Despite the rapid expansion of noninvasive (nonbiopsy) diagnosis, contemporary patients with cardiac amyloidosis too often present with advanced features of disease, such as diminished quality of life, elevated natriuretic peptides, and advanced heart failure. Therapeutics for transthyretin cardiomyopathy (ATTR-CM) are most effective when administered before significant symptoms of cardiac dysfunction manifest, making early identification of affected individuals of paramount importance. Community engagement and ensuring that a broad range of clinicians have working knowledge of how to screen for ATTR-CM in everyday practice will be an important step in moving disease identification further upstream. However, reliance on the appropriate and timely diagnosis by individual clinicians may continue to underperform. This review highlights how targeted screening of special populations may facilitate earlier diagnosis. Systems of care that operationalize screening of high-risk subpopulations and prospective validation of novel approaches to ATTR-CM identification are needed. In ATTR-CM, the native tetrameric form of TTR, produced mainly in the liver, pathologically dissociates to form amyloid fibrils that deposit in the myocardium, nerves, and soft tissues.3 AL-CM occurs when an abnormal clone of plasma cells produces a light chain that is prone to misfolding and forming amyloid fibrils that deposit in the myocardium, kidneys, GI tract, liver, nerves, and soft tissues. In both instances, the myocardium becomes progressively thickened, causing heart failure, conduction disease, and arrhythmias.5Cardiac amyloidosis (CA) is characterized by the extracellular deposition of amyloid fibrils in the heart with the distinctive histological property of green birefringence when viewed under cross polarized light microscopy after staining with Congo red.TTR gene (ATTRv) with more than 150 variants described to date; some present predominantly with cardiomyopathy, others with peripheral and/or autonomic neuropathy, and most with a mixed phenotype. The most common variant in the United States is the valine to isoleucine substitution at amino acid 122 (V122I or p.V142I), of which 3% to 4% of African Americans are heterozygote carriers, putting them at risk for the phenotypic expression of late onset ATTR-CM.6 Median survival after diagnosis in untreated patients is poor: 2.5 years for ATTRv-CM caused by the V122I variant and 3.6 years for ATTRwt-CM.10ATTR amyloidosis can occur as an acquired disease of aging—called “wild type” (ATTRwt)—which is most commonly found in older White males but has no known genetic cause. It also can be due to an autosomal dominant inherited pathogenic variant in the 14 Although awareness has increased, accurate and timely diagnosis remains suboptimal. This may be due in part to late recognition by clinicians as well as uneven access to noninvasive multimodality imaging tools and/or endomyocardial biopsy needed for the diagnostic evaluation. Furthermore, the attribution of the presenting symptoms and signs to aging, hypertension, hypertrophic cardiomyopathy, and other causes of heart failure and diastolic dysfunction contribute to missed and delayed diagnosis.7 Efforts to move the therapeutic window further upstream from the decline in quality of life, worsening renal function, elevation of natriuretic peptides, and shortened life expectancy associated with advancing ATTR-CM would have tremendous benefit to patients  highlight patient characteristics as well as multimodality imaging, electrocardiographic, and biomarker findings that should lead to a heightened clinical suspicion for ATTR-CM; (2) briefly touch on the differential diagnosis and diagnostic workup of ATTR-CM; (3) underscore the ongoing problem of missed or late diagnosis in the context of expanding therapeutic options for ATTR-CM; and (4) highlight opportunities for earlier diagnosis of ATTR-CM, including tissue screening during surgical procedures, noninvasive screening of high-risk subpopulations, electronic health record (EHR)-based strategies to bolster proactive diagnosis, and serial evaluation of asymptomatic carriers of 1 A history of hypertension with now normal blood pressure or requiring a decrease in the medication dose is a potential clue to the diagnosis of ATTR-CM.17 The evaluation of a patient with heart failure, arrhythmia, or conduction disease should begin with a review of the echocardiogram (echo) and electrocardiogram (ECG) in addition to a comprehensive history.It is important to recognize patient demographics, including sex, race, and age in the context of the clinical picture. Patients typically present with symptoms of heart failure but also can present with symptoms of atrial fibrillation and/or conduction disease.18 Witteles et al. put forth a schema recommending that CA be considered when a wall thickness of ≥ 14 mm is seen by echo. While this degree of wall thickening may be more specific, this cutoff may be less sensitive for the earlier diagnosis of ATTR-CM.4 Of note, the average wall thickness at diagnosis in most series and in the ATTR-ACT trial is 16 mm to 18 mm, well beyond that of what we are trying to achieve for earlier diagnosis.20 Furthermore, the presence of hypertension and/or aortic stenosis in a thickened ventricle does not exclude the coexistence of ATTR-CM. Other findings on echo are a nondilated left ventricle, mitral and tricuspid valve thickening, and biatrial dilatation.21 Additionally, while ATTR-CM is often considered a disease of heart failure with preserved ejection fraction (HFpEF), recent data from our institutional registry indicates that impaired LV function is an overlooked phenotype in patients with both ATTRwt-CM and ATTRv-CM. Nearly half of the patients presented with impaired LVEF < 50%, with more than a quarter of them presenting with HF with reduced EF (LVEF ≤ 40%).22 Finally, leveraging the application of longitudinal strain imaging in any patient with thickened LV walls can help differentiate CA from other causes of myocardial thickening. Diminished longitudinal strain with apical sparing is a pattern that is well described in advanced CA.23Increased wall thickness is a fundamental consequence of the development of ATTR-CM. Given that the upper limit of normal left ventricular (LV) wall thickness is 10 mm for males and 9 mm for females, it has been traditionally stated that a wall thickness of ≥ 12 mm in the absence of another clear etiology, such as uncontrolled hypertension or aortic stenosis, should prompt a suspicion for infiltrative heart disease.24 Indeed, in a cohort of 400 patients with ATTR-CM, low voltage was found in only 33%.18 Atrial fibrillation is a common rhythm disorder in elderly patients, but it has a very high prevalence in patients with ATTR-CM, particularly wild type.25 Therefore in an older patient with atrial fibrillation and a thickened LV wall and/or other red flags such as carpal tunnel syndrome, the index of suspicion for ATTR-CM should be heightened.26 Additionally, in the right clinical context, conduction disease on ECG such as PR prolongation, increased QRS duration with intraventricular conduction delay, left or right bundle branch block, or prior pacemaker are important clues to strengthen suspicion.28The echo evaluation should always take place with the concomitant review of the ECG. The discordance between LV wall thickness and QRS voltage has been noted as a finding to raise clinical suspicion of CA. Normal or even increased voltage should not provide reassurance when other concerning history, symptoms, imaging, or biomarker findings are suggestive of CA.3 Several common clinical conditions, including carpal tunnel syndrome, lumbar spinal stenosis, trigger finger, and bicep tendon rupture, are documented extracardiac manifestations of ATTRwt-CA.34 Bilateral carpal tunnel syndrome and lumbar spinal stenosis can precede the cardiac diagnosis by several years. A recent study found that bilateral carpal tunnel syndrome was present in approximately 50% of individuals with ATTRwt-CM in the 5 to 7 years preceding the diagnosis.35A thorough history is of paramount importance and should include questions regarding noncardiac symptoms or previous surgeries. Because ATTR amyloid deposits in the soft tissues, ligaments, joints and tendons, a comprehensive orthopedic history should be taken .3 Severa7Additionally, asking questions pertaining to peripheral and autonomic neuropathy symptoms  can signal systemic amyloidosis and is more commonly seen in ATTRv-CM. A comprehensive family history is of particular importance due to the inherited forms of transthyretin amyloidosis.27 Unfortunately, by the time these biomarkers are substantially elevated, the disease is typically more advanced.10 An elevated troponin in the absence of an acute coronary syndrome in a patient with thickened LV walls can be a clue to CA.Both serum troponin and natriuretic peptide levels are often elevated in cases of clinically apparent CA.36 Ultimately, neither a “negative” CMR in the context of a high clinical suspicion nor a characteristic CMR for CA obviates the need for confirmatory testing.Cardiac magnetic resonance imaging (CMR) is an extremely helpful tool to aid in the diagnosis of CA by helping to differentiate it from other causes of increased LV wall thickness. There are characteristic imaging findings regarding the pattern of late gadolinium enhancement (LGE) often showing a more diffuse subendocardial or transmural pattern as well as difficulty nulling the myocardium.99mTc-PYP scintigraphy.37 Recently, a simple clinical risk score including readily available demographic and echo data was validated in a largely White group of HFpEF patients.38 The implementation of pre-test risk scores has the potential to guide clinicians with more limited clinical experience and can be incorporated into EHR-based alerting. Once there is clinical suspicion of CA, a comprehensive approach to confirmatory testing should be undertaken.36The differential diagnosis of