Bicuspid aortic valve (BAV) is a congenital defect affecting 1–2% of the general population that is distinguished from the normal tricuspid aortic valve (TAV) by the existence of two, rather than three, functional leaflets (or cusps). BAV presents in different morphologic phenotypes based on the configuration of cusp fusion. The most common phenotypes are Type 1 (containing one raphe), where fusion between right coronary and left coronary cusps (BAV R/L) is the most common configuration followed by fusion between right coronary and non-coronary cusps (BAV R/NC). While anatomically different, BAV R/L and BAV R/NC configurations are both associated with abnormal hemodynamic and biomechanical environments. The natural history of BAV has shown that it is not necessarily the primary structural malformation that enforces the need for treatment in young adults, but the secondary onset of premature calcification in ~50% of BAV patients, that can lead to aortic stenosis. While an underlying genetic basis is a major pathogenic contributor of the structural malformation, recent studies have implemented computational models, cardiac imaging studies, and bench-top methods to reveal BAV-associated hemodynamic and biomechanical alterations that likely contribute to secondary complications. Contributions to the field, however, lack support for a direct link between the external valvular environment and calcific aortic valve disease in the setting of BAV R/L and R/NC BAV. Here we review the literature of BAV hemodynamics and biomechanics and discuss its previously proposed contribution to calcification. We also offer means to improve upon previous studies in order to further characterize BAV and its secondary complications. Bicuspid aortic valve (BAV) is the most common congenital heart malformation with an estimated prevalence of 1–3%, and a male predominance of 3:1 . The abnin situ, the two cell populations molecularly communicate through paracrine signaling to maintain ECM homeostasis and prevent disease , right coronary (R), and non-coronary (NC) . Each cu disease –7. Previ disease , 9. TogeIn cases of abnormal valve development, two of the three cusps fuse, leading to a bicuspid anatomy with unequal sized cusps that often contain a region of fibrous thickening at the fusion site, known as a raphe . The bicIn vivo models corroborated by human genetic studies have identified a few candidate genes potentially involved in the causal molecular mechanisms of BAV including NOTCH1, GATA family members  as well as ROBO4, eNOS, NKX2.5, and SMAD  and valvulopathy , aortic stenosis, and regurgitation) –18. The To date, the cause of secondary complications in BAV remains unknown. Proponents of a genetic theory hypothesize that the underlying gene mutations responsible for the structural malformation and heritability of BAV are the primary contributors to associated secondary complications. However, in addition there are a growing number of studies supporting the implications of altered hemodynamic and biomechanical influences in BAV. Abnormal mechanical stimuli imposed on the valve may disrupt the normal mechanoregulation of cellular processes responsible for valve homeostasis and subsequently lead to disease. Therefore, the role of hemodynamics and biomechanics in BAV cannot be neglected in the efforts to delineate causal mechanisms of BAV-associated complications.BAV, and associated complications present a large clinical burden for which there are inadequate treatment options currently available. Less invasive options for BAV patients with CAVD and aortic stenosis including balloon aortic valvuloplasty often lead to early symptomatic recurrence. While more invasive approaches such as transcatheter or surgical aortic valve replacement can be associated with suboptimal long-term outcomes , 26. FurThere is growing evidence from fluid and solid mechanics studies demonstrating the presence of an altered hemodynamic and biomechanical environment in patients with a normally functioning BAV, which may contribute to the pathogenesis of secondary complications, such as calcification at a later time , 28–35. in vivo), numerical simulations using image-based models , and bench-top approaches . The use of these methods have provided significant advancements toward our understanding of mechanical stimuli from BAV, although as discussed below, challenges remain in accurately replicating the associated complex hemodynamic and mechanical environment.Several methods have been employed to quantify the mechanical stimuli imposed on the BAV and uncover mechanisms influencing aortic and valvular pathology in this patient cohort. In this review, the approaches have been generally classified into three types: assessment through available clinical imaging modalities  methods including echocardiography, cardiac magnetic resonance imaging (MRI), and computed tomography (CT) often serve as the basis for computational modeling, and have been used independently in the past to describe patient-specific valve morphology and valvular performance  and laser doppler velocimetry (LDV) have been used in bench-top approaches to quantify velocity jet magnitude and direction as well as turbulence characteristics like vortical and helical formation , 43. BenNumerical methods including computational fluid dynamics (CFD) and fluid-structure interaction (FSI) simulations have been applied to BAV modeling with the goal of providing high spatial and temporal resolution , 33, 42.Multi-disciplinary studies coupling advanced imaging methods with state-of-the-art computational modeling are underway to create even better representations of the native and pathologic hemodynamic environments of the aortic valve. These studies attempt to address the limitations of the above approaches. A recent study by Kandail et al.  investig2 vs. TAV of 2.90–4.26 cm2)  , 51–53.  studies , 43, 54.atients,  which isatients, . Factorsatients, . This leatients, , 41, 47.In BAV, WSS indices are largely applicable to VECs that form an impermeable endothelium over the valve cusps. WSS indices act as mechanical stimuli that are ultimately transduced through signaling events via mechanotransduction . The vecAssociated literature point to several WSS indices of potential interest in BAV. WSS magnitude quantified as an average value over the cardiac cycle is most often denoted in the literature as time-averaged WSS (TAWSS), or temporal shear magnitude (TSM) , 58. Temin vitro study by Flemister et al.  when compared to TAV cusps (TSM from 5.8 to 19.9 dyn/cm2). TSG on the ventricularis was lower on the base and belly region of BAV cusps compared to TAV and greater in the tip region of BAV cusps. The BAV fibrosa was subjected to overall lower TSG than TAV with the exception of the base of the non-fused BAV cusp which exhibited higher TSG. These same regions on TAV and BAV R/L cusps experienced mostly unidirectional WSS (OSI < 0.06). While the base of the fused BAV R/L cusp experienced bidirectional WSS (OSI = 0.30), the base of the non-fused BAV R/L cusp and TAV cusps experience closer to unidirectional WSS (OSI < 0.14). Conversely, the fibrosa in all models were exposed to systemically lower WSS magnitudes than the ventricularis, but featured greater variability in regionality and directionality due to valve geometry. Compared to the fibrosa of TAV cusps (TSM from 0.8 to 3.5 dyn/cm2), the non-fused cusp in BAV R/L experienced elevated WSS (TSM > 3.8 dyn/cm2) while the fused cusp experienced sub-physiologic WSS (TSM < 1.3 dyn/cm2). WSS is bidirectional (OSI > 0.14) on the fibrosa of TAV cusps and BAV R/L fused cusps, but unidirectional on the fibrosa of the non-fused BAV type I (OSI < 0.03). Similar findings were attained in a highly sophisticated study conducted by Emendi et al. (2) and decreasing toward the belly and attachment regions. Further, it aligned with previous studies describing systemically lower WSS magnitude on the fibrosa of each cusp, where the fused cusp exhibited much lower WSS compared to the non-fused cusp. Although this study showed that coupling advanced cardiac imaging and computational modeling can provide realistic predictions of the in vivo flow environment, unfortunately results were not compared with a normal control valve to better appreciate the differences in values for WSS in diseased valves. Other computational models have outlined WSS alterations with respect to valve cusp angle and size  as well as non-physiologic WSS. To model non-physiologic WSS, the fibrosa and ventricularis were exposed to conditions normally experienced by the opposite surface. Inflammatory markers including VCAM-1, ICAM-1, TGF-β1 and BMP-2 were highly upregulated on the fibrosa when exposed to non-physiologic WSS, while expression of these markers remained relatively unchanged on the ventricularis. There were no significant changes under physiologic WSS conditions. The upregulation of pro-inflammatory pathways on the fibrosa only in response to non-physiologic WSS led this group to believe that disease initiation could be side-specific and influenced by altered WSS , pro-inflammatory paracrine signaling (indicated by elevated expression of TGF-β1 and BMP-4), and ECM remodeling . Following VIC osteoblast-like differentiation, elevated levels of the bone matrix protein osteocalcin were also detected on the fibrosa and spongiosa when exposed to fused BAV WSS. Additionally, despite immunoblotting data suggesting an increase in Runx2 and α-SMA expression upon exposure to fused and non-fused BAV cusp WSS, the results were not statistically significant when compared to fresh controls  experiences lower magnitude and more oscillatory WSS than the fibrosa of TAV cusps. Additionally, the ventricularis in BAV is subjected to overall higher magnitude WSS compared to TAV cusps. Biomechanics studies comparing the structural-induced alterations in BAV have shown increased strain and radial cusp stretch compared to TAV with little difference in circumferential stretch. Mechanobiology studies have revealed that the WSS and strain imposed on VECs can regulate inflammatory response, endothelial function, ECM remodeling, and VIC phenotype which are all contributing processes leading to calcification.Although we have a greater understanding of the hemodynamic and biomechanical environment of BAV from the clinical imaging studies, numerical modeling, and bench-top approaches employed in the studies above, the data available are not currently sufficient to directly show causality for BAV-associated secondary complications such as calcification. Deepening our understanding of these complex mechanisms will require more comprehensive studies using advanced cardiac imaging modalities and more realistic FSI simulations. For example, if the lack of data surrounding the BAV configuration most prone to developing calcification (BAV R/NC) were more extensively studied, resulting data may provide insight related to the underlying mechanisms of CAVD. Similarly, the pediatric population should now be studied using state-of-the-art computational studies to describe the hemodynamic and biomechanical environment of BAV in its earlier stages before the onset of calcification. Inclusion of these aspects in future investigations may elucidate the mechanisms responsible for secondary complications in BAV.HKaz performed literature review and drafted the article. HKan, JLi, and JLa contributed to revising and editing the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


