inserted into the left posterior superior iliac crest. The acquired images were transferred to the Stealth Station  for spinal navigation. Prior to the beginning of instrumentation, we used an image-guided ball tip probe to confirm the accuracy of navigation and locate the entry points. The entry points of CBT screws are generally over the cephalad lateral part of the pars interarticularis, slightly caudal to the sulcus of the facet complex. An image-guided high-speed drill was used to break through the cortex. The trajectory was chosen, using the navigation system, to allow the screw to course through the dense cortical bone with the screw tip barely penetrating the cortex of the vertebral body laterally. A 5.2 mm image-guided cannulated tap was used to create the screw tract. The Dynesys screws  were subsequently placed under navigation. A total of four CBT screws were inserted into the L4 and L5 bodies with bi-cortical purchase  and she was discharged from the hospital on post-operation day 5.A 58-year-old male presented with low back pain and left sciatica for 6 months. The symptoms were on and off several times and it got worse prior to a visit to our clinic.His radiographic evaluations demonstrated a huge L4–5 ruptured disc with caudal migration and left L4–5 foramen compression. Since his symptoms were compatible with nerve root compression by the L4–5 ruptured disc, surgical decompression, removal of sequestrated disc fragments and dynamic stabilization were recommended. The post-operative fluoroscopy demonstrated that 3.1 degree motility was preserved at post-op 12 months  for lumbar degenerative disease , 9, 10. The CBT used for screw insertion was first published by Santoni et al. in 2009 for posterior midline lumbar fusion surgery. Compared with the traditional pedicle-screw trajectory used, several biomechanical studies have demonstrated the superiority of CBT screws, which could yield a 30% increase in pullout strength and a 71% increase in insertional torque , 18. MorDynamic screw stabilization could be achieved with minimally invasive surgery via the CBT by a combination of microscopic decompression and spinal navigation. This innovative dynamic screw stabilization involves no discectomy, no interbody fusion, no facet violation, and little muscle dissection (not even of the Wiltse plane). As a minimally invasive surgery, CBT appeared to be a viable alternative to the conventional pedicle-screw-based dynamic stabilization. However, long term studies are needed to investigate such a minimally invasive surgical strategy.


BRCA2-driven breast cancer monitored for disease progression and acquired resistance using longitudinal multi-tissue genomic testing. Briefly, genomic testing was performed throughout the course of disease on tumor tissue from multiple sites, circulating tumor DNA from blood plasma, and matched normal tissue. Genomic analyses identified actionable variants for targeted therapies, as well as emerging resistance mutations over time. Two unique BRCA2 somatic alterations (p.N255fs and p.D252fs) were identified upon resistance to PARP inhibitor and platinum treatment, respectively. Both alterations restored the open reading frame of the original germline alteration, likely accounting for acquired resistance. This case exemplifies the evolution of multiple subclonal BRCA reversion alterations over time and demonstrates the value of longitudinal multi-tissue genomic testing for monitoring disease progression, predicting measures of response, and evaluating treatment outcomes in oncology patients.BRCA-mutant cancers often develop therapeutic resistance through several mechanisms. Here, we report a case of pathogenic germline  BRCA1/BRCA2 alterations have an increased risk of breast, ovarian, pancreatic, prostate, and other cancers. Tumors that arise in these patients typically exhibit a somatic mutation, loss-of-heterozygosity (LOH), or epigenetic silencing in the wild-type BRCA allele, resulting in truncated or absent BRCA proteins and defective homologous recombination DNA repair1. This homologous recombination deficiency (HRD) renders DNA particularly vulnerable to damage caused by double-strand breaks, resulting in an accumulation of mutations over time and increased carcinogenesis2. However, HRD also renders BRCA1/2-mutant cancers sensitive to DNA-damaging agents, such as radiation4, platinum-based therapies6, and poly ADP-ribose polymerase (PARP) inhibitors8.Individuals with pathogenic germline 9. Therefore, PARP inhibition targets DNA repair through multiple mechanisms of action, including PARP trapping11, inhibition of base excision repair of single-strand breaks8, and indirect activation of non-homologous end-joining14. In tumors with