ATTR-CM includes hypertensive heart disease (particularly when combined with chronic kidney disease), hypertrophic cardiomyopathy, Fabry’s disease, and others. The combination of biochemical (hs-cTnT), ECG (wide QRS), and structural  findings were highly predictive components of a pre-test probability risk score for patients with suspected ATTR-CM derived from a Japanese cohort undergoing 36 The appropriate tests to rule out AL amyloidosis are a serum free light chain assay and immunofixation of the serum and urine. The nuances of cardiac scintigraphy and the optimal approach to accurate imaging and incorporation of single photon emitted computed tomography (SPECT), and preferably SPECT/CT when available, are included in the American Society of Nuclear Cardiology (ASNC) guidelines.40 It is important to note that AL-CM can cause significant uptake on 99mTc-PYP scan.36 False positives may occur due to blood pool in the LV cavity as opposed to true myocardial uptake, which needs to be differentiated by SPECT imaging. Finally, endomyocardial biopsy should be pursued when there is conflicting data to confirm amyloid deposits with accurate typing by immunohistochemistry (only with experienced pathologists) or mass spectrometry.The appropriate steps for the diagnostic workup of CA has been well described, with published algorithms having in common the crucial need to rule out AL amyloidosis with simple laboratory testing and the pursuit of technetium-based cardiac scintigraphy.41 Two TTR silencing drugs that significantly reduce the production of TTR by the liver—patisiran and inotersen—are FDA approved for the treatment of neuropathy in ATTRv, with clinical trials of silencer therapy in ATTR-CM either in progress or recently completed.43A nuanced approach to earlier diagnosis of ATTR-CM is no longer an academic exercise since therapeutic options have expanded beyond cardiac and liver transplantation. The clinical use of the transthyretin stabilizer (tafamidis), the first ATTR-CM treatment approved by the United States Food and Drug Administration (FDA), has proven benefits with regard to mortality, hospitalization, and quality of life in both ATTRwt-CM and ATTRv-CM.TTR gene with a single infusion of NTLA-2001 in six patients with ATTRv.44 A trial in patients with ATTR-CM is in the planning phases.The top-line announcement from APOLLO-B, a phase 3 double-blind placebo-controlled study examining the impact of patisiran on the primary end point of change in 6-minute walk distance at 12 months, showed benefit in patients with ATTR-CM. The full manuscript detailing the results is not available at the time of this review. Additionally, CRISPR-Cas9 in vivo gene editing for the treatment of TTR-related polyneuropathy has proven safe and effective at reducing TTR protein concentrations through the targeted knockout of the 8 A staging system built on natriuretic peptide and renal function thresholds developed by the National Amyloidosis Center (NAC)—with stage I defined as N-terminal pro-brain natriuretic peptide (NT-proBNP) ≤ 3,000 pg/mL and estimated glomerular filtration rate (eGFR) ≥ 45 mL/min/1.73 m2, stage III defined as NT-proBNP > 3,000 pg/mL and eGFR < 45 mL/min/1.73 m2, and the remainder of patients at stage II—has significant prognostic value with divergent survival across the three stages.9A longitudinal registry study from 2000 to 2017 catalogues the substantial diagnostic delay, high use of inpatient hospital services, and persistent diminishment of quality of life in the period prior to confirmatory diagnosis of both ATTRwt- and ATTRv-CM. Strikingly, the diagnosis of ATTRwt-CM was delayed > 4 years after initial presentation of cardiac disease in 42% of cases.45 Despite a rapid increase in the volume of cardiac scintigraphy performed at amyloidosis centers, the median time from symptom onset to diagnosis of ATTRwt-CM has not changed in large registry studies.47Contemporary data from a large referral center in the post-tafamidis period suggests that NAC staging is still highly prognostic in patients on transthyretin stabilizers; however, a large proportion of patients are still significantly advanced in their disease presentation, with 56% of patients with ATTR-CM being diagnosed with NAC stage II or III disease .45 Despi19 Given that current therapeutics do not address existing deposition, misdiagnosis and delays result in potentially irreversible damage both in the heart and systemically.27Early diagnosis of ATTR-CM is paramount in preventing undue morbidity and mortality from the continued deposition of amyloid fibrils. The ATTR-ACT study showed that patients with New York Heart Association (NYHA) class I and II derived the most benefit of treatment when compared with NYHA III.29Because carpal tunnel syndrome and lumbar spinal stenosis have been associated with amyloid deposition in patients who later develop ATTR-CM, investigators have sought to understand the yield of active ascertainment strategies, which involves evaluating surgical pathology for the presence of amyloid fibrils.48 One patient with TTR amyloid deposits in the tenosynovium developed asymptomatic subclinical ATTR-CM at 4-year follow-up   undergoing carpal tunnel release surgery, Congo red staining of tenosynovial tissue detected amyloid deposits in 10.2% (10/98) of patients. After evaluation of the 10 biopsy positive patients , it was found that 2 patients (1 AL and 1 ATTR) had cardiac involvement.ollow-up .49 Curreed data) . At the 52 In a recent publication, 34% of patients undergoing spinal stenosis surgery had amyloid deposition. Unfortunately, of those with amyloid deposition, more than one-third could not be accurately typed.53 Though sampling and analysis of surgical tissue at the time of surgical procedures may be a low-risk opportunity to identify patients who are at risk for developing ATTR-CM, how to best define the surgical screening cohort for the highest-risk patients, as well as the optimal plan for surveillance of patients with amyloid deposition, merits further investigation.Although surgical pathologic data has been mixed, 324 patients in one study underwent surgery for spinal stenosis, and 43 (13%) had ATTRwt in the ligamentum flavum, with 2 cases of confirmed cardiac involvement.54Other subpopulations considered at high pre-test risk include outpatients and inpatients presenting with heart failure and elderly patients referred for transcatheter aortic valve replacement (TAVR).13 There was no significant difference in the proportion of men and women who had ATTRwt-CM, raising the possibility of ascertainment bias in registries that have consistently shown a dramatic predominance of males.55 Additionally, in patients presenting to a dedicated HFpEF clinic, 14% of consecutive patients who underwent endomyocardial biopsy were found to have CA.12 Pathways to identify and screen inpatients and ambulatory HF patients with risk factors for ATTR-CM may be a high-yield opportunity.One study prospectively screened consecutive patients ≥ 60 years old admitted with acute HFpEF and LV wall thickness ≥ 12 mm. Of the 120 patients, 16 (13.3%) had cardiac scintigraphy (99mTc-DPD) consistent with CA, and all were ATTRwt.56 Some have proposed implementing “red-flag” screening and evaluation prior to enrolling patients in HFpEF trials to prevent off-target use of heart failure therapeutics.57Lastly, given the heterogeneity of HFpEF, there is mounting evidence that a significant proportion of amyloid patients may have been inadvertently enrolled in prior HFpEF clinical trials.58 Patients referred for TAVR at three international sites underwent cardiac scintigraphy and 11.8% (48/407) were scan positive. The authors developed a clinical risk score utilizing LV remodeling (hypertrophy/diastolic dysfunction), age, injury (high-sensitivity troponin T), and electrical abnormalities (right bundle branch block/low voltages) with reasonable sensitivity and specificity for the diagnosis of AS with concomitant ATTR-CM.54Severe calcific aortic stenosis (AS) represents an elderly population for which targeted screening has had variable but significant yield.59 Again, patient characteristics including thicker LV walls, higher NT-proBNP, and lower ECG voltage were present in a higher proportion of patients with ATTR-CM compared with non-ATTR-CM AS. Outcomes did not differ between groups in the latter study over a median 19-month follow-up period. Importantly, modified screening criteria will be needed as the population referred for TAVR continues to evolve.Another study found a slightly higher prevalence of ATTR-CM (13%) in an older population referred for TAVR.60 In one study, having the V122I variant was associated with increased risk of incident heart failure, heart failure hospitalization, and chronic myocardial injury manifested by elevation in cardiac troponin compared with control patients.61 Community-based efforts aimed at earlier identification of ATTRv-CM in Black patients is an area that merits further exploration.62 Studies such as SCAN-MP , a prospective cohort study using bone scintigraphy recruiting Black and Hispanic patients with heart failure, will aim to detect ATTR-CM in Black patients regardless of genotype.63Given the prevalence of the V122I variant (3% to 4%) in the self-identified Black population in the United States, there may be significant opportunity to proactively identify early heart failure phenotypes through imaging, biomarker-based, and genetic screening for ATTRv-CM.64 In 1,235 patients without screening outreach, 1.3% were diagnosed with ATTR-CM. Meanwhile, in the proactive screening cohort of 286 patients who consented to cardiac scintigraphy, 6.3% were diagnosed with ATTR-CM.64 Importantly, EHR-facilitated clinical suspicion for CA can expand the diagnostic pathway beyond the tertiary referral center.Given the broad use of EHR, the ability to identify patients with risk factors for ATTR-CM using diagnosis codes and imaging reports has emerged as a powerful tool to reduce the diagnostic inertia associated