With increasing use of minimally invasive parathyroidectomy (PTx) over traditional bilateral neck exploration in patients with primary hyperparathyroidism (PHPT), accurate preoperative localization has become more important to enable a successful surgical outcome. Traditional imaging techniques such as ultrasound (US) and sestamibi scintigraphy (MIBI) and newer techniques such as parathyroid four-dimension computed tomography (4D-CT), positron emission tomography (PET), and magnetic resonance imaging (MRI) are available for the clinician to detect the diseased gland(s) in the preoperative workup. Invasive parathyroid venous sampling may be useful in certain circumstances such as persistent or recurrent PHPT. We review the diagnostic performance of these imaging modalities in preoperative localization and discuss the advantages and weaknesses of these techniques. US and MIBI are established techniques commonly utilized as first-line modalities. 4D-CT has excellent diagnostic performance and is increasingly performed in first-line setting and as an adjunct to US and MIBI. PET and MRI are emerging adjunct modalities when localization has been equivocal or failed. Since no evidence-based guidelines are yet available for the optimal imaging strategy, clinicians should be familiar with the range and advancement of these techniques. Choice of imaging modality should be individualized to the patient with consideration for efficacy, expertise, and availability of such techniques in clinical practice. Primary hyperparathyroidism (PHPT) is an endocrine disorder resulting from excess secretion of parathyroid hormone (PTH), from one or more of the parathyroid glands. It accounts for most cases of hypercalcemia and has a prevalence of ~3 in 1000 within the general population. It is most commonly seen in postmenopausal women ), a smaller proportion are as a result of a double adenoma or hyperplasia (multigland disease [MGD]) ,9.Localizing the abnormal parathyroid gland (or glands) is critical to successful PTx and knowledge of the typical and atypical anatomical locations of the parathyroid glands is important in successful localization. Generally, there are four normal glands in place, although supernumerary glands can occur infrequently , up to 7Paired superior glands arise from the fourth brachial pouch and are relatively more consistent in its anatomic location, due to the shorter descent and migration with the thyroid gland. Typically, they are located behind the mid-to- upper pole of the thyroid gland, 1cm above the junction between the inferior thyroid artery and the RLN at the level of the cricoid cartilage gland, posterior to the plane of the RLN . It is iOver the past two decades, unilateral minimally invasive parathyroidectomy (MIP) has largely replaced traditional bilateral neck exploration (BNE) as the surgery of choice for PTx, with the rationale that most patients (>80%) will have SGD, precluding the need for more extensive surgical exploration ,17. CompMIP is performed in patients with SGD (solitary adenoma) detected on preoperative imaging, while BNE is performed in patients with MGD  or with negative imaging .99mTechnetium-sestamibi (MIBI), parathyroid four-dimensional computed tomography (4D-CT), parathyroid venous sampling, magnetic resonance imaging (MRI), and positron emission tomography (PET). While preoperative imaging is generally accepted as standard of care and recommended by society guidelines . Co. Co13]. The conventional 4D-CT imaging acquisition protocol consists of CT images acquired from the angle of the mandible down to the carina, which includes all potential areas of eutopic and ectopic parathyroid glands . Prior tInterpretation of 4D-CT begins with reviewing the arterial phase in all three planes, as adenomas and hyperplastic glands are usually most conspicuous on this phase. Review should be targeted first towards eutopic anatomic locations of parathyroid glands near the thyroid gland and then expanded to ectopic locations , if not As in other modalities, reported diagnostic accuracy of 4D-CT should be interpreted in light of the study patient population, biochemical profiles, and MGD rates. Two meta-analyses on preoperative imaging have included 4D-CT ,52. CheuThe main advantage of 4D-CT is the high spatial resolution and excellent anatomic detail for precise localization and delineation of important anatomic landmarks for the surgeon. 4D-CT is performed very rapidly (~2 min) compared to MIBI (~3 to 4 h), which is more convenient and easier for patients. Unlike ultrasound, 4D-CT is not user dependent and allows for visualization of deeper and ectopic parathyroid glands . Disadva4D-CT has been increasingly utilized in clinical practice as a first-line and adjunct imaging modality for preoperative localization. Recent society guidelines now propose 4D-CT as a potential option to consider in addition to ultrasound and MIBI ,19.Selective parathyroid venous sampling (PVS) is an invasive localization technique, which is now typically reserved for patients who have persistent or recurrent PHPT after PTX and/or who have had negative or discordant imaging studies. Usually performed by an experienced interventional radiologist who obtains various venous samplings from veins surrounding the thyroid bed and draining the anterior mediastinum. Sampling includes the internal jugular, brachiocephalic, internal mammary, vertebral, and thymic veins is and performed via peripheral vein catheterization. Sampling of the superior, middle and inferior thyroid veins may also be attempted to provide more precise localization. Laboratory support with use of rapid PTH assay is helpful to facilitate this procedure contemporaneously. Detailed knowledge of the anatomy of the typical and variant venous drainage of eutopic and ectopic parathyroid glands is paramount. The ultimate aim of the procedure is to regionalize the abnormal gland(s). An abnormal gradient is defined as ≥2-fold increase in PTH levels compared to the inferior vena cava  and the Correct lateralization (left or right) is deduced when abnormal PTH gradient is derived from each of the four quadrants of the neck. Subsequently, more precise localization can be mapped from where the abnormal gradient occurs around the thyroid bed. Superior thyroid veins drain into the upper internal jugular veins and an abnormal gradient here suggests the superior parathyroid gland as the source of excess PTH. Middle thyroid veins drain into the lower internal jugular veins, suggesting a superior or inferior parathyroid lesion. Inferior thyroid veins drain into the brachiocephalic vein, suggesting an inferior parathyroid lesion. An important variant to note is, paired inferior thyroid veins drain may into a common trunk before draining into the left brachiocephalic vein and thus, an abnormal PTH gradient here may not lateralize to the correct side. Another important caveat is that contralateral flow of blood may occur within the thyroid veins and inference about the lateralization may confounded by this. Abnormal PTH gradient from the vertebral vein suggests an inferior, intra-thyroidal, or retroesophageal parathyroid lesion. If venous sampling from thymic vein or internal mammary vein is abnormal, then a mediastinal or thymic parathyroid lesion is likely .Given the variability of sampling protocols, venous drainages patterns, as well as indications for selective parathyroid venous sampling, its diagnostic accuracy may be difficult to determine. A recent meta-analysis of 12 pooled studies showed a pooled sensitivity of 74%, specificity of 41%, and positive likelihood ratio of 1.55 and for negative likelihood ratio was 0.47 . HoweverThe advantages of selective parathyroid venous sampling are that it can guide surgery for patients who have failed a previous PTx or localize a culprit parathyroid gland if non-invasive imaging modalities have been negative or inconclusive . This isPositron emission tomography (PET) is a non-invasive diagnostic imaging technique in nuclear medicine that can provide high quality images following intravenous injection of a radioactive tracer . Unlike 11C-Choline) or 18-Fluorine (18F-Fluorocholine or FCH) . I. I100]. Diffusion-weighted imaging (DWI) is an MR imaging technique used in tumor imaging that measures the random motion of water molecules within a voxel of tissue. In general, highly cellular tissues or those with cellular swelling demonstrate bright signal with corresponding low signal on the corresponding apparent diffusion coefficient (ADC) map. Parathyroid lesions usually demonstrate higher (brighter) signal on DWI with increasing diffusion strength which can allow their differentiation from other soft tissues structures of the head and neck region ,99,104.The diagnostic accuracy of MRI for detecting parathyroid lesions varies with magnet field strength of the MRI scanner: For 1.5-Tesla scanners, MRI has a sensitivity of approximately 80% in most studies ,107,108.Eliminating the use of ionizing radiation is the main advantage of MRI. Compared to 4D-CT (which can be associated with radiation doses of up to 10 mSv) ,110,112,MR imaging of parathyroid glands also has some disadvantages. First, MRI often takes longer time to acquire images and is susceptible to artifacts from breathing and motion . EffortsThe armamentarium of available imaging tests for PHPT has undergone significant advancement in recent years, including the adoption of PET/CT and MRI. The evolution of hybrid imaging techniques such as PET/MRI combining both these modalities to provide excellent anatomical resolution as well as physiologic information will likely further advance this field . FCH PETRecent advances in preoperative localization of abnormal parathyroid glands in PHPT have allowed more accurate and potentially curative minimally invasive surgical treatments for patients. With these advancements, endocrinologists and surgeons treating patients with PHPT now have a battery of imaging tests and techniques to choose from. As such, physicians should familiarize themselves with this array of techniques to provide the most suitable imaging studies to optimize patient care, while also considering local expertise and experience with these different modalities.