HRD, such as those with BRCA alterations, PARP inhibition is especially effective because multiple DNA repair pathways are simultaneously impaired, resulting in synthetic lethality8.PARP inhibitors target the highly abundant proteins PARP1 and PARP2, which play an important role in transcription, chromatin modification, and DNA repair17. Recent studies suggest response rates continue to improve with combined treatment regimens. However, despite initial effectiveness, BRCA-mutant cancers often develop resistance to PARP inhibition19. While many potential mechanisms for this resistance have been described, BRCA reversion mutations have emerged as a key resistance mechanism and have been described in a number of recent cases23. BRCA reversions occur when acquired somatic mutations, typically insertions/deletions (indels) or base substitutions, restore the open reading frame of the altered BRCA allele, resulting in a functional protein that restores efficient homologous recombination DNA repair. As a result, PARP inhibition no longer causes synthetic lethality, leading to drug resistance and disease progression.In BRCA-mutant breast cancers, single-agent PARP inhibitor treatment induces partial response rates in as high as 47% of patients, and complete response lasting 60 weeks in up to 33% of patientsBRCA2-driven breast cancer who acquired resistance to the PARP inhibitor olaparib. The resistance likely resulted from an acquired somatic reversion mutation, which was detected by matched tumor-normal genomic analysis. A second reversion mutation was also detected after carboplatin treatment through genetic sequencing of circulating tumor DNA (ctDNA) in blood plasma. This case highlights the benefits of longitudinal genomic testing, using multiple assay and tissue types including both tissue and blood plasma, to track the evolution of tumor mutations in order to provide the best treatment options for each patient based on their unique genomic profile.Here we report the case of a patient with pathogenic germline BRCA2 germline alteration, and the patient opted for a bilateral oophorectomy 2 years after starting tamoxifen. Her anti-estrogen therapy was changed to anastrozole, which she maintained for an additional 5 years. The patient palpated a mass in right axilla, but imaging workup did not show definitive evidence of malignancy or target for biopsy ).At the age of 50, a female patient without regular mammography screening presented with a mass in her left breast . Patient timeline is presented in Fig. BRCA2 alteration  was not part of a typical clinical workup. Due to a difference in 10 years between primary diagnosis and metastatic disease, with evidence of clinical utility and a change in hormone status, the metastatic lesion was sent for NGS sequencing. The tumor-normal matched genomic analysis of the metastatic bone lesion and blood sample confirmed the presence of the known germline BRCA2 at a variant allele frequency (VAF) of 18.1%, which was in cis  with the pathogenic germline alteration. This somatic alteration  in combination with the germline frameshift resulted in an in-frame indel and subsequent restoration of the BRCA2 reading frame , likely accounting for the resistance to PARP inhibition , as well as an additional unique somatic BRCA2 alteration  at 0.9% VAF. The secondary somatic BRCA2 mutation was also in cis with the pathogenic germline alteration, but in trans with the first somatic reversion mutation. The second somatic mutation in combination with the germline frameshift resulted in an in-frame indel and thus represented a second subclonal reversion mutation  and TP53 . Due to elevated tumor mutational burden (TMB) in the patient’s first sequencing results, and the possibility that previous therapies may have contributed to the development of neoantigens, the patient briefly underwent immunotherapy with one cycle of ipilimumab and nivolumab therapy. However, liver function worsened and so the therapy was discontinued. Shortly after discontinuation, the patient passed away at the age of 63.A liquid biopsy from blood plasma was obtained 5 months later for ctDNA sequencing. The genomic analysis identified the known somatic and germline BRCA2 reversion mutation that restored the open reading frame of a germline frameshift alteration. This case is consistent with recent reports of BRCA reversions in both germline and somatic alterations reported in prostate cancer, ovarian cancer, and breast cancer after treatment with PARP inhibitors, which were associated with resistance to treatment25. However, in most studies to date, the timing and mechanism of the reversion alterations remain unclear due to a lack of longitudinal testing that started before the reversion