with ATTR-CM. In an illustrative population-based study conducted through an integrated EHR-derived cohort in the United States, patients with a validated HF diagnosis, age ≥ 60 years, ejection fraction ≥ 40%, and ventricular wall thickness ≥ 12 mm were assessed for ATTR-CM.65 International Classification of Diseases tenth revision codes were used to train a random forest machine learning model, which was validated with multiple external cohorts. Various combinations of cardiac and noncardiac diagnoses that demonstrated higher prevalence in ATTR-CM compared with non-ATTR-CM controls were described. For example, the combination of AF, joint disorders, and HFpEF was 29.7% prevalent in ATTR-CM compared with 7.0% in non-amyloid HF. The authors envision future studies that prospectively validate their modeling by utilizing automated laboratory testing, echocardiography , and cardiac scintigraphy in patients at risk for ATTR-CM.65Machine learning may be additive to traditional EHR-based cohorts, offering a novel approach to identifying patients with high pre-test risk of ATTR-CM. A recent study from Huda et al. used a large claims dataset to develop a cohort of patients with heart failure with ATTR-CM and non-ATTR-CM HF serving as the control population.67 Lastly, artificial intelligence (AI) may enhance our ability to detect CA earlier through AI modeling that predicts early disease, utilizing the combination of electrocardiographic and echocardiographic data as inputs.69Learning algorithms also may aid the expansion of imaging-based surveillance beyond the formal clinic/hospital setting and enable earlier disease detection. There is pilot data to suggest that imaging algorithms can enable novices without experience in ultrasonography to obtain basic diagnostic transthoracic echocardiographic images. This could facilitate a future state wherein targeted community-based screening could be scaled using algorithmic image acquisition as well as interpretation.70 EHR-linked biobanks have recently been leveraged to track associations between the TTR V122I variant and heart failure as well as polyneuropathy in patients with African or Hispanic/Latino ancestry.72 Given that serial imaging and biomarkers, particularly in patients with a known TTR gene variant, can diagnose presymptomatic disease, the use of EHR-linked biobanks to better understand natural history and facilitate early disease intervention has significant potential.70Patient education, genetic counseling, and patient advocacy could increase the identification of TTR variants in the first-degree relatives of patients with known disease. Furthermore, gene biobanks linked to EHR have emerged as effective tools for the characterization of rare genetic variants.Once a patient with a TTR variant is identified, how to surveil for phenotypic expression remains unclear. However, it should be tailored to the specific variant’s organ tropism and the age at which the proband developed disease. Recommended baseline cardiac and neurologic testing repeated at various intervals should be done in consultation with amyloidosis experts.73 The Scripps Research Institute has developed a peptide-based probe that specifically labels and quantifies non-native TTR levels in the blood (TTR oligomers). The elevation of these TTR oligomers can suggest that the TTR misfolding process is active and might predate the subclinical or clinical phenotypic expression of ATTR amyloidosis. The “Monitoring of Early Disease Progression in Hereditary Transthyretin Amyloidosis” (MED-hATTR) Study is currently testing the longitudinal monitoring of these non-native TTR levels in asymptomatic carriers of TTR variants to predict the development of early disease.74Innovative work in determining whether circulating non-native TTR levels (which are breakdown products of the TTR tetramer) correlate with clinical status and treatment response in patients with ATTRv amyloidosis is currently underway and has shown promising mechanistic potential.75 Therapy for ATTR-CM is most effective when administered before significant symptoms of cardiac dysfunction manifest; therefore, early identification of affected individuals is paramount.7 Community engagement and ensuring that a broad range of clinicians have working knowledge of how to screen for ATTR-CM in everyday practice will be an important step in moving disease identification further upstream  are most effective when administered before significant symptoms of cardiac dysfunction manifest, making early identification of affected individuals essential.Targeted screening of special populations may facilitate earlier diagnosis.Systems of care that operationalize screening of high-risk subpopulations and prospective validation of novel approaches to ATTR-CM identification are needed.


It is highly challenging for pathologists to distinguish small B-cell lymphomas from reactive lymphoid tissue and to accurately diagnose common histological subtypes of such lymphomas. This is due to overlapping morphological features and limitations of current ancillary testing, which itself involves many further tests. Hence, there is a pressing need for better biomarkers for accurate diagnosis and subtyping of small B-cell lymphomas as better diagnosis can lead to better treatments and clinical outcomes for patients. In this study, we identified and validated two sets of microRNA biomarkers that can distinguish small B-cell lymphomas from reactive lymphoid tissue and distinguish between four subtypes of such lymphomas, respectively. This study suggests that miRNA expression profiling may serve as a promising tool to aid in the diagnosis of small B-cell lymphomas.n = 100) to screen for differentially expressed miRNAs and a validation cohort (n = 282) to develop classification models. The models were evaluated for accuracy in subtype prediction. MiRNA gene set enrichment was also performed to identify differentially regulated pathways. 306 miRNAs were detected and quantified, resulting in 90-miRNA classification models from which smaller panels of miRNAs biomarkers with good accuracy were derived. Bioinformatic analysis revealed the upregulation of known and other potentially relevant signaling pathways in such lymphomas. In conclusion, this study suggests that miRNA expression profiling may serve as a promising tool to aid the diagnosis of common lymphoid lesions.Accurate diagnosis of the most common histological subtypes of small B-cell lymphomas is challenging due to overlapping morphological features and limitations of ancillary testing, which involves a large number of immunostains and molecular investigations. In addition, a common diagnostic challenge is to distinguish reactive lymphoid hyperplasia that do not require additional stains from such lymphomas that need ancillary investigations. We investigated if tissue-specific microRNA (miRNA) expression may provide potential biomarkers to improve the pathology diagnostic workflow. This study seeks to distinguish reactive lymphoid proliferation (RL) from small B-cell lymphomas, and to further distinguish the four main subtypes of small B-cell lymphomas. Two datasets were included: a discovery cohort ( Lymphoma diagnosis is well recognized as one of the most difficult areas of diagnostic pathology. The pathological diagnosis of lymphoma hinges largely upon recognition of morphological abnormalities on a well-represented tissue section. However, to many pathologists who have limited experience in examining lymphoid tissues, identification of normal and pathological changes in different lymphoid compartments and recognition of neoplastic lymphoid entities may be highly challenging. Lymphoid tissue often appears to be a morass of small and large lymphoid cells that defies recognition of cell types and functional compartmentalization. Compounding this is the complexity of lymphoma classification, often necessitating the incorporation of additional testing with a plethora of immunostains and molecular genetic investigations for definitive diagnosis, making lymphoma diagnosis one of the most complicated tasks encountered by pathologists worldwide.Therefore, it is not surprising that errors in lymphoid tissue diagnosis are prevalent . MisdiagIn this study, we investigated the potential utility of microRNA (miRNA) expression signatures as an adjunctive ancillary test in the diagnosis and classification of small B-cell lymphomas. MiRNAs, a family of short, evolutionarily conserved, non-coding RNA of approximately 18–25 nucleotides ,4, have Formalin-fixed paraffin-embedded (FFPE) tissue samples from RL and the 4 histological subtypes of small B-cell lymphomas were included: SLL, FL, MCL, and MZL. The diagnosis was based on the criteria established in the World Health Organization (WHO) classification . Samplesn = 23), FL (n = 21), MCL (n = 20), MZL (n = 19), and RL (n = 17). Whole tissue sections of the recruited samples were used, and the percentage of tumor cells was estimated to be 50% or more for each sample. A validation set of 282 FFPE tissue samples comprising SLL (n = 20), FL (n = 74), MCL (n = 22), MZL (n = 74), and RL (n = 92) was further collected from three different institutions: NUH, Tan Tock Seng Hospital (TTSH), Singapore and University Malaya Medical Centre (UMMC). Samples were classified and analyzed using miRNA expression profiling and the results were compared to the reference diagnosis.A total of 382 subjects were included in this study. A discovery set of 100 FFPE tissue samples was obtained from the Department of Pathology, National University Hospital (NUH), Singapore: SLL  according to the manufacturer’s protocol. Three synthetic short RNA species (spike-ins) with sequences distinct from endogenous human miRNAs were added into the lysis buffer as controls to monitor and normalize for workflow variations. The miRNA was eluted using 50 µL nuclease-free water. Total RNA quantity and quality were measured by NanoDrop 2000 . For each sample, 900 ng total RNA was used for subsequent reverse-transcription and PCR reactions. TM Thermal Cycler  according to the manufacturer’s instructions. For each RT reaction, a standard panel comprising a series of six 10-fold dilutions of synthetic miRNA and two no-template controls (NTCs) were included on the same plate. cDNA was then pre-amplified using a 14-cycle PCR reaction with Augmentation Primer Pools  on the VeritiTM Thermal Cycler. Single qPCR was performed on the amplified cDNA samples using a miRNA-specific qPCR assay and ID3EAL miRNA qPCR Master Mix according to the manufacturer’s instruction . The qPCR reaction for each sample was performed with technical duplicates on the QuantStudio 5 Real-Time PCR System . Raw threshold cycle (Ct) values were calculated using the QuantStudioTM Design and analysis software with an automatic baseline setting and a threshold of 0.4. MiRNA profiling was performed using a multiplexed RT-qPCR platform following an established protocol . IsolateThe synthetic spike-ins added at various stages were used to correct for variations in RNA isolation and RT-qPCR efficiency. The Ct values generated from the panel of 6 serially diluted synthetic miRNAs were then used to generate a standard curve. The absolute expression of each miRNA (number of copies present) was calculated by interpolation of the standard curve. MiRNA with a Ct value higher than NTCs (no template controls) was deemed undetectable and removed from the analysis. 