Purpose: To describe the feasibility of a new one-step approach to aspirate the aqueous and apply melphalan in a single-go without repeated entries into the anterior chamber.Methods: This retrospective non-comparative study was conducted at a referral center and included 12 patients. The one-step approach is described in a step-wise manner. No complications were observed among the patients.Results: One single injection of intracameral melphalan proved to be a successful treatment in nine cases. Two patients required a second injection, which was administered two weeks after the first one following the same technique.Conclusions: This proved to be a reasonable technique for the smooth application of melphalan in the anterior chamber studded with retinoblastoma seeds. Our outcomes revealed that it is an effective, quick, and cost-effective technique. Longer-term data collection is underway, though initial findings are encouraging. RB has the highest survival rate, nearly 95% in the developed countries, compared to other pediatric intraocular malignancies .Retinoblastoma (RB) is the most common ocular childhood malignancy [4]. In the management of intraocular RB, adequate chemoreduction and subsequent adjuvant treatment are usually planned instead of external beam radiotherapy (EBRT) . For children affected by extraocular RB, systemic chemotherapy and radiation therapy (EBRT or plaque radiation therapy) are likely to be effective [7]. Otherwise, extraocular disease usually requires intensive systemic chemotherapy and may need additional high-dose myeloablative chemotherapy and blood-forming stem cell transplant with or without radiotherapy [4]. Multidisciplinary care in RB management is mandatory. Treatment of RB is based on saving the patient’s life through a customized and risk-adapted approach to reduce systemic exposure to chemotherapy, optimize ocular drug administration, and safeguard vision [7]. Other indications for enucleation include the presence of pseudohypopyon, hyphema, iris nodules, vitreous hemorrhage impeding posterior segment examination, phthisis bulbi, and staphyloma [7]. Many attempts have been carried out to control the aqueous humor seeding, yet it still represents a real therapeutic challenge in RB management. Histopathology confirmation of anterior chamber involvement is also considered a criterion for enucleation, classified as stage E disease  [8]. The latter requires adjuvant chemotherapy [1]. Enucleation is often necessary and is the treatment of choice in localized tumors, posterior surface of lens invasion, raised intraocular pressure (glaucoma) and buphthalmos [9]. However, it may be only avoided when globe-sparing therapies do not compromise the patient’s survival [10]. Recently, brachytherapy showed successful outcomes in the treatment of diffuse anterior RB not involving the retina . Eye salvage through the combined use of intravitreal and intracameral melphalan is nowadays a successful technique and is being practiced [9]. Shields et al. reported that systemic chemotherapy and EBRT to control vitreous seeding showed a 5-year eye preservation rate of 47% and 53.4%, respectively [10]. Recently, a new intra-arterial chemotherapy technique has been developed, which showed a complete resolution of vitreous seedings in 67% of cases .Enucleation is hard to indicate in children, especially with only one functional eye .Nevertheless, diffuse RB infiltration to vitreous and anterior chamber that showed resistance to all available eye salvaging treatments inevitably leaves enucleation as the only option .At present, a lot of work is being carried out and different techniques have been described, aiming at globe salvage treatments for patients with RB [15]. They delivered iodine plaque therapy to the entire anterior segment, including the ciliary body, in three eyes affected by anterior diffuse RB without retinal or vitreous involvement. At 35-months follow-up examination, no recurrence occurred, and satisfactory visual acuity was preserved. It should be pointed out that radio-induced cataract, corneal limbal stem cell insufficiency and radiation-induced glaucoma could be long-term side effects [8]. Over the past few years, intravitreal chemotherapy has been developed in an attempt to reduce or complete elimination of vitreous tumoral seeding and to decrease the size of the lesion. When applied, intraocular chemotherapeutic drugs result in secondary salt and pepper changes at the site of lesion or in close proximity [16]. Further investigations include the combination of intravitreal chemotherapy and intraarterial chemotherapy together with transpupillary thermotherapy and brachytherapy [17]. Another chemotherapeutic drug, topotecan, displays an excellent rapid and potent antitumor effect in refractory or recurrent vitreous seeding [18-20].The first successful treatment procedure to control aqueous seeding was reported by Shields et al. [7].The choice of local chemotherapy grants a higher effective dose at the tumoral site, allowing an effective treatment while reducing chemotherapy’s systemic side effects [We proposed a novel technique of local chemotherapy, consisting of the intracameral injection of melphalan in patients with a diagnosis of group E retinoblastoma with anterior chamber seeding. We have designed a special gadget and described the technique that allowed the administration of the drug in a safe, controlled, quick, and cost-effective manner. The main benefit of that gadget was that it allowed the surgeon to aspirate the aqueous humor and to inject melphalan in a single go without repeated entries into the anterior chamber, hence avoiding the risk of spillage of tumor cells. Conflict of Interest statementThe authors state no conflict of interest.Informed Consent and Human and Animal Rights statementInformed consent has been obtained from all individuals included in this study.Authorization for the use of human subjectsEthical approval: The research related to human use complies with all the relevant national regulations, institutional policies, is in accordance with the tenets of the Helsinki Declaration, and has been approved by the review board of Lahore General Hospital/ PGMI, Lahore.AcknowledgementsNone.Sources of FundingNone.DisclosuresNone.


Adult diffuse glioma, particularly glioblastoma (GBM), is a devastating tumor of the central nervous system. The existential threat of this disease requires on-going treatment to counteract tumor progression. The present outcome is discouraging as most patients will succumb to this disease. The low cure rate is consistent with the failure of first-line therapy, radiation and temozolomide (TMZ). Even with their therapeutic mechanism of action to incur lethal DNA lesions, tumor growth remains undeterred. Delivering additional treatments only delays the inescapable development of therapeutic tolerance and disease recurrence. The urgency of establishing lifelong tumor control needs to be re-examined with a greater focus on eliminating resistance. Early genomic and transcriptome studies suggest each tumor subtype possesses a unique molecular network to safeguard genome integrity. Subsequent seminal work on post-therapy tumor progression sheds light on the involvement of DNA repair as the causative contributor for hypermutation and therapeutic failure. In this review, we will provide an overview of known molecular factors that influence the engagement of different DNA repair pathways, including targetable vulnerabilities, which can be exploited for clinical benefit with the use of specific inhibitors. Therapeutic resistance is a known phenomenon that continues to be a formidable foe in the search for a curative treatment. Based on the observation of recurring tumors, early investigation supports the idea that tumors are initiated and maintained by a small population of cancer cells that share similar biological traits to normal adult stem cells. This led to the discovery of a distinct subset of CD133+ cells that endows malignant brain tumors with radioresistant properties . EvidencPDGFRA aberration and are associated with younger patients which have a high prevalence of IDH1 mutation. For the classical subtype, amplification of EGFR and Chr.7 is correlated with the frequent loss of Chr.10 and CDKN2A. Conversely, the mesenchymal subtype has an angiogenic phenotype and is characterized by NF1 mutation with extensive upregulation of necrotic and inflammatory factors. Moreover, the neural subtype was also identified during the initial analysis but has been withdrawn due to the presence of non-tumorigenic cells. Among the different subtypes, tumors with the mesenchymal profile have the worst prognosis, whereas the proneural subtype shows a more favorable outcome. From these studies, it is evident that GBM subtypes are reliant on various independent signaling networks to permit tumor growth. One interesting observation is the broad shift in tumor heterogeneity. Current estimates indicate two-thirds of all diagnosed GBM undergoes subtype switching upon treatment [EGFR is lost upon recurrence, leading to the establishment of other tumor subtypes. Likewise for the transition from the proneural to mesenchymal subtype, PDGFRA deficient tumors are subjected to NF1 loss.Such attributes are largely influenced by genetic fitness, where malignant cancer cells comprise or acquire specific alteration in the genome that permits the rewiring of signaling cascades to gain new modes of survival. In an attempt to unravel the relationship between driver mutations and resistance, large-scale molecular profiling by The Cancer Genome Atlas (TCGA) has taken up the role of defining the genomic landscape of malignant brain tumors . Here, treatment . For insThe shift in gene alteration is crucial for post-therapeutic survival. In this discovery, approximately 60 different mutations in various assorted efforts can contribute to the gain of malignancy in primary GBM. The same tumor at recurrence can acquire >500 different mutations in excess as a result of treatment intervention . Most ofDNA lesion comes in various forms, including mismatch, chemical base modification, and single (SSB) and double strand break (DSB). Among the different lesions, SSB is the most frequently occurring and is characterized by the loss of individual nucleotides on one strand of the DNA. While SSBs are relatively harmless, they can be fatal during S-phase progression, causing replication fork collapse and subsequent strand breakages . These DIn the presence of DSBs, hierarchical recruitment of different DDR factors is initiated. The MRN (MRE11-RAD50-NBS1) complex first senses and tethers to the break site, bringing ATM along via protein interaction with NBS1. Tyrosine kinase C–Abl then activates Tip60 acetyltransferase, which methylates adjacent histone H3 lysine–9 (H3k9me3) to ensure an open–relaxed chromatin structure, which guarantees subsequent DDR factors access to the break site . Co-locaGiven that DNA lesions are resolved in a cell-cycle dependent manner, two downstream effector kinase proteins, namely CHK1 and CHK2, are essential for cell-cycle arrest. Their activation promptly degrades CDK complexes and stalls the cell-cycle for the timely removal of faulty lesions. For a rapid G1/S-phase arrest, the ATM–CHK2 axis initiates the ubiquitination of CDC25A, which prevents CDK2 and Cyclin–E interaction . If DNA When the appropriate cell-cycle arrest occurs, SSB and DSB repairs come into play where each process focuses on a specific class of DNA lesions . The priEGFRvIII mutation can mediate radioresistance by hyperactivating DNA-PKcs to increase NHEJ activity [MYC mutation also drives the interaction between CDK18 and ATR to promote a more conducive cell-cycle environment, wherein enhanced HR can render DSBs ineffective [BCR-ABL has a positive stimulation on CtIP to augment SSA repair [Notably, each of the four pathways leads to a different genetic outcome that can be manipulated by oncogenic factors to establish preferential repair. For instance, GBM patients that carry the activity . Conversffective . Even fuA repair . A reasoA repair . Hence, A repair .DDR acts in concert to protect the genome from detrimental harm. Under normal circumstances, the recruitment of various