alteration appeared. Indeed, many patients receive different lines of treatment or neoadjuvant therapies before PARP inhibitor treatment that may induce the accumulation of mutations27. It is possible, a small population of a clone with a reversion mutation could exist in a patient for years, and once PARP inhibition is initiated that clone is selected for and becomes the dominant clone.Here we report a case of longitudinal diagnostic testing methodology using multiple assay and tissue types. This patient acquired resistance to PARP inhibitor olaparib, a result of a somatic BRCA reversion alterations over time and highlights the utility of combined tissue/normal/blood biopsies in routine care of patients with cancer. For example, genomic analysis of tumor-normal matched samples enables a more thorough understanding of germline and somatic alterations and can identify co-existing actionable variants that may have been overlooked in standard genetic tests. In addition to the previously identified germline BRCA alteration in this case, CDK4 and MYC copy number alterations were identified by NGS of matched tumor-normal tissue, informing subsequent treatment decisions. Indeed, CDK4 inhibition with palcociclib allowed for a year of successful treatment before treatment with PARP inhibitor olaparib began.This case exemplifies the evolution of multiple subclonal Like many patients with BRCA-mutant cancers, this patient initially responded favorably to PARP inhibition. However, after 9 months of PARP inhibitor therapy, the patient was diagnosed with progressive disease and metastasis to the liver. Genomic analysis of the liver metastasis revealed a somatic reversion mutation absent from the previous bone metastasis biopsy. This somatic reversion restored the open reading frame in tumor cells, enabling the synthesis of an in-frame BRCA2 protein and efficient DNA repair through homologous recombination, which is consistent with the resistance to PARP inhibitor treatment this patient experienced.28. While reliable at identifying driver alterations in solid tumors, due to the heterogeneous nature of tumors and emerging resistance mutations, genomic solid tissue analysis can be somewhat limited in detecting the diversity of mutations associated with advanced cancers. Indeed, many studies have identified actionable mutations in metastatic lesions that were not present in the primary tumors31. Because tumor cells from multiple locations can shed DNA into the blood, liquid biopsies can detect alterations present in distant metastases. As such, analysis of ctDNA from liquid biopsies is increasingly used in combination with solid tissue analyses.Additionally, this patient received a liquid biopsy that identified a second reversion alteration several months after identification of the first somatic reversion in a solid tumor biopsy. It is unclear whether the second subclonal mutation was present in another tumor site when the first reversion was identified, if it was acquired after discontinuation of PARP inhibition, or in response to the platinum treatment as has been previously documentedCritical questions remain in the treatment of BRCA-mutant cancers. For example, for which patients should serial genomic testing be performed, and how should reversion alterations impact clinical decision-making? While ideally all patients would have access to serial genomic testing, in reality this is unlikely to be feasible in the near future due to cost and availability. However, serial testing will be particularly relevant for patients taking drugs with known resistance mechanisms, such as PARP inhibitors and platinum-based therapies. How reversion alterations may affect clinical decision-making likely depends on whether the reversion was detected in the tumor tissue or in blood plasma (liquid biopsy). For example, if a reversion alteration is detected through liquid biopsy, but not in the tumor tissue biopsy, continuation of PARPi treatment seems reasonable. However, the patient would benefit from being more closely monitored, as the identification of the reversion alteration in cfDNA indicates imminent progression.BRCA reversions, the genetic source of resistance, as well as additional actionable variants for targeted therapy. This demonstrates the value of routine genomic testing in clinical care of oncology patients for monitoring disease progression, predicting measures of response, and evaluating treatment outcomes.In conclusion, serial NGS sequencing with multiple assays and sample types, including paired solid tumor/normal and liquid biopsy, revealed the evolution of Overall tumor content and percent tumor cellularity as a ratio of tumor-to-normal nuclei verified specimens met a 