2 copy numbers in each sample. In the discovery cohort, miRNAs that were not detected in >10% of samples were removed, resulting in 306 miRNAs in 100 samples. Biological normalization was performed by the global normalization approach demonstrated by Mestagh et al. [t-test was used to compare the miRNA expression between the subtype of interest and other subtypes (one-vs.-rest). The top 3 up- and 3 down-regulated miRNAs were selected based on the most significant p-value, resulting in a total of 30 subtype biomarkers for five subtypes. For each pair of the five subtypes, the same Student’s t-test was performed and the top 3 up- and 3 down-regulated miRNAs between the two subtypes (one-vs.-one) were identified, adding up to 60 miRNA markers that differentiated the two subtypes. Finally, 10 miRNAs that displayed the lowest variance across samples were selected as the housekeeping miRNAs. A 100-miRNA customized panel including 30 one-vs.-rest biomarkers, 60 one-vs.-one markers, and 10 housekeeping miRNAs was constructed and used for the FFPE samples in the validation cohort. miRNA expression levels were calculated as logh et al. , followe2 copies/sample. The mean expression level of the 10 housekeeping miRNA was used to normalize both the discovery and validation cohort to ensure comparability. As the FFPE samples in the validation cohort were collected from three different sites , the batch correction was performed using the ComBat approach [t-test was used to perform a one-vs.-rest and pairwise comparison for each subtype in the validation cohort. For all the 90 previously identified markers, the expression differences between the two groups of interest in the validation cohort were compared with the findings in the discovery cohort. miRNA markers that showed the same trend of expression changes were considered successfully validated.RNA from additional 282 FFPE tissues were profiled using the 100-miRNA customized panel and all expression levels were obtained as logapproach , settingHousekeeping gene normalization was applied to the raw expression levels in both discovery and validation cohorts, followed by ComBat batch correction  for diffUsing the combined dataset, we first developed a classification model to differentiate reactive control (RL) and lymphoma  samples. The combined dataset was subjected to 100 iterations of 4-fold cross-validation in which 3 folds were used for training and the last fold was used for testing. Support vector machine (SVM) with radial kernel was used for model training in the training datasets, and 3-fold cross-validation was performed to tune the cost parameter from 1 to 10. We applied the best-tuned model to the testing dataset, and a confusion matrix was created based on the predicted types (reactive or cancer) vs. actual samples’ types. The model’s performance was assessed by the prediction accuracy on the testing set . Using the miRNA expression and tissue site data from the lymphoma tissue samples, we further trained classification models to differentiate the four lymphoma subtypes FL, MCL, MZL, and SLL. Similarly, 100 iterations of 4-fold cross-validation were applied to the combined dataset. One-vs.-one classification strategy was used for the multi-class classification, by employing SVM model with radial kernel to the training set for model building, and another 3-fold cross-validation was performed for the determination of the optimal cost parameter from 1 to 10 based on the highest cross-validated accuracy . The besp-value for hypergeometric distribution was used to calculate the enrichment of miRNA targets in any given pathway in the universe of targets. MiRNA fold change together with the p-value for pathway targeting was used to calculate the regulation of pathway with weighted Kolmogorov–Smirnov-like statistics. Pathways targeted by less than 10 miRNAs or more than 500 miRNAs were ignored. The p-value of any pathway regulation was calculated by randomly permutating miRNAs 10,000 times. False discovery rate correction was carried out by using the null distribution of all pathways and the enrichment of unpermutated dataset. Pathway analysis was performed with all samples pooled together through miRSEA method. Briefly, miRNA and mRNA linkages were curated with miRTarBase Release 7.0 based solely on strong experimental evidence support. Pathway database was curated from Broad Institute C2 pathways sets including Kegg, Reactome, Pathway Interaction Database and Biocarta. miRNA and pathway were correlated together by identifying the specific strength of the miRNA targeting the pathway. The Current gold standard for lymphoma diagnosis is based on histopathological evaluation where tissue morphology is the foundation. However, morphological similarities between common subtypes of small B-cell lymphomas, which predominantly consist of small lymphoid cells with condensed chromatin A, mandatTo assess miRNAs for their potential utility as diagnostic biomarkers for diagnosis and subtyping of small B-cell lymphomas using routine FFPE samples, we first profiled the expression of 306 miRNAs in a discovery cohort of 100 samples. Out of the 306 miRNAs quantified, a 100-miRNA profiling panel comprising 10 housekeeping miRNAs and 90 candidate miRNAs was assembled for validation in the second cohort of 282 samples. Hierarchical clustering performed using the expression of the 90 candidate miRNAs showed distinct clusters representing the four subtypes of small B-cell lymphomas and RL in both the discovery and validation cohorts A,B. PrinTo differentiate lymphoma from reactive lymphoid proliferation, a 90-miRNA and tissue information classification model was established by training and testing on a combined cohort of samples, resulting in a mean area under the ROC curve (AUC) of 0.959 (95% CI: 0.922 to 0.988) A. Other In addition to distinguishing neoplastic from reactive lymphoid proliferation, we explored if miRNA expression profile could also be used for subtyping lymphomas. To further differentiate the four subtypes of small B-cell lymphoma, a 90-miRNA and tissue information classification model was built by training and testing using samples from both cohorts, resulting in a sensitivity of 86.8% for FL,87.8% for MZL, 85.2% for MCL and 84% for SLL and overall accuracy of 86.3% A. We sel12/13 signaling pathway  was performed to identify differentially regulated pathways between lymphoma and reactive lymphoid tissues. Using a cutoff q-value of 0.01, 13 KEGG pathways A and 20  pathway D.Given that distinguishing small B-cell lymphoma from reactive lymphoid proliferation represents a frequent diagnostic dilemma confronting many practicing pathologists, herein we propose a two-staged algorithm where miRNA-based classifiers instead of a wide panel of IHC markers is used to diagnose and subtype lymphoid proliferations that are morphologically suspicious of small B-cell lymphoma .In this era of individualized medicine, precise diagnosis and subtype classification of lymphomas have become increasingly important with the availability of disease-specific protocols and new targeted agents that inhibit specific pathways for different lymphoma subtypes. However, practicing lymphoma pathology is highly challenging. Accurate lymphoma diagnosis often requires the availability of hematopathologists with deep knowledge and experience in evaluating lymphoid lesions, high-quality laboratory infrastructure, as well as easy accessibility to a wide panel of immunohistochemical stains and additional molecular genetic testing such as fluorescence in situ hybridization (FISH) and clonality studies, all of which may not be available in resource constrained nations. In addition, pathologists have to make do with increasingly smaller samples, which do not permit the application of a large number of immunostains. Given that small B-cell proliferation is one of the most common lymphoid lesions encountered by general pathologists, we investigated the biomarker potential of miRNA expression signatures as a diagnostic adjunct to improve the identification and subtyping of small B-cell lymphoma. We used a novel high throughput qPCR platform to develop miRNA-based classifiers to distinguish neoplastic from reactive lymphoid proliferation and to subtype the four most common histological subtypes of small B-cell lymphomas. We then proposed a two-staged diagnostic process where the miRNA classifiers are incorporated to complement morphological evaluation . MiRNAs have previously been reported to be aberrantly expressed in almost all human cancers , includiThe application of miRNA expression profiling