repair enzymes occurs in a DNA lesion-specific manner, yet alteration of the same process permits tumor cells to have a more aggressive stance. In the treatment of such cancers, chemical inhibition that targets the enzymatic activity or interaction of DNA repair proteins can be detrimental. The rationale of exploiting “pathway addiction” is to convert endogenous damage to fatal DNA lesions so that selective killing can be established. The benefit of utilizing this strategy also reduces collateral toxic accumulation of radiation and DNA-damaging chemotherapies to normal tissues.The aspect of the tumor’s DDR is different because the majority has lost one or more associated pathways, leading to a higher dependency on the remaining pathway/s. They are susceptible to specific DDR inhibition , top as PTEN deficiency is frequently associated with GBM development [P53 deficiency [P53 are likely to benefit from this treatment strategy.In addition to DNA sensors, downstream cascade (transducers and effectors) also carries significant importance in amplifying damage signals and coordinating the appropriate response for transcriptional activation, checkpoint arrest and DNA repair. This series of events are first initiated by transducer proteins, DNA-PK, ATM and ATR. For DNA-PK, recruitment to the damage site promotes the inward sliding of KU70/80 to allow NHEJ core proteins to process (if required) and re-ligate the ends of the DNA. The versatility of this repair machinery in restoring different DSBs is a valuable target for cancer therapy. Early inhibitors such as Quercetin and LY294002 were promising, with the subsequent development of NU7427 and NU7441. Both inhibitors are potent at the nanomolar concentration. A surprising advancement is the dual DNA-PK/PI3K inhibitor, KU-0060648 with approximately a 500-fold increase in efficacy. Importantly, targeting DNA-PKcs is effective in MMR-deficient tumors, and is often identified in recurrent GBM with acquired TMZ resistance ,51. ATM elopment . ATR is elopment ,56, follAs DDR signaling converges downstream to a more specific role, effector proteins are required to act as intermediaries. One notable example is the presence of DSBs in activating checkpoints arrest to facilitate DNA repair. The delay in cell-cycle progress allows the timely removal of lesions prior to replication or division. Depending on the nature of the injury and cell-cycle phase in which the lesions are encountered, extensive arrest could be initiated at G1/S, intra-S-phase or G2/M transition, all of which are governed by CHK1 or CHK2. Checkpoints failure is therapeutically effective at circumventing the engagement of DNA repair. Hence, independent CHK1 (UCN-01), CHK2 (CCT241533) or dual CHK1/2 (AZD7762) inhibitors have been extensively tested in GBM with promising outcome ,45,57. TSince the completion of the Human Genome Project, advances in sequencing technology have enabled rapid discoveries on genetically targeted cancer therapies. Imatinib , EncorafBRCA1) or 2 (BRCA2) mutations [BRCA1/2 plays a vital role in HR repair which is essential for genome protection. Loss of PARP promotes replication fork stalling and is dependent on BRCA-dependent HR repair for recovery. BRCA-deficient tumors subjected to PARP inhibition promote selective killing but spare normal cells with functional BRCA1/2. Success of this outcome has spawned numerous attempts to identify new interacting partners with synthetic lethal properties  inhibitor against tumor with breast cancer gene 1  production for tumorigenesis. Elevation of this metabolic enzyme impacts chromatin formation by inhibiting KDM4B and inadvertently hypermethylates H3K9 to compact the genome. When DNA breaks occur within these heterochromatin regions, DDR factors are unable to access the damage sites and perform genome restoration, leaving tumor cells highly sensitive to alkylating-based treatments . Targetie damage  or lack e damage . In a ree damage . Likewise damage . Early ee damage ,78,79. Se damage . As hypoe damage .Prospective therapeutic interventions of DDR are in support of a more dynamic approach, using the imprint of genes (or “signature”) to unveil vulnerable sites within biological processes, instead of employing classic radiologic and histological analysis. During the progress of tumor malignancy, exposure to exogenous and/or endogenous reactive agents allow individual cancer cells to accumulate somatic mutations. While these alterations have been previously ignored as non-oncogenic drivers, it is clear that passenger mutations and structural events represent underlying defects in the DNA repair machinery. These counteracting processes are jointly shaping genomic changes and are best exemplified by the interplay of nucleotide misincorporation between DNA polymerases and MMR pathway. When the fidelity of DNA synthesis is compromised, post-replicative MMR mediates damage removal by excision to provide subsequent opportunity of an error-free synthesis prior to cell division. In the event of a malfunction, MMR deficiency brings about the full spectrum of replication errors which can be annotated by the high prevalence of transversion, an interchange of purine to pyrimidine bases. The pattern of mutations bears clinical value as predictors in therapeutic targeting and can be broadly categorized using base substitution or indel to determine DDR dysregulation.In base substitution, there are six different nucleotide modifications (C∙G→A∙T/G∙C/T∙A or T∙A→A∙T/C∙G/G∙C), which consist of 16 additional sequence changes for each neighboring base , thus giving rise to a theoretical 96 different trinucleotides combination in mutational patterns. The COSMIC platform houses a reference catalogue of all curated mutational signatures across different cancer types, including GBM . Here, iAlthough traditional chemotherapeutics are imperative for first-line treatment, there has been a growing interest in targeted approaches. Driven by omics advancement, the premise of precision oncology is to utilize therapeutics to address patient-to-patient tumor heterogeneity. Personalized therapeutics offer a compelling advantage over conventional one-size-fits-all treatment. Furthermore, challenges exist in areas of high attrition rates for drug development. These shortcomings can be mitigated by the use of genome editing , or the RNA targeting  toolkit, to understand functional vulnerabilities. Inhibitors based on these targetable genes can subsequently be developed. Combined with the use of preclinical models to evaluate drug safety profiles, the overall strategy will allow a more accurate prediction of success in clinical trials.For instance, patient-derived xenograft (PDX) has been routinely adopted to evaluate patient-specific drug response, as it retains the genetic, transcriptional and histological features of the parental tumor. In this context, transplanting the biopsied specimen subcutaneously allows direct monitoring of tumor growth. Importantly, subcutaneous xenograft favors vasculature studies due to the availability of blood vessels while allowing different modes of drug administration; intratumoral, intraperitoneal and intravenous. The main drawback is the lack of BBB to observe therapeutic permeability. Intracranial PDX, on the other hand, is suited to examine tumor response in its native microenvironment but risks genetic drifting due to the need for cultivating biopsy tissue in vitro prior to implantation. Moreover, engraftment is slow, and lacks quantitative measures for malignant growth. Animal symptoms are the only reference for tumor progression. Despite its disadvantages, orthotopic xenograft closely mirrors cancer progression in humans. A third intraocular tumor model with the merits of both subcutaneous and intracranial approaches has also been proposed as an alternative means to evaluate therapeutic efficacy .Regardless of the models used, PDXs are essential in providing accurate estimates of a compound’s pharmacokinetics and pharmacodynamics for allometric scaling in human studies. In phase-I trial, patients are usually enrolled instead of healthy subjects because of the risk-to-benefit factor. which favors the former due to the exhaustion of existing therapeutics, and drug assessment that involves higher cytotoxic compounds. This is relevant to DDR inhibitors, as they rely on RT and/or other alkylating agents to achieve synergism, which can lead to overlapping toxicity. One strategy for developing these inhibitors for phase-I study is to determine their BBB penetrance, followed by efforts to adjust dose intervals to limit cumulative side effects. Additional diagnostic work-up on radiographic imaging should be included for real-time assessment to better differentiate between tumor progression and/or observe unforeseen neurotoxicity by white matter changes. So far, only a handful of DDR inhibitors have entered early trials but none has progressed to phase-III . The chaBRCA1/2 mutation. While radiotherapy and TMZ have proven to be the most effective treatment for GBM to date, there are also clear signs of progressive resistance, with accompanying treatment failure. The use of DDR inhibitors is becoming apparent as the next viable strategy for cancer therapy, both to eliminate DNA repair pathways that are responsible for counteracting treatment efficacy, and to exploit defects within the DDR cascade, which renders tumor cell dependency to a limited choice of repair pathway/s for survival. The latter is clinically attractive because of its advantage to promote selective killing while minimizing genotoxic build-up in normal cells. Additionally, DDR defects, as a result of genetic aberrations, bear prognostic values as biomarkers for targeted therapy. Apart from deciphering druggable targets, preclinical animal models are important translational tools for modern clinical trial designs in guiding patient selection, drug scheduling, and treatment response. There is no doubt that a multitude of opportunities still exist within the DDR network waiting to be exploited for GBM treatment (Great strides have been made in targeting DDR with newer and more potent inhibitors. The approval of PARP inhibitor represents the first DDR breakthrough for patients with reatment .


Acinetobacter, Pseudomonas and Sphingopyxis to be highly associated with carcinogenesis. Functional characterization of microbial genes suggested the potential to induce pathogen-related inflammation. Host-microbiota interplay analysis provided new insights into the tumorigenic role of specific microbiome compositions and demonstrated the influence of host genetics in shaping the tumor microbiome. Taken together, these findings indicated that the tumor microbiome is closely related to PDAC oncogenesis and the induction of inflammation. Additionally, our data revealed the microbial basis of PDAC heterogeneity and proved the predictive value of the microbiome, which will contribute to the intervention and treatment of disease.Despite the uniform mortality in pancreatic adenocarcinoma (PDAC), clinical disease heterogeneity exists with limited genomic differences. A highly aggressive tumor subtype termed ‘basal-like’ was identified to show worse outcomes and higher inflammatory responses. Here, we focus on the microbial effect in PDAC progression and present a comprehensive analysis of the tumor microbiome in different PDAC subtypes with resectable tumors using metagenomic sequencing. We found distinctive microbial communities in basal-like tumors and identified an increasing abundance of  Guo, Zhang, Guo et al. investigate microbiome alterations in different molecular subtypes of PDAC and report that distinct signatures exist in basal-like tumours, characterized by an increase in certain bacteria, such as Acinetobacter, Pseudomonas and Sphingopyxis. The authors associate increases in these bacteria with increased inflammation and carcinogenic potential, as suggested by pathway analyses. Most patients with PDAC have a dismal prognosis, with a 5-year overall survival of only 9%. Surgical resection remains the main option for PDAC treatment; however, the recurrence rate is relatively high, and most patients will eventually die due to metastasis3. Despite uniform mortality, clinical heterogeneity among patients has been discovered. Among patients with surgically resectable tumors, some progress to an advanced stage within months, while others stabilize for long-term survival5. A molecular subtype of PDAC termed ‘basal-like’, which has worse outcomes and a more aggressive phenotype, was identified by transcriptome analysis in a recent publication6. Many genomic studies have attempted to elucidate the mechanism of distinct disease progression; however, limited mutational variations have been found8. The cause of the clinical heterogeneity of tumors remains unclear.Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and the third most lethal cancer worldwide9. Both innate and adaptive immune cell subsets cooperate through various mechanisms to promote tumorigenesis10. Consistently, an activated inflammatory stromal response has been considered responsible for PDAC progression11. However, the precise mechanisms underlying the pathogenic role of inflammation are still unknown. Increasing evidence suggests that the human microbiome is likely to play a role in activating immune receptors and inducing cancer-associated inflammation12. Past studies focusing on the gut microbiome have confirmed the critical effect in intestinal tract malignancies, including esophageal, gastric and colorectal cancers13. Recently, the presence of intratumor bacteria in human PDAC has been confirmed14. The presence of Gammaproteobacteria in pancreas was proven to modulate tumor resistance to gemcitabine. The diverse microbiota alterations have been found in patients with PDAC compared to healthy subjects at oral, gastrointestinal and intrapancreatic tissues15. Many studies have found that oral or gut microbiota can translocate to pancreas and play a pivotal role in pancreatic carcinogenesis through several pathways including inflammation, immunity, metabolism, and hormonal regulation16. Pushalkar et al. reported a 1000-fold increase of intrapancreatic bacteria in PDAC and demonstrated that the tumor microbiome promotes oncogenesis by inducing immune suppression17. Moreover, Riquelme et al. demonstrated that tumor microbiome composition is able to influence pancreatic cancer outcomes by activating antitumor immune response18. The intratumor bacteria in pancreatic cancer were also confirmed by Nejman et al., and they found that bacteria are mostly intracellular and are present in both cancer and immune cells19. Besides, the fungal mycobiome was shown to be implicated in the pathogenesis of PDAC via activation of MBL by Aykut et al. study20. The essential role of the microbiome in immune regulation and oncogenesis was highlighted in recent years that modulations of the microbiome can be profound for the immunotherapy against pancreatic cancer21. These findings suggest the implication of the pancreatic microbiome in tumorigenesis, inspiring us to investigate the effect of the microbiome on the clinical heterogeneity of tumor subtypes.In the past two decades, pancreatic cancer has been recognized as an inflammation-driven cancer in which patients suffering from chronic pancreatitis carry a 13-fold higher risk of PDAC developmentThe microbiome composition in human PDAC remains incompletely studied, and whether it produces a favorable or adverse contribution to disease progression demands further exploration. In this study, we presented a comprehensive analysis of the tumor microbiome in different molecular subtypes from 62 resected PDAC. In addition, we performed functional characterization of microbial genes to elucidate the potential of a specific microbiome in inducing inflammation. Our data demonstrated the host-microbiota interplay and found a close association between the microbiome and tumor progression. We also discovered the influence of host genetics in shaping the tumor microbiome. Overall, our work shows that specific microbial compositions promote pancreatic cancer and contribute to aggressive phenotypes by inducing inflammation.24. Different tumor subtypes exhibit distinctive prognoses, which motivated us to explore the cause of tumor-specific progression. Following the subtyping scheme by Chan-Seng-Yue et al.23, we performed tumor subtype clustering using published gene signatures on our cohort. For the 62 PDAC samples in the cohort, clear clustering was revealed by 4 groups of tumor-related signatures , Shannon index of alpha-diversity, and Bray-Curtis metric distance of beta-diversity. As shown in Fig. p = 0.0017 and p = 0.0026), while the Shannon index was significantly decreased in basal-like tumors relative to that in classical tumors (p = 0.017). Increased richness with a decreased Shannon index indicated a microbial community with diverse members but dominated by a few species with extremely high abundance. We also observed that the Bray–Curtis dissimilarity within basal-like tumors was much higher than that within classical or hybrid tumors. A clear clustering between tumor subtypes  was revealed by PCoA, suggesting that the tumor microbiome showed phylogenetic closeness within each tumor subtype  and five bacterial species which belong to Sphingopyxis genus  were identified to be enriched in basal-like subtypes  PDAC samples of our cohort were applied to the fluorescence in situ hybridization (FISH) using a specific probe that against bacterial 16S rRNA sequences. 16S rRNA FISH indicated the presence of bacterial DNA within PDAC tumors, and also demonstrated an increased load of bacteria in basal-like tumors compared with classical/hybrid subtypes  with genetic dissimilarity (Jaccard distance), and a significantly positive correlation was found , and 39 genes were identified from these loci. Next, we performed GO term and KEGG pathway enrichment analyses against the background of immune response-related genes. The functional enrichment for GO terms demonstrated that the potential microbial-regulatory genes are involved in interferon-γ-mediated signaling pathway and peptide antigen binding. Moreover, enrichment in KEGG pathways of Kaposi sarcoma-associated herpesvirus infection, natural killer cell-mediated cytotoxicity and TNF signaling pathways was also observed . A total of 134 association pairs involving 97 SNVs and 29 genera were identified at d P < 10−, and 39 ved Fig. . A simil32. However, emerging evidence has shown that the microbiome also makes substantial contributions to some types of cancer; for example, the gut microbiota was demonstrated to play a regulatory role in liver cancer via bile acid metabolism33. In other sites beyond the gut, microbial dysbiosis is being increasingly recognized for its role in oncogenesis34. Our study presents the first research to explore the influence of the tumor microbiome in different PDAC subtypes. Current subtyping schemes can identify prognostic subgroups by transcriptome analysis23. Guided by these studies, our samples were split into three subtypes labeled ‘basal-like’, ‘classical’, and ‘hybrid’. Basal-like tumors are thought to be more aggressive, with worse outcomes6. The main distinguishing genes (signature 2) are related to squamous differentiation, suggesting that basal-like tumors may involve the squamous malignancies. Squamous expression programs usually accompany TP53 and KDM6A mutations and exhibit poor prognosis35. Comparatively, the default path of pancreatic pathogenesis is considered to cause the classical phenotype. The signatures for classical tumors are related to pancreatic lineage differentiation. Clinically, classical tumors are more frequent in early stage23. The single-cell analysis demonstrated that most tumors harbor both basal-like and classical programs, which lead to a mixed transcriptional profile at the bulk RNA-seq level, and hybrid tumors may be the outcome of this issue23. Consistent with these findings, we found that basal-like tumors were enriched for DNA replication, TGF-β signaling, and EMT. Interestingly, our data demonstrated that the inflammatory response and interferon-γ response were elevated in basal-like tumors. The immune infiltration analysis also showed an increasing level of activated mast cells and M1 macrophages in basal-like tumors. M1 macrophages are able to secrete pro-inflammatory cytokines and play roles in direct host-defense against pathogens36. Additionally, mast cells interact directly with bacteria and are involved in allergic inflammation37. All these signs indicate that pathogen-induced inflammation is critical for aggressive progression in basal-like tumors.Cancer is classically considered a disorder attributed to aberrations in the genome39. We inferred that the tumor microbiome may produce procarcinogenic effects through perpetual inflammation induction rather than direct mutagenic effects. In this study, we identified distinctive microbial communities in basal-like tumors, and notably increasing levels of Acinetobacter, Pseudomonas and Sphingopyxis were observed. Acinetobacter and Pseudomonas are common opportunistic pathogenic bacteria and are able to cause serious infections41. Notably, the increased abundance of Pseudomonas was also observed in PDAC patients with short-term survival by Riquelme et al. study, which confirmed the protumorigenic effect of Pseudomonas as revealed in our study18. Besides, Pushalkar et al. identified Pseudomonas as the most abundant bacterial genus that translocated from gut to pancreas in PDAC patients17. As demonstrated by the comparison of pancreatic microbiome between PDAC and wild-type mice, higher level of Acinetobacter was observed in PDAC mice. The presences of predominant genera such as Elizabethkingia, Delftia, Agrobacterium, and Sphingomonas by our analysis were also detected in PDAC patients by previous studies18. The microbial family Sphingomonadaceae (the upper level of Sphingopyxis) is a gram-negative bacterium that contains glycosylceramides in the outer membrane and can be detected in some human fecal samples but is not abundant or highly pathogenic42. NKT cells were shown to be stimulated after exposure to bacterial glycosphingolipids from Sphingomonadaceae, suggesting a classical innate immune response by pattern antigen recognition44. In addition, we performed functional characterization of the microbial genes enriched in basal-like tumors and found enrichment of metabolism, energy production, replication, cell motility, defense mechanisms and cell membrane/envelope biogenesis, indicating improved microbial activity and pathogenicity45. These findings revealed the inflammation-inducing role of the specific microbiome in basal-like tumors. Importantly, we found few enrichments of microbiata in classical/hybrid subtype, which may imply that abundant presence of microbiota in pancreas likely generates a protumorigenic effect rather than a common commensal role.Inflammation is relevant as a risk factor for PDAC development and progression. Lipopolysaccharide (LPS) can hyperstimulate Kras and lead to the initiation of carcinogenesis27. We next aimed to explore the relationship between the host and tumor microbiome. Our data demonstrated that the abundance of specific bacteria, including Acinetobacter, Pseudomonas and Sphingopyxis, positively correlated with host functions of the antibacterial immune response , presenting the interaction between microbial invasion and host immunity. Moreover, many cancer-associated signaling pathways, such as Kras signaling, MAPK signaling and the Wnt/β-Catenin pathway, also displayed significant correlations with these microbiota constituents. The facilitation effect of inflammatory insults, such as LPS in Kras signaling, has been described previously39. Experimental evidence also suggested that bacterial infection can induce malignant transformation by activating the AKT and MAPK signaling pathways46. Interestingly, our data demonstrated that the pancreatic tumor microbiome may give rise to dysfunction of bile acid metabolism. The role of bile acid in liver cancer has been widely studied, and the gut microbiota has an important impact on the composition of bile acids33. However, the role of bile acid in pancreatic cancer remains unclear. Our work provided some insights into the effect of the pancreatic microbiota on bile acid metabolism and pancreatic cancer. Collectively, this study depicted a detailed relationship