20% threshold. Solid tumor total nucleic acid was extracted from FFPE tissue sections using Chemagic 360 sample-specific extraction kits (Perkin Elmer) and digested by proteinase K.33. Briefly, 100 ng of DNA was mechanically sheared to an average size of 200 base pair (bp) using a Covaris Ultrasonicator. KAPA Hyper Prep Kit was used to prepare DNA libraries, which were then hybridized to the xT probe set, and amplified with the KAPA HiFi HotStart ReadyMix. Library preps were hybridized to xGEN Exome Research Panel v1.0 (Integrated DNA Technologies) and target recovery was performed using Streptavidin-coated beads. This was followed by amplification with the KAPA HiFi Library Amplification Kit. The amplified target-captured library was sequenced using 2 × 126 bp paired-end (PE) reads to an average unique on-target depth of 500× (tumor) and 150×  on an Illumina HiSeq 4000. Samples were evaluated for uniformity and verified to have 95% of all targeted bp sequenced to a minimum depth of 300×.DNA sequencing of 596 genes was performed as previously describedWhole blood samples were collected in Streck Cell-Free DNA BCT (blood collection tubes) and centrifuged to separate plasma and buffy coat. Plasma was centrifuged a second time to remove any cellular or platelet carryovers. Plasma hemolysis was scored and reviewed by a pathologist prior to cfDNA extraction. cfDNA was isolated using the Qiagen QIAamp MinElute ccfDNA Midi Kit and the QIAcube system. The Fragment Analyzer was used to evaluate the quality of the extracted cfDNA. Quality was verified by the presence of a primary peak at approximately 150 bp and minimal to no genomic DNA (gDNA) peaks.® Ultra™ II DNA Library Prep Kit for Illumina®, hybridized to the xF probe set, captured using Streptavidin-coated beads, and amplified with the KAPA HiFi Library Amplification Kit. The amplified target-captured cfDNA library was sequenced using 2 × 151 bp PE reads to an average unique on-target depth of 4500× on an Illumina NovaSeq 6000.The xF assay is a 105-gene hybrid capture NGS panel designed to detect actionable oncologic targets in plasma. Briefly, a minimum of 30 ng of cfDNA is required as input for library preparation. cfDNA libraries were prepared using the New England BioLab’s NEBNext33. Briefly, adapter-trimmed FASTQ files were aligned to the 19th edition of the human reference genome build (hg19) using Burrows-Wheeler Aligner (BWA). Following alignment, reads were grouped by alignment position and UMI family, and collapsed into consensus sequences using fgbio tools. SNV and indel variants were detected using VarDict. Copy number variants (CNVs) were analyzed using CNVkit15 plus a Tempus CNV annotation and filtering algorithm. Rearrangements were detected using the SpeedSeq analysis pipeline. Gene rearrangements were analyzed by LUMPY. Data were visualized using Integrative Genomics Viewer (IGV)34. Individual reads in IGV were colored pink or blue, based on forward or reverse orientation, respectively. Reads containing the deletion variants were sorted to the top for visualization and cis and trans calls were made manually.Variant detection, visualization, and reporting were performed as previously describedWe have complied with all ethical regulations. This is a case report of one patient, and she did not receive any experimental procedures or treatments as part of this study. As this is a retrospective analysis of data obtained through standard treatment protocols, this case does not require IRB approval. Written informed patient consent for clinical testing, analysis, and publication was obtained by Tempus Laboratories.Further information on research design is available in the Reporting Summary


However, the efficacy of venetoclax in myeloma patients with the t IGH::CCND3 translocation remains less investigated.Methods: In this study, we conducted a retrospective analysis to investigate the efficacy of venetoclax-based therapy in relapsed/refractory myeloma patients with t translocation. The treatment courses of three patients, that included previous therapies and responses to venetoclax, were assessed. Clinical data, laboratory results, and adverse events were analyzed to evaluate treatment outcomes.Results: Our findings demonstrated remarkable therapeutic responses in three consecutive patients with t translocation-associated myeloma who received venetoclax-based therapy. Patient 1, a lenalidomide-bortezomib-daratumumab and alkylator treatment refractory patient, achieved sustained stringent complete remission (sCR) after combining carfilzomib-dexamethasone with venetoclax, which was his best response ever. Similarly, Patient 2, refractory to frontline bortezomib-thalidomide-dexamethasone therapy, attained CR following a