in the field of molecular cancer diagnostics requires a practical and reliable method that works on routinely available clinical materials. miRNAs can be robustly detected in FFPE tissue samples because they are small and less susceptible to degradative processes, and have been reported to be stable in FFPE archival tissue specimens that have been stored for close to 30 years . RemarkaAlthough miRNAs have unique attributes that render them suitable biomarkers in clinical practice, their accurate detection and quantification can be challenging because of their small size and sequence similarity among various members. For biomarker discovery and genome-wide expression analyses, most investigators deployed high-throughput hybridization-based methods, such as microarray technology for global gene expression profiling . AlthougThe current gold standard for quantitative gene expression measurement is quantitative PCR. qPCR is a robust and reproducible technology that can detect low levels of miRNAs with high sensitivity and specificity ,40, and The main challenge of PCR-based miRNA biomarkers discovery work lies in the design of individual primers required for specific amplification of each miRNA gene included in large-scale analyses. Due to the short length of miRNAs (roughly the size of a PCR primer), primer design for specific PCR amplification poses significant difficulty. As such, most commercially available high throughput qPCR platforms employ only one or two miRNA-specific primers with selective incorporation of universal primers. In the current study, we performed multiplex comparative analyses of 360 miRNAs based on a unique method that uses three miRNA-specific primers , obviating the use of universal primers altogether. All the primers of each miRNA analyzed have been carefully designed to detect single nucleotide differences. We believe that this platform offers a unique advantage to detect both low- and high-abundance miRNAs with unparalleled specificity. The combination of high sensitivity and specificity, broad dynamic range and multiplexing capability of this assay hold great promise in delivering highly reliable, reproducible, and representative disease-specific miRNA profiles. With this novel miRNA RT-qPCR profiling platform, we found two unique miRNA-based classifiers, each comprising a small set of 14 miRNAs, that can help to diagnose and subtype the four most common entities of small B-cell lymphomas with reasonably high accuracy. We believe that the proposed two-staged diagnostic workflow incorporating miRNA-based classifiers can potentially serve as a cost-effective and practical tool to complement traditional morphological diagnosis, especially in the resource-constrained nations. Typically, when confronted with the differential diagnoses of reactive lymphoid hyperplasia versus one of the low-grade B-cell lymphomas, pathologists will order a panel of 7–8 immunostains, sometimes with additional fluorescence in situ hybridization (FISH) and B-cell clonality analyses. Using a curated panel of miRNA targets, the cost of our RT-qPCR assay is economical, especially when it is reactive in nature and only classifier 1 is needed . The turConventional wisdom may hold that IHC may be easier to perform and accessible compared to molecular techniques. In fact, optimization and validation of IHC is technically challenging and the range of antibodies available is limited due to cost constraints in developing nations. On the other hand, due to the need for COVID testing during the global COVID-19 pandemic, RT-qPCR machines have become widely available even in countries with limited resources, hence rendering our assay feasible in such countries. Our study has its limitations. The number of MZL and FL cases are more than three times greater than that of SLL and MCL cases for the validation cohort, even though the number of cases for each subtype in the discovery cohort is roughly the same . Our miRBesides their potential utility as biomarkers, deregulated miRNAs in the tumors may point us to relevant cancer biology under their regulation and provide insights into B-cell lymphomagenesis and progression. Most of the top 14 differentially expressed miRNAs between the small B-cell lymphomas and reactive lymphoid samples in our study are in concordance with existing literature.The downregulation of miR-29b-2-5p in our samples corroborates a finding on the repression of miR-29, a tumor suppressor, in mantle cell lymphoma and other aggressive Myc-driven lymphomas . This reSeveral differentially expressed miRNAs are in concordance with studies done on other hematological malignancies that may share common underlying mechanisms in lymphomagenesis. For instance, miR-342-5p reduces cancer cell proliferation by targeting a cell cycle regulator Cyclin D1 (encoded by the CCND1 gene), and is reported to be downregulated in chronic myeloid leukemia patients . SimilarMiR-139-5p has been reported to be a tumor suppressor by repressing EIF4G, a factor in the initiation of protein translation, and is downregulated across various subtypes of acute myeloid leukemia (AML) . EIF4G, Yet, another tumor suppressor, miR-126, suppresses T and B-cell migration—a key lymphomagenesis event—by targeting the G protein-coupled receptor (GPCR), S1PR2 . Low expAdditionally, in DLBCL, a circular RNA circ_0003645 acts as a miRNA sponge to miR-335-5p, hence abolishing miR-335-5p’s tumor suppressive effect in targeting NFIB . The dowTo the best of our knowledge, not much is known yet about miR-143-5p and miR-224-5p—both downregulated in our lymphoma samples. However, studies by association appear to correlate miR-143 polymorphisms to reduced incidence of non-Hodgkin lymphomas in caucasian populations  and highWith the ability to bind to thousands of target mRNAs by complementary base pairing, miRNAs could potentially play diverse regulatory roles in many processes, including tissue and cancer development like lymphomagenesis. Interestingly, three highly significant and upregulated KEGG pathways—cytosolic DNA sensing pathway, RIG-I-like receptor signaling pathway, and NOD-like receptor signaling pathway—are functionally related. These pathways underlie the sensing of foreign matter that may be introduced during infections, in the form of single or double-stranded DNA, from viruses and other pathogens . Hence, While molecular mechanisms linking infection and these pathways are still unclear in the context of small B-cell lymphoma, these pathways have been known to eventually lead downstream to NF-κB signaling, which is heavily implicated in lymphomagenesis . ConstitAdditionally, inactivating somatic mutations or deletions have been reported in the tumor suppressor gene, ubiquitin-editing protein A20 (or TNFAIP3) across many types of lymphomas including MALT and FL . OverexpInterestingly, a recent study reported on the absence of the expression of STING  specifically in B-cell non-Hodgkin lymphomas, including various small B-cell lymphomas like FL, MCL, MZL and SLL, but not in T- and NK-cell lymphomas . This fi12 and Gɑ13 are members, consist of G proteins, which are G-alpha subunits of heterotrimeric GTP-binding proteins. G proteins serve as the intermediary between GPCRs on the cell membrane and downstream signaling, and they work by binding to guanine nucleotides. G12 proteins, together with other G protein subfamilies, form the most diverse group of receptors, playing a wide range of important roles in normal physiology. In the context of this study, Gɑ12 and Gɑ13 have been demonstrated to regulate the maturation of B cells in the marginal zone in a murine model [12 and Gɑ13 has been linked to several cancers. However, G12 proteins still remain one of the least understudied subfamily in cancer biology, especially in hematological malignancies. However, the few studies done in lymphomas do point towards significant roles that Gɑ12/13 signaling play in lymphomas in general [The G12 subfamily, of which Gɑne model . Unsurpr general ,67,68. i and Gɑ12, and not other G proteins. The resulting signaling complex then initiates a cascade of events involving non-canonical signaling complexes, ultimately leading to the activation of NF-κB signaling [12 could play an important enabling role in lymphomagenesis by mediating the activation of NF-κB signaling.Constitutive NF-κB signaling is known as an important hallmark of lymphomas and much work has been done on pathways that drive its activity. Enhanced NF-κB activity has been associated with increased hedgehog signaling. Smoothened (SMO), yet another GPCR and also an essential signal transducer of the hedgehog signaling pathway, has been shown to recruit and activate Gɑignaling . This st13, along with associated receptors S1PR2 and P2RY8, appear to promote the confinement of B cells to ensure a physiologically normal germinal center. Gɑ13 deficiency has been shown to give rise to germinal center B-cell-derived lymphoma in mice [13), S1PR2, or P2RY8—found in GCB-DLBCL patients—have been demonstrated to cause the dissemination of germinal center B-cells , hence also leading to germinal center B-cell-derived lymphoma [12, Gɑ13 plays a tumor suppressive role in orchestrating the proper development of the germinal center.Conversely, Gɑ in mice . Similarlymphoma . Unlike 12/13 signaling events in lymphomas, we hypothesize that Gɑ12 signaling could be a significant player in small B-cell lymphomagenesis.Taking together the limited knowledge gathered on GɑmiRSEA analysis also identified pathways that are regularly implicated in lymphomas, hence validating the relevance of biomarker miRNAs that differentiate small B-cell lymphomas from RL . SignifiOverall, our results demonstrate that miRNA expression profiling may serve as a promising biomarker and practical tool to aid the diagnosis of common lymphoid lesions. Specifically, we identified and validated two unique miRNA-based classifiers that can help to diagnose and subtype the four most common diagnostic entities of small B-cell lymphomas.