between host cell function and the tumor microbiome, which helps to elucidate the tumorigenic role of the specific microbiota.It has been reported that microbiota dysbiosis can induce transcriptional alterations in intestinal epithelial cellsAn essential question is what determines the microbiome composition. We wondered whether host genetics have impacts on shaping diverse microbial communities, which may confer a higher risk for the colonization of the specific pathogenic microbiota and result in carcinogenesis. Our findings demonstrated the remarkable relevance of host genetic factors and microbiome compositions, proposing a possible role of host genetics in shaping the microbiome. In addition, we found that genetic variations related to immune dysfunction, such as compromised function of interferon-γ signaling or NK cell-mediated cytotoxicity, are associated with the abundance of bacteria. This analysis led to a possible hypothesis that independent of microenvironment conditions, host antimicrobial immunodeficiency may induce microbiome imbalances and give rise to the invasion of a specific pathogenic microbiota that promotes carcinogenesis.17. However, several studies suggested that the presence of microbiome in gut/tumor may boost the cancer immunity and be linked to favorable immunotherapy responses or tumor-fighting efforts26. The complex interplay among microbiome, immune cells and pancreatic tumorigenesis remains unsolved. It is quite difficult to answer the precise mechanisms of microbiome in immunity by our results. In this study, we focused on the effect of persistent inflammation induced by tumor microbiome in PDAC, and suggested that the activated inflammatory response in basal-like subtype serves as a facilitator in pancreatic carcinogenesis. In conclusion, our study delineated the intrapancreas microbiome profile of resected PDAC and found that the distinctive microbial communities in basal-like tumors may play an inflammation-inducing role and contribute to pancreatic carcinogenesis. We detailed the interplay between host cell function and the tumor microbiome, suggesting the tumorigenic role of specific microbiome compositions. We also demonstrated the effect of host genetics on PDAC microbiome. Our work highlighted the microbial basis of PDAC heterogeneity and supported the potential that microbial signatures may be useful as predictors of patient outcomes. Treatment targeting the PDAC microbiome may be promising.It is important to note that our cohort is comprised only of resectable pancreatic tumors due to difficulty in obtaining specimens from metastatic patients. This caused a limitation that our finding may be not applicable to advanced stage PDAC with metastatic tumor. Further study is required to take entire cancer population into the consideration. Interestingly, the tumor microbiome seems to have the dual effect of promoting immune suppression or activation by previous studies. Pushalkar et al. reported that ablation of the tumor microbiome was protective against cancer progression via immune activationPatients with a confirmed PDAC diagnosis were recruited from ChangHai Hospital, Shanghai. All patients gave written informed consent for collection and use of the samples. In this study, patients who received antibiotic treatment within the past month were excluded. A total of 62 resectable tumors were obtained from surgical specimens and immediately frozen at −80 °C prior to nucleic acid extraction. All samples were collected between 2015 and 2016, and only fresh tumors were used in this study. All procedures conformed to the Code of Ethics of the World Medical Association (Declaration of Helsinki) and were complied with the guidelines of the institutional review committee of the Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences.Frozen tumor tissues were aseptically sliced into appropriate sizes and placed in 1.5 ml sterile tubes. We utilized β-mercaptoethanol and a rotor-stator homogenizer to disrupt the tissue and homogenize the lysate. Metagenomic DNA and total RNA were simultaneously extracted using QIAGEN Pathogen Lysis Tubes and QIAGEN AllPrep DNA/RNA Mini Kit. DNA/RNA was quantified for concentration and purity using a NanoDrop spectrophotometer and stored at −20 °C. DNA samples were applied to library construction and whole-genome sequencing on the Illumina platform and yielded 2 × 150 bp paired-end reads. After discarding the adaptor contamination and low-quality reads, an average of 80 Gb of clean data per sample was obtained. Meanwhile, samples of RNA were sequenced for the whole transcriptome on the Illumina platform  and yielded an average of 15 Gb of clean data per sample.47, and then we used HTseq to count reads for each gene based on the GTF file, which provides gene structure information48. The gene count matrix was normalized using DESeq2 size factors determined by median ratios49. Differential gene expression analysis between different subtypes  was performed with DESeq2 based on the negative binomial distribution model. The significantly differentially expressed genes  were ranked based on the log2 fold change. Gene set enrichment analysis (GSEA) was next performed against the hallmark gene set from MSigDB using the R package clusterProfiler51. The normalized enrichment score was assessed for each set.Clean RNA sequencing data from 62 pancreatic tumors were aligned into human genome reference (GRCh38) using HISAT2 with default options23, we performed consensus clustering to identify tumor subtypes using the tumor expression signatures from their non-negative matrix factorization (NMF) results. We selected the top 25 ranked gene signatures from each of the four malignancy-related NMF components  and utilized the R package ConsensusClusterPlus for tumor subtyping52. For clustering, we used Pearson correlation distance and the hierarchical cluster algorithm with 10,000 resamplings to generate the final consensus. According to the cumulative distribution function (CDF) plot, a flatter middle segment represented a lower value of the proportion of ambiguous clustering (PAC). Therefore, we chose k = 3 as the optimal clustering number. Finally, three main subtypes were identified in our cohort.Guided by the Michelle et al. study6. The top 25 ranked genes from each of two NMF components  were selected. Consensus clustering was performed with the same parameters as in the previous method, and the samples were divided into 3 different subtypes based on the optimal CDF.To validate the clustering result, we performed another subtyping using gene signatures from the Moffitt et al. study53. Combined with the leukocyte gene signature matrix, which is termed LM22, CIBERSORTx was used to estimate the fractions of 22 immune cell types in our study. The differential expression proportion of immune cell types was evaluated using the Kruskal-Wallis test across tumor subtypes.CIBERSORTx is a computational approach that accurately resolves the relative fractions of diverse cell subsets using gene expression dataOverall survival was defined as the time from tumor resection to death from any cause. Patients who did not experience death were censored at date of last follow-up. The survival information of PDAC patients were collated, and some clinical data were obtained . We utilized the log-rank test to assess the difference in survival between different groups.Acinetobacter, Pseudomonas and Sphingopyxis. The primer sequences used in this study were: 5′-CGGACGGGTGAGTAATGCTT-3′, 5′-CAGACCCGCTACAGATCGTC-3′ (Acinetobacter); 5′-CAAGGCGACGATCCGTAACT-3′, 5′-ATGGCTGGATCAGGCTTTCG-3′ (Pseudomonas); 5′-AGGCGACGATCTTTAGCTGG-3′, 5′-ACGCCCAGTAATTCCGAACA-3′ (Sphingopyxis). We introduced four types of negative controls to address contaminations in this study, including DNA from pancreatic adjacent tissues using the same extraction process, environmental control (samples from the same freezer), DNA extraction control (buffers of extraction kit), and PCR no-template control.For 16S rDNA PCR, metagenomic DNA from PDAC tumors was detected using genus-specific primers targeting 54. FFPE tumor tissues were hybridized to detect the bacterial colonization within pancreatic tisues.Ribosomal RNA (rRNA) fluorescence in situ hybridization (FISH) was executed using the EUB338 16S rRNA gene probe labeled with the fluorophore 5’-Cy3 FFPE PDAC tissues were stained for bacterial LPS  with the automated slide stainer BOND RXm (Leica) using the Bond polymer refine detection kit. Heat induced epitope retrieval was done by a 20 min heating step with the epitope retrieval solution 1 (BOND).55 and Super-deduper (https://github.com/dstreett/Super-Deduper), the passing reads were aligned to the human reference (GRCh38) using Bowtie256 to preliminarily remove the host DNA sequences. The remaining reads were processed for taxonomic assignment using Kraken257. A customized database, which consists of reference libraries of bacteria, viruses, fungi, archaea, plasmids, UniVec and human from the NCBI database, was constructed for taxonomic classification in Kraken2. To avoid mistaken matching generated by repeated sequences, a confidence score threshold of 10% was set to improve the accuracy of classification. Combined with the Kraken2 classifier, we utilized Bracken58 to estimate the counts of reads originating from every taxon present in the sample based on Bayesian probability and produced a relatively accurate abundance profile at the species, genus, family, order and class levels. The microbial count matrix was normalized by DESeq2 sizefactors49, and the relative abundance of the microbiome was calculated. Alpha-diversity (Shannon index) and beta-diversity (Bray–Curtis metric distance) analysis and principal coordinates analysis (PCoA) utilized the R package vegan59. Differential abundance analysis of the microbiome utilized the Kruskal-Wallis test among tumor subtypes. The false discovery rate (FDR) was used to correct for multiple hypothesis testing.After sequence trimming and duplicate filtering by Fastp60. We utilized MetaGene61 to predict prokaryotic genes in contigs with lengths larger than 300 bp. A nonredundant gene catalog was constructed by removing redundancies that aligned to others with over 95% identity and more than 90% coverage using Cd-hit62. Taxonomic assignment of predicted genes was performed using Diamond63 with the BLASTX algorithm against the NR database of NCBI. Alignment hits with e-values larger than 1e−5 or query coverage less than 80% were filtered, and the best match was retained for the subsequent annotation of each gene. If one gene was equally matched to multiple alignments with the same score, the taxonomic assignment was determined by the lowest common ancestor (LCA) algorithm. The abundance of the predicted gene was estimated using an in-house pipeline in which nonhuman reads were aligned against the customized nonredundant gene catalog using Bowtie2, and the gene length was used to calculate the abundance by dividing counts. Differential abundance analysis of predicted microbial genes was performed using a two-sided Wilcoxon rank-sum test, and p values were adjusted with the FDR algorithm. The functional characterization of predicted genes was performed on the basis of KEGG ortholog and eggNOG ortholog groups with the application of BlastKOALA and eggNOG-mapper, respectively65. Due to the very low number of basal-like depleted genes, we compared the gene proportion of each functional category between the basal-like enriched gene set (n = 8960) and background gene set . Significance was determined by Fisher’s exact test.Reads without human-associated contaminants were assembled by the MEGAHIT assembler, which shows improved performance for large and complex metagenomics assembly51. The level of functional modules was identified in three steps. First, the significant enrichment categories were selected according to the p values (p < 0.05). Second, for each enrichment category, the enriched genes were used to build the expression profile. Third, we estimated the first principal component (PC1) to represent the general expression level of the functional module. Microbial genera with an abundance of zero in more than 80% of samples were excluded, and 122 genera were left for