transition to bortezomib-dexamethason-venetoclax treatment. Patient 3, who was immunomodulatory (IMID)-intolerant, showed a highly favorable response to venetoclax-dexamethasone therapy after his first relapse following autologous stem cell transplantation. No significant adverse effects were observed in any of the patients.Discussion: Our study provides compelling preliminary evidence for the efficacy of venetoclax in t translocation-associated myeloma. The outcomes observed in our patients suggest that venetoclax-based therapy holds substantial promise as an effective treatment option for this specific genetic subgroup. Furthermore, the similarities in treatment response between t and t translocation subgroups highlight the importance of personalized approaches targeting specific genetic abnormalities to optimize therapeutic outcomes. Multiple myeloma is a complex hematological malignancy characterized by the clonal proliferation of plasma cells in the bone marrow. It is considered a quintessential disease of aging, as it is typically diagnosed at an average age of 70 years . The incDespite the therapeutic benefits observed with venetoclax –18, concThe expression of Bcl-2, a key anti-apoptotic protein and the molecular target of venetoclax, has emerged as an important predictor of therapeutic response to venetoclax in myeloma patients. Multiple myeloma is characterized by significant genomic heterogeneity . ExpressRecognizing the striking similarity in gene expression profiles between patients with t and t translocations –25, our Within this context, our present pilot study aims to investigate the safety and efficacy of venetoclax in a distinct subset of multiple myeloma patients. By analyzing the clinical data of a meticulously selected relapsed/refractory patient cohort exhibiting the t translocation, our study seeks to elucidate the therapeutic potential of venetoclax for this particular genetic subtype. Furthermore, our research endeavors to provide valuable insights into the identification and selection of patients who are most suited to receive venetoclax-based therapy, thus maximizing therapeutic outcomes while ensuring that their safety remains paramount.We conducted a retrospective analysis of the medical records of three patients diagnosed with multiple myeloma characterized by the presence of the t founding translocation, who received treatment that included venetoclax. The publication of this report was approved by the ethics committee of Central Hospital of South Pest/National Institute of Hematology and Infectious Diseases, and written informed consent was obtained from the patients. Each individual therapy was a preapproved “off-label” therapy authorized by the Hungarian National Institute for Pharmacy and Public Health (OGYÉI/22147-2/2021).Data were collected from the patient’s medical records, which included comprehensive information such as laboratory test results, imaging studies, surgical reports, and medication administration records. The patients’ clinical courses were meticulously tracked from their initial admission to our department up to the present day. Descriptive statistics were employed to analyze the patient’s clinical data, including laboratory test results and medication regimens. The changes in M protein and λFLC (free light chain) over time were graphically depicted to illustrate the treatment response. Assessment of therapy response additionally encompassed monitoring clinical symptoms, laboratory test results, and the occurrence of adverse events.IGH::CCND3 translocation and monosomy 13 based on fluorescence in situ hybridization (FISH) analysis.Patient Information: A 56 year-old male was diagnosed with IgG kappa myeloma in June 2013. He presented with an International Staging System (ISS) score of III and demonstrated the per os Melphalan treatment. In 2017, he was administered daratumumab monotherapy, followed by daratumumab, bortezomib, and dexamethasone triplet treatment in 2020. Despite the various treatment approaches, the patient’s myeloma remained refractory to available therapies, as evidenced by the serial measurements of the M protein, a key marker of myeloma  translocation, further contributing to the complexity of the case.Treatment History: The patient underwent multiple treatment lines over the course of his disease. From 06/14/2013 he received 5 cycles of VDT , followed by high-dose cyclophosphamide  mobilization and autologous stem cell transplantation . In subsequent treatment lines, the patient received 4 cycles of Len-Dex . The 5th and 6th cycles of Len-Dex were unsuccessfully combined with  myeloma . He alsoKD-Venetoclax Treatment: Given the patient’s refractory myeloma, the presence of a plasmacytoma, and the