The global health emergency caused by the COVID-19 pandemic has put healthcare professionals in an unprecedented challenge, considering them extremely at risk population.To estimate the prevalence of clinically significant mental disorders and to assess associated factors among Consorci Sanitari del Maresme workers during the COVID-19 pandemic.We made a descriptive cross-sectional study. All workers were invited to participate in an online survey during May 2021. Individual characteristics and frequency of direct exposure to COVID during professional activity were assessed. We used three Spanish versions of psychometric scales: the Patient Health Questionnaire to evaluate depressive symptomatology, the Generalized Anxiety Disorder scale, which detects anxiety, and the 4-item version of the PTSD checklist for DSM-5 for PTSD screening purposes. Chi-square tests and logistic regression were used to analyze the data.A total of 355 workers participated. Overall, 31% met the criteria for Major Depressive Disorder (PHQ-9> 9), 36% for Generalized Anxiety Disorder (GAD-7> 9) and 22% for Post-traumatic Stress Disorder (PCL-5> 7). It has been found that young adult, women, those with prior mental disorders or those with greater exposure to COVID-19 are risk factors for any current mental disorder.There are large mental healthcare needs among healthcare professionals. There is a clear need to closely monitor the extent to which these needs are adequately met. In the design of measures and interventions to reduce this impact, an individualized approach should be considered while taking into account sociodemographic variables, psychiatric history and the frequency of direct exposure to COVID-19.No significant relationships.


Complex carcinogenic mechanisms and the existence of tumour heterogeneity in multiple myeloma (MM) prevent the most commonly used staging system from effectively interpreting the prognosis of patients. Since the microenvironment plays an important role in driving tumour development and MM occurs most often in middle-aged and elderly patients, we hypothesize that ageing of bone marrow mesenchymal stem cells (BM-MSCs) may be associated with the progression of MM.  In this study, we collected the transcriptome data on MM from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. Differentially expressed genes in both senescent MSCs and MM tumour cells were considered relevant damaged genes. GO and KEGG analyses were applied for functional evaluation. A PPI network was constructed to identify hub genes. Subsequently, we studied the damaged genes that affected the prognosis of MM. Least absolute shrinkage and selection operator (lasso) regression was used to identify the most important features, and a risk model was created. The reliability of the risk model was evaluated with the other 3 GEO validation cohorts. In addition, ROC analysis was used to evaluate the novel risk model. An analysis of immune checkpoint-related genes, tumour immune dysfunction and exclusion (TIDE), and immunophenotypic scoring (IPS) were performed to assess the immune status of risk groups. pRRophetic was utilized to predict the sensitivity to administration of chemotherapeutic agents.  We identified that MAPK, PI3K, and p53 signalling pathways were activated in both senescent MSCs and tumour cells, and we also located hub genes. In addition, we constructed a 14-gene prognostic risk model, which was analysed with the ROC and validated in different datasets. Further analysis revealed significant differences in predicted risk values across the International Staging System (ISS) stage, sex, and 1q21 copy number. A high-risk group with higher immunogenicity was predicted to have low proteasome inhibitor sensitivity and respond poorly to immunotherapy. Lipid metabolism pathways were found to be significantly different between high-risk and low-risk groups. A nomogram was created by combining clinical data, and the optimization model was further improved. Finally, real-time qPCR was used to validate two bortezomib-resistant myeloma cell lines, and the test confirmed that 10 genes were detected to be expressed in resistant cell lines with the same trend as in the high-risk cohort compared to nonresistant cells.  Fourteen genes related to ageing in BM-MSCs were associated with the prognosis of MM, and by combining this genotypic information with clinical factors, a promising clinical prognostic model was established. Multiple myeloma (MM) is a cancer entailing heterogeneous clonal proliferation of plasma cells and accounts for more than 10% of all haematologic malignancies . AlthougSmouldering multiple myeloma (SMM) is very likely to progress to active multiple myeloma (with a probability of approximately 73%) . Mice wiMesenchymal stem cells (MSCs) play an important role in the bone marrow microenvironment. Bone marrow mesenchymal stem cells (BM-MSCs) are adult stromal cells of mesodermal origin with the ability to modulate immune system responses, self-renewal, injury repair, and multipotent differentiation . HoweverBM-MSCs could be more than victims; they may constitute the cause of microenvironmental inflammation in the development of myeloma . The infhttps://www.ncbi.nlm.nih.gov/geo/) database and The Cancer Genome Atlas (TCGA) database . GEO accession number GSE7888 contains cultivated BM-MSCs samples from 6 healthy humans and 6 matched senescent stage samples obtained from an in vitro culture. Multiple myeloma expression profiles were obtained from accession number GSE6447, with 125 MM (excluding MUGS) and 15 healthy donor plasma cell samples. The MSC senescence-related gene prognostic signature was reconstructed based on the training dataset (MMRF-CoMMpass downloaded from TCGA) (n = 858). The other MM data were obtained from GEO accession numbers GSE57317 (n = 55), GSE83503 , and GSE4581 (n = 414) to validate the outcome. A flowchart of the investigation is depicted in Transcript levels and matched clinical information were collected from the GEO  were identified using adj. p value <0.05 as the screening condition.The relative gene expression of MSC senescence-related genes was normalized and identified using the “limma” package with adj. The intersection of genes differentially expressed between MM and normal plasma cells with MSC senescence-related genes was examined. The roles of intersecting genes in biological pathways were explored using the “clusterProfiler” package. Intersected DEGs were entered into the STRING database to obtain a protein-protein interaction (PPI) network, which was visualized using Cytoscape software. Differential gene enrichment analysis for high and low-risk groups was obtained from the Metascape database.The samples in TCGA-MMRF-COMMPASS were used as the training set, and genes significantly associated with survival were identified using univariate Cox proportional hazards analysis. The candidate genes obtained from one-way Cox regression analysis were subjected to lasso regression analysis using the R package “glmnet.” The risk model was constructed based on the expression of genes obtained from lasso regression. Risk scores were calculated, and patients were classified into high-risk and low-risk groups according to the median risk score. Differences in survival between patients in high-risk and low-risk groups were analysed using Kaplan–Meier (KM) curves. ROC curve analysis was used to evaluate the performance of risk scores for predicting patient survival status.The constructed model was validated using the validation set , and based on the model, the risk score was calculated for each patient. According to the median risk score, patients were divided into a high-risk group and a low-risk group, and survival differences between patients in the high-risk and low-risk groups were analysed using Kaplan–Meier (KM) curves. ROC curve analysis was also used to evaluate the performance of risk scores in the prediction of patient survival status.The 50% inhibition concentration (IC50) values of 138 drugs were inferred using the “pRRophetic” algorithm, comparing the IC50 of chemotherapeutic agents in the high-risk and low-risk groups.n = 858) from the TCGA dataset after log2 (TPM+1) transformation. The data were normalized using the mean of all samples as a control, as required by official documents. Processed data were uploaded to the TIDE website (https://tide.dfci.harvard.edu). TIDE analysis results were obtained. An analysis of correlations between TIDE and risk scores was conducted, and the differences in TIDE scores between the high-risk and low-risk groups were examined. Moreover, immunophenotypic scoring (IPS) of all 858 tumour samples was performed using the “IOBR” package.A total of 282 immune checkpoint-related genes (ICRGs) were collected based on a previous study. The differences in ICRGs between the high-risk and low-risk groups were analysed. Tumour immune dysfunction and exclusion (TIDE) was used to evaluate the prognostic effect of immune checkpoint inhibition therapy. Analytical data were based on TPM data of all tumour samples . Differences between the two groups were evaluated using the Wilcoxon test. Kaplan–Meier analysis was applied to evaluate survival differences between the low-risk and high-risk score groups. A value of p < 0.05 was considered statistically significant.Using the “rms” package in R, we built clinical prognostic models based on the TCGA dataset using the risk scores generated in the previous phase and clinical data from patients, including sex, ethnicity, and stage. Prognostic models were constructed using univariate Cox analysis and multivariate Cox regression, respectively. The accuracy of the nomogram was predicted by plotting calibration curves over time. Multivariate Cox regression analysis was also performed to determine whether the prognostic risk score model could be employed as an independent predictor of OS in MM. The nomogram's prognostic value was then computed using AUC values from online ROC curves. All data analyses employed In this experimental study, we used the RPMI-8226 and OMP-2 cell lines from Fourth Military Medical University, and RPMI-8266 bortezomib-resistant cells and OMP-2bortezomib-resistant cells were previously developed and cultured in the presence of bortezomib (Selleckchem PS-341) .