the correlation analysis. The correlation coefficient was calculated using the Pearson algorithm in the R package psych, and p values were adjusted by FDR for multiple comparisons. Visualization was performed using the R package corrr.On the basis of differentially expressed genes identified in RNA-seq analysis, we performed gene enrichment analysis against several functional categories, including KEGG pathways, GO terms and the hallmark gene set, using a hypergeometric test by clusterProfiler66, and we utilized Picard tools to process the postalignment procedures, including sorting and indexing (https://broadinstitute.github.io/picard/). Briefly, the bam files yielded by alignment were submitted to mark duplicates, base quality score recalibration (BQSR), SNV calling, and variant filtration by the Java programs in GATK. Over 3,500,000 SNVs per sample were identified after quality control, and we utilized ANNOVAR67 to perform gene annotation based on the ENSEMBL database.Genetic profiles in the form of single nucleotide variants (SNVs) and short insertion/deletions (InDels) were identified using the Genome Analysis Toolkit (GATK) version 4.0 pipeline. Clean DNA sequencing data from 62 pancreatic tumors were aligned to human reference (GRCh38) using BWA-MEM with default parameters59. We used linear regression to evaluate the correlation between Jaccard distance and microbial Bray–Curtis metric distance in R.First, a large integrated genotype matrix of all 62 PDAC samples was constructed based on the SNV profiles, which consisted of 14,692,240 rows (SNVs) and 62 columns (samples). We excluded SNVs with minor allele frequencies (MAFs) less than 0.1, resulting in a smaller genotype profile with 4,778,023 SNVs. Jaccard distance, also known as the complement of the intersection over union, was calculated based on the genotype matrix in R using vegan68, which provides ultra-fast eQTL analysis for large matrix operations. We chose a linear model based on the assumption that genotypes have only additive effects on microbial abundance. To reduce the multiple test burden, we defined a region of 250 kb around the immune-related genes according to the gene list  supplied by NanoString69. We selected SNVs located at this region for the targeted association analysis. After filtering, a final genotype matrix consisting of 171,561 targeted SNVs was yielded for downstream analysis. Only microbial genera present in at least 20% of samples were included. For the association analysis, about 107 pairwise tests were performed between roughly 105 SNVs and approximately 100 microbial genera. We identified significant associations using a stringent threshold of P < 5 × 10−8, which corresponds to the FDR < 1%. We found 134 associations at this level of significance.To link the microbial genera to genetic variation, we treated the abundance of genera as quantitative traits, and quantitative trait locus (QTL) mapping was carried out using the R package MatrixEQTLP < 10−6, and a total of 340 associations were found. The SNVs were annotated to genes based on their chromosome locations, and a total of 39 potential microbial-regulatory genes were identified. We performed GO term and KEGG pathway enrichment analyses against the particular background gene list from NanoString69, which was used for screening in the previous steps. We linked the functional category to microbial genera if the enriched genes from this functional category were associated with the microbial genera. The correlation network was visualized by Cytoscape version 3.670.In order to investigate the impact of identified SNVs on function. We re-screened the associations with a less stringent cutoff of P values < 0.05 were considered significant. The FDR was used to adjust p values for multiple hypothesis testing. The selections of statistical methods were described in each steps.All statistical analyses were carried out using R v3.6.3 with corresponding packages. Further information on research design is available in the Transparent Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


Glioblastoma (GBM) is a highly malignant brain tumor with a dismal prognosis. Standard therapy for GBM comprises surgical resection, followed by radiotherapy plus concomitant and adjuvant temozolomide (TMZ) therapy. The methylation status of the O6-methylguanine DNA methyltransferase (MGMT) promoter is one of the most essential predictive biomarkers for patients with GBM treated with TMZ. Patients with an unmethylated MGMT promoter (umMGMT), who comprise 60% of patients with GBM, present an even worse prognosis because of TMZ resistance. Radiotherapy with various fractionation, chemotherapy compensating for TMZ, targeted therapy against diverse oncogenic pathways, immunotherapy of vaccine or immune checkpoint inhibitor, and tumor treating fields have been studied in umMGMT GBM patients. However, most efforts have yielded negative results or merely minimal improvements. Therefore, effective patient subgroup selection concerning precision medicine has become the focus. By assigning different treatments to the corresponding patient subgroups, a better curative effect and subsequently prolonged survival can be achieved. In this review, we re-evaluate the value of standard TMZ therapy and summarize the new clinical strategies and attempts to treat patients with umMGMT, which yielded positive and negative results, to provide alternative treatment options and discuss future directions of umMGMT GBM treatment. Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults, accounting for 54% of all adult gliomas TMZ is an alkylating agent that methylates the guanine base of DNA, inducing futile mismatch repair Great debate over the use of TMZ in umMGMT patients has existed since the discovery of MGMT as a biomarker. In this review, we summarize clinical studies that aimed to treat patients with umMGMT GBM and reported both positive and negative outcomes, and discuss current treatment strategies and future treatment directions for patients with umMGMT GBM.To date, the MGMT status has been measured at the protein level using immunohistochemistry (IHC), RNA level using quantitative real-time PCR (Q-RT-PCR), promoter methylation level using pyrosequencing, or as methylation profiling with the Illumina BeadChip arrayThe assessment of the MGMT status at the protein or RNA level was unable to predict survival. IHC has yielded inconsistent results Pyrosequencing is currently considered the most reliable approach to quantitatively measure the methylation status In recent years, studies have attempted to measure MGMT methylation in cell-free DNA (cfDNA) using methylation-specific PCR or pyrosequencing when the tumor tissue is not available. Although the results of pyrosequencing of cfDNA correlated with OS and PFS, both methods have low sensitivity and high negative predictive value 3 is already of better prognosis for umMGMT patients 3, respectively). However, a study by Sales et al. focusing on umMGMT patients suggested that complete resection of the CE-TV did not result in improved survival Surgical resection has been considered imperative for newly diagnosed GBM (nGBM) and possesses a certain value for recurrent GBM (rGBM). A greater extent of surgical resection of the contrast-enhancing tumor volume (CE-TV) is strongly associated with both prolonged OS and PFS Laser Interstitial Thermal Therapy (LITT) is a new technique available as an alternative for deep, irresectable GBM patients, which utilizes the laser heat effect to destroy tumor cells with minimal invasion. LITT has been reported to annihilate deep-seated nGBM, rGBM, recurrent brain metastasis and radiation necrosis 2/d) and adjuvant (150-200 mg/m2 for 5 days each 28-day cycle) TMZ therapy was able to improve OS and PFS for nGBM patients compared with radiotherapy alone. This treatment, known as the Stupp's protocol, is now deemed the standard therapy for nGBM patients, especially for those with a methylated MGMT promoter. UmMGMT patients also benefit from radiotherapy plus TMZ, with a prolonged OS and PFS The phase III EORTC-NCIC trial indicated that radiotherapy (60 Gy/30) plus concomitant  plus standard concomitant and adjuvant TMZ and proved that it significantly prolonged the OS and PFS of umMGMT patients. The median OS increased from 8 months to 14 months, and PFS increased from 5 months to 9 months. This study was the first to moderately escalate radiation specifically in patients with umMGMT and was confirmed effective 18F-DOPA) PET guided dose-escalated radiotherapy(51~60~76 Gy/30)Dose-escalated radiotherapy was also co-registered with positron emission tomography (PET) to better delineate the target volume. A prospective phase II clinical trial utilized 3,4-dihydroxy-6-[18F]-fluoro-L-phenylalanine (2) Among all nGBM patients, elderly patients appear to rely more on radiotherapy, since elderly patients are likely to be more sensitive to the toxicity of intensive chemotherapy. The phase III NOA-08 trial suggested that the event-free survival (EFS) was longer in elderly umMGMT patients treated with radiotherapy alone than in patients treated with TMZ alone  and standard radiotherapy In summary, for adult umMGMT patients, radiotherapy plus concomitant and adjuvant TMZ was still more effective than radiotherapy alone. For patients older than 65 years of age, hypofractionated radiotherapy plus TMZ is an available choice. If one of the schedules must be eliminated from the plan, umMGMT patients should choose radiotherapy alone first, while mMGMT patients should consider otherwise.Despite all these improvements, the prognosis of umMGMT patients remains poor. Therefore, investigators have paved the way for clinical trials of novel or traditional chemotherapies and other therapies that could substitute or compensate for TMZ. However, the results were generally unsatisfying. Only a few treatments resulted in slight improvements. A list of clinical trials with both positive and negative results is summarized in Table Tumor treating fields (TTF) is an external device that can eliminate tumors regardless of the MGMT methylation status. TTF utilize an electric field to interfere with the mitosis of tumor cells and thus inhibit tumor progression. In a randomized phase III clinical trial, EF-14, including 695 nGBM patients TTF appear to be a promising treatment method apart from the limitations of chemotherapy. It has been officially included in National Comprehensive Cancer Network (NCCN) guideline of CNS tumors. TTF is independent of known molecular biomarkers of GBM, and can combine swiftly with standard therapy. The hindrance of TTF is that its effectiveness relies heavily on the put-on time per day, which cannot be reached in some patients due to intolerable skin reaction or poor patient compliance.Recently a phase II clinical trial recruited 47 nGBM patients with 31 umMGMT and applied the concomitant and adjuvant combination of temozolomide, vincristine and interferon Dianhydrogalactitol (VAL-083) is another kind of DNA-alkylating agent independent of MGMT repair and temozolomide pathway. In the halfway report of this phase II clinical trial targeting umMGMT nGBM, 10 patients remained progression free, while another 12 patients progressed with PFS of 9.9 months 2) was administered during the radiotherapy session on the first day of each week to enhance tumor sensitivity to radiotherapy. Adjuvant TMZ therapy was commenced after radiotherapy. However, this study showed no clinical benefit for umMGMT patients.Paclitaxel poliglumex (PPX) is a polymer agent derived from the traditional chemotherapy agent paclitaxel. In phase II clinical trial (BrUOG 244) The significant efforts lie in targeted therapy. However, the study of cilengitide, an integrin inhibitor, has reached a dead end. Integrins are important proteins that mediate angiogenesis in nGBM and cell apoptosis. A previous phase I/II study of the CORE trial revealed that both standard and intensive cilengitide treatments were able to prolong the OS and PFS of umMGMT patients. Further analysis also showed that high αvβ3 integrin expression in umMGMT nGBM was associated with prolonged PFS independent of the treatment type 2) plus procarbazine (50-100 mg) during and after radiotherapy. However, this