identification of t translocation, a decision was made to initiate KD (carfilzomib plus dexamethasone)-Venetoclax (400 mg) therapy in 2021. This therapeutic approach aimed to target the identified genetic abnormality and overcome the patient’s refractoriness to previous treatments. Regular monitoring of treatment response, disease progression, and potential adverse effects was carried out to assess the efficacy and safety of the KD-Venetoclax regimen.Best Response Ever: Stringent Complete Remission: Remarkably, the patient demonstrated an unprecedented response to the KD-Venetoclax treatment. Serial measurements of the M protein revealed a complete remission, indicating the absence of detectable levels  and normConclusion: Patient #1, a 56 year-old male with IgG kappa myeloma, faced multiple treatment lines with limited success. The presence of cervical spine plasmacytoma and the identification of t translocation posed significant challenges in managing his refractory disease. The initiation of KD-Venetoclax therapy aimed to address these complexities, providing a targeted approach against the identified genetic abnormality. The remarkable sustained complete remission observed following KD-Venetoclax treatment represents a significant milestone in the patient’s journey and highlights the potential of tailored therapies in improving treatment outcomes. Continuous monitoring and further investigation are required to assess the durability and long-term benefits of this exceptional treatment response.+ selected plasma cells revealed an atypical IGH::CCND3 fusion with the absence of CCND3::IGH on the derivative chromosome 6. Notably, three intact FGFR3 genes (possible trisomy 4) and three intact CCND1 genes (possible trisomy 11) were also detected by interphase FISH.Patient Information: A 78 year-old male was diagnosed with IgG kappa myeloma in January 2022. The ISS score was determined as I, and FISH analysis from CD138Treatment History: The patient initially received VDT  treatment, but not even minimal response (MR) was observed. The patient’s condition and the refractoriness to VDT treatment necessitated salvage treatment. Due to the presence of t translocation, the treatment was switched to a combination of VDT with Venetoclax (400 mg) in March 2022 and to a combination of VD (bortezomib and dexamethasone) with Venetoclax (400 mg) from September 2022 to March 2023. Notably, the patient achieved a rapid VGPR concluding into complete immunohematological response following the treatment combination with Venetoclax/dexamethasone (bortezomib was stopped at month 7 of therapy). Serial measurements of the M protein indicating complete remission are shown in Patient Information: A 73 year-old male was diagnosed with IgA lambda myeloma in September 2020, accompanied by significant light chain excretion. The ISS score was determined as II, and FISH analysis revealed the presence of a t translocation, as well as gains of chromosomes 1q21, 11, and 17.Treatment History: The patient initially received VDT. However, due to intolerance to thalidomide (adverse skin reaction), the treatment line was discontinued. From November VD treatment was combined with lenalidomide, however, due to again adverse skin reactions, this treatment line had to be discontinued. From the end of November, VD treatment (bortezomib and dexamethasone) was administered until February. At the end of February 2021, the patient received high-dose cyclophosphamide (HDCy) priming treatment for peripheral stem cell mobilization, and in March 2021 autologous stem cell transplantation (ASCT) was performed with standard melphalan conditioning. The patient achieved a complete immunohematological response following this treatment combination. Serial measurements of the M protein and λFLC indicating a temporal complete remission are shown in The three cases presented in this report underscore the potential efficacy of venetoclax treatment in myeloma patients with the t translocation. These findings further extend the initial observations from the BELLINI study, emphasizing the importance of targeted therapies in specific genetic subtypes of multiple myeloma. Importantly, our study contributes novelty by specifically evaluating the response to venetoclax in patients with the t translocation, a relatively understudied area.CCND3 (Cyclin D3) gene, which is involved in cell cycle regulation. This dysregulation results in increased expression of Cyclin D3, leading to uncontrolled cell proliferation and contributing to the pathogenesis of myeloma with t translocation. Prognostically, myeloma patients with t translocation tend to exhibit distinct molecular patterns, clinical features, and outcomes compared to those without this