−ΔΔCT to calculate the fold change for relative expression. Data were presented as a mean ± standard deviation. Statistical analysis was performed with the GraphPad Prism 8.0.3 software. The mean values of two groups were compared by Student's t-tests. P < 0.05 was considered statistically significant.Total RNA was isolated from the bortezomib-resistant and nonresistant RPMI-8226 and OMP-2 according to the TRIzol manufacturer's protocol . RNA was reverse transcribed to cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics) according to the manufacturer's protocols. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with SYBR Green PCR Master Mix (Roche Diagnostics). n = 125) and plasma cells from healthy individuals (n = 15) in data from GEO accession number GSE6477. A total of 3568 differentially expressed genes (MM vs. control) were identified, of which 1842 were upregulated and 1726 were downregulated. The volcano map and the gene heatmap of MM DEGs are plotted in Figures Differentially expressed genes were identified in CD138+ plasma cells from multiple myeloma patients , with 128 genes upregulated and 89 genes downregulated. A volcano map and a heatmap of genes differentially expressed in ageing MSCs are shown in Figures The intersection of differentially expressed MM and MSC genes included a total of 48 genes. Venn diagram mapping is shown in A total of 48 damage-related genes obtained were subjected to GO enrichment. These genes were found to be enriched in biological processes (BPs), such as activation of MAPK activity, oestrogen response, positive regulation of epithelial cell proliferation, response to magnesium ions, and positive regulation of actin cytoskeleton reorganization. Furthermore, lattice protein vesicles, inhibitory synapses, voltage-gated sodium channel complexes, sodium channel complexes, lattice protein vesicle membranes, and other cellular components (CCs) were enriched. The main molecular functions (MFs) included voltage-gated sodium channel activation, sodium channel activity, catalytic-specific chromosome binding, and activation of RNA polymerase II-specificDNA-binding transcriptional activators .According to KEGG enrichment analyses, differentially expressed genes were mainly involved in the MAPK signalling pathway, PI3K-Akt signalling pathway, acute myeloid leukaemia, p53 signalling pathway, chronic myeloid leukaemia, and other signalling pathways .Damage-related genes were entered into the STRING database to obtain the protein-protein interaction (PPI) network . Genes wA total of 858 samples with clinical information from TCGA-MMPF-COMMPASS were used as the training set, and a one-way Cox analysis was performed on 48 damage-related genes to screen out 16 genes associated with prognosis. The forest diagram is shown in Lasso regression is a statistical method for obtaining a more refined model by constructing a penalty function, compressing some regression coefficients, reducing data dimensionality, and avoiding multicollinearity and overfitting in multiple regression models Figures . Sixteeni=1nβi∗xi, where βi is the coefficient and xi is the sample expression corresponding gene i. The coefficients for all 14 genes are calculated and shown in The risk model was constructed based on the expression of 14 genes obtained by lasso regression. The coefficient from the linear term of the lasso regression model was used to calculate the risk score Risk score = ∑n = 429) and the low-risk group (n = 429) based on the median risk score. The distribution of patients' risk scores and survival status is shown in Figures The formula was used to calculate risk scores for 858 patients, and patients were divided into the high-risk group (p < 0.05). Additional validation with two datasets. There was no median follow-up time in this dataset GSE83503, we removed the 16 samples of the missing visit data and used the chi-square test to calculate the survival rate in the high-risk and low-risk groups, and the results showed that the mortality rate was significantly higher in the high-risk group than in the low-risk group (p < 0.05). GSE4581 (n = 414) showed significant differences in survival between the high-risk and low-risk groups distinguished by the model constructed in this study  curve. As shown in  Figures  and S1B.ROC curve analysis was also used to evaluate the performance of risk scores in the prediction of patient survival status, and ROC curves were plotted for 1, 3, and 5 years. The results are shown in n = 55). Combined with the model, risk scores were calculated for each patient. Patients were divided into the high-risk group (n = 27) and the low-risk group (n = 28) based on the median risk score. The distribution of risk scores and survival status of patients are shown in Figures The constructed model was validated using the validation set (p < 0.05), as shown in The Kaplan–Meier (KM) curve was used to analyse the difference in survival between the high-risk and low-risk groups of patients in the validation group. Log-rank analysis showed a significant difference in survival between the two groups , 20 immune checkpoint molecules were found to differ significantly between the high-risk and low-risk groups: ADORA2L, BTLA, BTNL2, C10orf54, CD200, CD274, CD28, CD40, CD70, CTLA4, IDO2, KIR3DL1, LAG3, LAIR1, TIGIT, TMIGD2, TNFRSF14, TNFRSF4, TNFRSF8, and TNFRSF9  in the high-risk group and short survival after receiving ICB treatment. Furthermore, TIDE scores were significantly different between the high-risk and low-risk groups, as shown in A correlation analysis was conducted between the TIDE scores and risk scores of the TCGA-MMRF-COMMPASS dataset, and the differences in TIDE scores between the high-risk and low-risk groups were examined. Immunophenotypic scoring (IPS) was performed on all 858 tumour samples. The results were plotted on a mountain plot ; overallThe half-maximal inhibitory concentration (IC50) was calculated to predict the treatment response to chemotherapy drugs in the cohort from TCGA. 137 chemotherapeutic agents were evaluated in the high-risk and low-risk groups of patients in the TCGA dataset, and MG.132 (a proteasome inhibitor) was found to be least sensitive to tumour cells in the high-risk group, while some CDK inhibitors and multitarget kinase inhibitors had lower drug sensitivity in the high-risk group . AdditioThe risk score for the TCGA-MMRF-COMMPASS dataset was calculated, and the variation in risk scores with clinical indicators was analysed. The results showed that risk scores differed significantly among ISS stages and sexes , which iUnivariate Cox analysis screened for clinical factors associated with prognosis, and the effect of race was not significant . Among fWe evaluated mRNA levels of 14 genes in myeloma cell lines. In agreement with the gene model, compared with bortezomib-sensitive myeloma cell lines (negative control), there was an increase in mRNA expression of SCN9A, RGS7, NTF3, HSPB8, and ZBED1 and a decrease in CCND1, COBLL1, EGR1, OCLN, and ZBED1 mRNA expression of bortezomib-resistant cell lines . The expMultiple myeloma has a complicated pathogenesis, and mechanisms underlying its occurrence and progression remain largely unknown. For decades, resistance to drugs (particularly bortezomib), relapse, refractory disease, and inconsistency in the treatment of older individuals have been bottlenecks that hinder myeloma treatment. Furthermore, most of the genetic complexity of MM might be present at asymptomatic stages, but how initiating plasma cell clones acquire the potential for oncogenic transformation to MM is a crucial factor that has eluded researchers . There iA growing number of studies confirm that the bone marrow microenvironment is not always passive in the development of tumours . The infIn this study, we discovered a set of gene signatures that are essential in predicting MM progression and linked to MSC senescence, suggesting that early prevention of ageing in MSCs could help slow disease progression. The MAPK pathway and DNA repair pathway-related genes have been shown to be independent prognostic factors for high-risk SMM , and theThe following are some of the study's limitations: First, all of the patient records included in the study were obtained from the GEO database; second, the lack of some crucial clinical data hindered further investigation. BM-MSCs from healthy individuals, cultured in vitro and proven to be senescent, were chosen as targets for differential gene selection in this work because their transcriptomes were influenced by tumour cells in vivo. Only one study's selection of the MSC gene for ageing may have resulted in biased gene selection, while several studies may have introduced batch-to-batch variation.This is the first study to use the intersection of genes in ageing MSCs with differential genes in myeloma to find biomarkers that represent prognosis. We found genes in tumour cells with the same expression trend observed in MSC senescence that may provide targets for developing therapies capable of treating tumours while improving the ageing status of MSCs. Our research may help advance the development of drugs that not only kill MM cells but also improve the microenvironment. We offer new insights into the clinical diagnosis and treatment of MM, as well as suggestions for further research into the pathogenic mechanisms of drug resistance in myeloma. Overall, this research marks a step towards the ideal of data-driven clinical decision-making, in which better MM treatment options are determined using statistical models.This research uncovered a collection of genes associated with ageing in MSCs that were linked to MM prognosis. The prognostic model was also constructed by incorporating clinical characteristics, which will facilitate individualized treatment. Moreover, the combination of drugs targeting lipid metabolism is expected to be a better treatment option for our predicted high-risk patients.