combination of targeted treatments did not produce a positive outcome.Another phase II trial specifically targeting umMGMT patients added procarbazine to cilengitide Another novel medication that has been extensively studied was bevacizumab, which resulted in prolonged PFS but not OS The combination of bevacizumab and other medications did not improve OS either. In the phase II GLARIUS trial, TMZ was substituted by bevacizumab plus irinotecan Although studies of bevacizumab have all shown improvement in PFS but not OS, bevacizumab is well effective in reducing brain edema and improving symptoms, as a substitute of glucocorticoids, but without the extensive long-term adverse effects. It has also shown potential benefit to the immune microenvironment of GBM Approaches to intervene in other oncogenic pathways have also been researched. In a phase II clinical trial In the phase II clinical trial EORTC 26082 2 on days 1, 4, 8, and 11 of the 28-day cycle) In addition to traditional chemotherapy, protease inhibitors have also been considered. However, a phase II clinical trial declared that the umMGMT subgroup had a relatively poor prognosis after treatment with standard radiochemotherapy plus bortezomib (1.3 mg/mVeliparib is a poly ADP-ribose polymerase (PARP) inhibitor that kills tumor cells and sensitizes radiotherapy in preclinical studies Likewise, nimotuzumab, an EGFR antibody, was a beneficial treatment with predictive biomarkers from the Akt and mTORC signalling pathways. In an open-label phase III trial DCVax-L was a phase III clinical trial that utilized the autologous tumor lysate-pulsed dendritic cell (DC) vaccine to treat nGBM patientsRecently, the checkpoint inhibitor nivolumab was also reported to have failed in a phase III trial in umMGMT (Checkmate 498) and mMGMT (Checkmate 548) patients. Nivolumab was added as an adjuvant to standard radiochemotherapy. The primary endpoint, OS, was not reached by the time of the announcement, although toxicity was well tolerated. On the other hand, the use of a neoadjuvant PD-1 inhibitor in GBM may be promising. In a phase II clinical trial analyzing neoadjuvant PD-1 in 27 rGBM and 3 nGBM patients, increased immune activity was detected According to previous studies, patients with GBM are often diagnosed with cytomegalovirus infection, potentially a treatment target. In the phase III clinical trial ASPECT, adenovirus-mediated gene therapy called sitimagene ceradenovec was commenced to treat nGBM patients A retrospective study of GBM patients receiving valganciclovir in addition to standard radiochemotherapy has yielded positive results for umMGMT patients. Valganciclovir exerts an anti-cytomegalovirus effect and has significantly improved the OS for umMGMT patients from 11.6 months to 21.1 months. Although this retrospective study only included some eligible patients for the MGMT methylation analysis, valganciclovir represents a promising treatment option and future direction.The first oncolytic virus therapy in GBM has been approved by the Japan Ministry of Health, Labour and Welfare in 2021. Teserpaturev (Delytact/G47) is a herpes simplex virus 1 (HSV-1) oncolytic virus that has been genetically modified to selectively replicate in cancer cells Unlike nGBM, a well-defined standard therapy is not available for rGBM patients. Strategies, such as a second resection, re-irradiation, bevacizumab, lomustine and TMZ rechallenge, are generally considered beneficial.A second resection should only be performed when a survival benefit will be obtained. A retrospective study has found that rGBM patients who underwent GTR and STR of the tumor experienced longer OS than patients with biopsy or without surgery 2/d) was able to increase the PFS-6 for rGBM patients regardless of MGMT status. Though other studies still regarded MGMT methylation as a predictive biomarker of rGBM.At the time of relapse, the MGMT promoter methylation status is changed bidirectionally in a range of 8-37% GBM patients due to selective pressure and intratumor heterogeneity In addition to TMZ rechallenge, antiangiogenic therapy is one of the most commonly used therapies for treating rGBM patients. For umMGMT patients, one promising treatment combines onartuzumab, an anti-MET antibody, with bevacizumab Ongoing phase II or phase III clinical trials either specifically designed for patients with umMGMT or a cohort of umMGMT patients are listed in Table In the future, precision medicine may lead to higher therapeutic effectiveness. In a phase I/Ib clinical trial published in Nature in 2019, a personalized neoantigen vaccine was shown to trigger the immune response mediated by neoantigen-specific T cells in umMGMT GBM patients Another study design was published based on the idea of precision medicine. The phase I/II NOA-20 trial intended to match patients to the molecular subgroups The umMGMT status is a lynchpin factor that leads to TMZ resistance in GBM patients. However, only 40% of GBM patients carry hypermethylated MGMT, leaving 60% of patients with minimal benefits. In addition to surgical resection and standard TMZ radiochemotherapy, bevacizumab plus irinotecan, enzastaurin plus TMZ and TTF also benefit umMGMT patients to a certain extent. For elderly patients with umMGMT, hypofractionated radiotherapy plus TMZ is preferred when tolerable. If not, standard or hypofractionated radiotherapy alone should be considered superior to TMZ alone. For umMGMT GBM patients, recent clinical trials have failed to yield promising outcomes. Researchers have been studying the ways to reverse TMZ resistance or to overpass MGMT pathways. The success of TTF was an example of therapy or even external device with novel anti-tumor mechanisms. Because of the high inter- and intra-tumor heterogeneity and dynamic transformation of MGMT methylation status, precision medicine is currently of particular therapeutic value.


The clinical diagnosis of asbestosis is primarily based on chest radiographic evidence of pleural thickening and interstitial fibrosis combined with a history of exposure to asbestos. We report herein the case of a 65-year-old man with asbestosis pathologically diagnosed after surgical lung biopsy. He had a work history including farming, cementing, and casting and was admitted with dyspnea. Chest computed tomography revealed multiple well-defined nodules in both lungs and a 4.1 cm peribronchial consolidation with fibrotic changes in the right lower lobe. We suspected metastatic lung cancer and video-assisted thoracoscopic biopsy was performed in the lung lesion of the right lower lobe. Asbestosis was confirmed following histological examination. The patient is currently completing outpatient visits without significant changes. Asbestosis is a fibrosing interstitial lung disease that can develop following occupational asbestos exposure and a lengthy latency period . The cliA 65-year-old man with a 20 pack-year history of smoking visited the emergency department with dyspnea for two days. The patient had several symptoms including cough and purulent sputum. He had worked in dairy farming for 20 years, cementing for 8 years, and casting for 9 years. In addition, he had participated in construction, replacing asbestos ceilings for 1 year at the age of 63 years without wearing a mask.2 of 37.8 mmHg, PaO2 of 67 mmHg, and SaO2 of 93.0% (room air). Laboratory results revealed an increase in the serum levels of C-reactive protein (6.1 mg/dL), while other laboratory findings were within their normal limits. Chest X-ray imaging revealed features of bilateral nodular opacities with lung nodules and pleural thickening compared to a previous chest X-ray 12 years prior  was 2.26 L , the forced volume vital capacity (FVC) was 3.01 L , and the patient’s FEV1/FVC was 75% of the expected value. His pulmonary diffusion capacity of carbon monoxide (DLco) was within the normal range at 13.8 mL/min/mmHg . Fiberoptic bronchoscopy did not reveal any significant endobronchial lesions. Bronchial washing for bacterial culture, acid-fast bacilli (AFB), tuberculosis polymerase chain reaction (PCR), and cytology were done in the right lower lobe, which showed consolidation on chest CT imaging; however, the results were all negative, and we excluded the possibility of pulmonary tuberculosis. Additionally, we excluded atypical pneumonia because serum Although the levels of the tumor markers including carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCC) were normal, we suspected metastatic lung cancer due to the following: (1) the patient’s old age, (2) history of heavy smoking, and (3) well-defined multiple lung nodules on chest CT. Thus, video-assisted thoracoscopic (VATS) biopsy was performed in the peribronchial consolidation of the right lower lobe, and intra-alveolar asbestos bodies with pleural fibrosis were observed upon performing iron staining A,B. FinaA follow-up chest CT scan performed at 3 and 12 months after the diagnosis of asbestosis showed no interval changes, with multiple well-defined nodules remaining apparent in both lungs C,D. The This is an uncommon case detailing the diagnosis of asbestosis that presents with multiple lung nodules that can be easily mistaken as metastatic lung cancer. Although pleural plaques on chest CT imaging are more frequently noted in asbestosis, an uncommon feature, i.e., multiple lung nodules, was observed in our patient. We initially planned surgical lung biopsy due to favorable findings for lung cancer on chest CT imaging, but asbestosis was ultimately confirmed with the observation of intra-alveolar asbestos bodies.The current clinical diagnosis of asbestosis is mostly based on a history of exposure to asbestos combined with chest radiographic evidence of interstitial fibrosis and pleural plaques . HoweverClinical history is important for differentiating asbestosis and lung cancer. In patients with a history of asbestos exposure, asbestosis is the likely primary diagnosis, but lung cancer should be considered because asbestos is associated with lung cancer or malignant mesothelioma . Lung caMultiple pulmonary nodules on chest CT are a sign of various benign inflammatory or infectious conditions, such as fungal infections, tuberculosis, and sarcoidosis. However, asbestosis is not generally considered a possible cause of multiple pulmonary nodules. In our patient, the presence of focusing lesion on chest CT (a consolidation in the right lower lobe) was more indicative of primary lung cancer than of distant metastasis from another primary malignant condition. However, primary lung cancer often spreads to the intrathoracic lymph nodes before spreading to other parts of the body including bilateral lungs. This finding without lymphadenopathy made a diagnosis of metastatic lung cancer uncertain. Bronchoscopic biopsy was not available because there were no endobronchial lesions or lymphadenopathy; thus, we planned surgical lung biopsy to ensure an accurate diagnosis. Presumably, the multiple lung nodules were anthracofibrotic nodules because no significant interval changes between the chest CT scans at admission and follow-up visits, respectively, were observed after the diagnosis of asbestosis. Establishing an accurate diagnosis is important for introducing earlier preventive therapy such as discontinuing asbestos exposure if ongoing and stopping smoking to improve the patient’s condition . At the 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) because it is not currently covered by Korean national health insurance before histologic malignant confirmation and does not typically change the lung biopsy plan due to the potential overlap between benign and malignant conditions.This case study was limited in terms of available information regarding the details of the patient’s work environment associated with exposure to asbestos. In particular, we did not assess the asbestos concentration and exposure period in his workplace. In addition, we did not perform It is important to consider asbestosis in the case of unusual chest radiograph findings if the patient has a history of exposure to asbestos. Early surgical biopsy may be helpful to ensure accurate diagnosis and the timely introduction of preventive therapy including the discontinuation of both asbestos exposure and smoking.