translocation. Studies with limited power have suggested that myeloma cases with t translocation may be associated with a higher risk of disease progression, shorter survival times, and poorer responses to conventional treatments. The presence of this translocation may correlate with a more aggressive disease course and with a more challenging clinical scenario. Given the unique gene expression profile and uncertain prognosis associated with myeloma harboring t translocation, there is a need for targeted therapeutic approaches.The t translocation is rarely seen in multiple myeloma, accounting for approximately 1%–3% of cases. This translocation leads to distinct alterations in gene expression profiles. One of the notable consequences of the t translocation is the dysregulation of the The BELLINI study provided valuable insights into the therapeutic potential of venetoclax in multiple myeloma. It demonstrated promising efficacy and improved progression-free survival in patients receiving venetoclax in combination with standard treatment regimens . HoweverThe treatment outcomes observed in our three patients with t translocation-associated myeloma provide compelling evidence of the rapid and deep therapeutic responses achieved with venetoclax-based therapies. Patient 1, who had been battling myeloma for nearly a decade and experienced partial responses alternating with relapses during previous treatments, achieved sustained complete remission (CR) when carfilzomib-dexamethasone treatment was combined with venetoclax. Similarly, Patient 2, who exhibited complete refractoriness to frontline VDT therapy, attained CR after transitioning to VD-venetoclax treatment. Patient 3, an IMID-intolerant individual with the t translocation, demonstrated a highly favorable response to Venetoclax-Dexamethasone therapy in his first relapse after ASCT. These outcomes highlight the efficacy of venetoclax in this specific genetic subgroup and reinforce its potential as a valuable therapeutic option. Notably, no significant adverse effects were observed in any of the patients.The collective evidence derived from these cases strongly suggests that venetoclax holds substantial promise as an effective treatment option for myeloma patients harboring the t translocation. The notable efficacy of venetoclax in both t and t translocation subgroups can be attributed to the striking similarity in gene expression profiles observed between these two subgroups. Recent studies suggest that venetoclax-sensitive myeloma cell lines retain a B cell–like pattern of gene expression and chromatin accessibility . It is aIn addition to the treatment outcomes observed in our three patients, our study plays a pivotal role in addressing a significant gap in the existing literature by specifically investigating the efficacy of venetoclax in the context of t translocation-associated myeloma. By focusing on this specific genetic abnormality, we provide novel insights into the management of a subgroup that has historically faced limited treatment options. Through our ongoing research, we aim to contribute to the growing body of knowledge surrounding this specific genetic alteration, ultimately advancing the understanding and therapeutic management of t translocation-associated myeloma. A comprehensive investigation of venetoclax's efficacy in t translocation-associated myeloma will provide valuable information for clinicians and researchers, offering potential new avenues for improving patient outcomes.It is important to acknowledge certain limitations associated with this retrospective analysis. These include the potential for missing or incomplete data within the medical records and the lack of Bcl-2 expression analysis, which would have provided valuable insight into the relationship between Bcl-2 expression levels and treatment response. Additionally, these case reports represent the experiences of a limited number of patients and may not be entirely generalizable to other individuals with similar conditions. Further studies involving larger patient cohorts and prospective designs are required to confirm and expand upon our findings.IGH::CCND3 translocation-associated myeloma, thus extending the existing evidence from the BELLINI study. However, further research and larger studies are necessary to validate these findings and optimize treatment strategies specifically tailored to this genetic subtype. The exploration of personalized approaches targeting specific genetic abnormalities, including the t translocation, holds great potential for improving outcomes in patients with multiple myeloma [In conclusion, our findings provide preliminary evidence supporting the targeted efficacy of venetoclax in t  myeloma –33. Ther