Retinal vein occlusion (RVO) is the main cause of retinal vascular blindness. Laser photocoagulation therapy is the regarded as the standard treatment for branch retinal vein occlusion (BRVO) in the guidelines, but it is not effective for macular edema (ME) secondary to central retinal vein occlusion (CRVO). As anti-VEGF  or steroids monotherapy has been used to treat RVO, but each has its advantages and disadvantages. Our purpose was to evaluate the efficacy and safety of intraocular injection of anti-VEGF combined with steroids versus anti-VEGF or steroids monotherapy for ME secondary to RVO.We systematically searched trials on Pubmed, Embase, Cochrane Library, Web of Science and China National Knowledge Infrastructure (CNKI) for RCTs  or non-RCTs, comparing anti-VEGF or steroids monotherapy to their combination. The primary outcomes were changes in best-corrected visual acuity (BCVA), central macular thickness (CMT) and intraocular pressure (IOP). The pooled data was analyzed by random effects model.P = 0.01} or steroids  alone on changes of BCVA. Compared with anti-VEGF monotherapy group, the combination therapy also had a better effect {pooled MD (mean difference), 95% CI, -9.62 , P = 0.01)} at improvements on CMT. On the changes of IOP, assessment favored that combination therapy was associated with a better relief of IOP compared to steroids monotherapy group . What’s more, the incidence of ocular hypertension was lower in the combined treatment group compared with control group treated with steroids alone . Results also showed that the combination group can prolong the average time to first anti-VEGF reinjection  compared to control group treated with anti-VEGF alone.A total of 10 studies selected from 366 studies were included in this meta-analysis. Our results favored anti-VEGF with steroids combination therapy in comparison with anti-VEGF {pooled SMD (standardized mean difference), 95% CI, -0.16 , Anti-VEGF with steroids combination treatment can enable a better achievement of improving BCVA, CMT, reducing the risk of increased IOP and improving patient prognosis compared to anti-VEGF or steroids therapy alone, lengthening the average time to anti-VEGF reinjection with reducing the injections during follow-up.The online version contains supplementary material available at 10.1186/s12886-022-02682-7. Retinal vein occlusion (RVO) is the second most common retinal vascular disease after diabetic retinopathy, leading cause of retinal vascular blindness. It can be classified into branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO) according to the location of vascular occlusion . MacularThe occurrence and development of RVO is caused by multiple whole and local pathogenic factors. Current researches show that cytokines are involved in the pathophysiology in RVO. Noma et al.  have conAccording to the European Society of Retina Specialists (EURETINA), vitreous injection of anti-VEGF drugs is currently the primary treatment for RVO secondary to ME, no matter it is CRVO or BRVO. Once ME is found, vitreous injection should be carried out in time. Continuous injection should be at least 3 months, once a month, until the vision acuity has been stable, debating whether 3 + PRN or 6 + PRN should be chosen for application . Due to Inflammatory processes has been considered to be crucial in the pathogenesis of ME . SteroidSingle or repeated injection of TA or Ozurdex can significantly improve the visual acuity of patients with RVO, especially in the treatment of recurrent RVO macular edema , but it Therefore, intravitreal injection of anti-VEGF drugs is a first-line approach for ME secondary to RVO, and steroid sustained-release drugs also represent an effective alternative for ME in RVO . CombinaIn this systematic review and meta-analysis, we aimed to explore the efficacy and safety of anti-VEGF or steroids monotherapy vs. combination treatment of anti-VEGF plus steroids for ME secondary to RVO, and to verify the differences in the effects of treatment approaches on BRVO and CRVO, so as to provide clinical reference for the treatment of RVO.This meta-analysis is reported in accordance with the Systematic Reviews and Meta-Analyses (PRISMA) Statement. The registration number in PROSPERO is CRD42022332751.Trials were identified by a comprehensive search on Pubmed, Embase, Cochrane Library, Web of Science and CNKI. For studies published in Chinese journals, only journals indexed by The key magazine of China technology were considered to reduce publication bias. Trials on the ClinicalTrials.gov website, including those had been terminated, were excluded. We selected studies without date limits and language restrictions. The main search strategy were as follows: #1: vascular endothelial growth factor OR VEGF OR bevacizumab OR ranibizumab OR aflibercept OR pegaptanib OR avastin OR conbercept; #2: glucocorticoid OR steroids OR Ozurdex OR triamcinolone acetonide OR dexamethasone; #3: retinal vein occlusion; #4: Macular edema; #5: Controlled Trial; #6: #1 AND #2 AND #3 AND #4 AND #5.We considered studies as eligible for inclusion if they were clinical trials including randomized and non-randomized controlled trials, comparing control group as monotherapy of steroids or anti-VEGF treatment with experimental group as a combination of both, including patients with ME secondary to RVO. We recorded the diverse types of RVO in the included cases, but we didn't make that distinction when searching. Trials that included laser therapy in therapeutic scheme and provided data lacking specific mean or standard deviation (SD), as well as non-clinical trials, were excluded.The outcomes we assessed included changes on BCVA and changes on CMT, as well as side effects such as IOP, cataract, macular epiretinal membrane formation, endophthalmitis, retinal detachment, vitreous hemorrhage and glaucoma. Two investigators  independently screened all trials retrieved by search strategy described above, with one investigator reviewing the titles and abstracts and the other assessing the full texts without disagreement.The data of included studies that we extracted or calculated were as follows: study ID (first author), year, design, sample size (participants), male/female, age, etiological factor, case/control (monotherapy/combination therapy), details of injection, follow-up periods, selection criteria and evaluating parameters including changes in BCVA (mean [SD]), changes in CMT (mean [SD]), changes in IOP (mean [SD]).Due to the different study types of RCTs and non-RCTs, we respectively adopted different methods for risk of bias assessment. Two independent reviewers  assessed the risk of bias of RCTs based on PRISMA recommendations. The risk of bias of non-RCTs  was assessed according to “Methodological Index for Non-randomized Studies” (MINORS) , accordi2 testing was also performed to assess the heterogeneity, with values greater than 50% considered to indicate moderate to high heterogeneity. There was moderate heterogeneity between the studies of a few subgroups. Due to the small sample content of each of them, we conducted a sensitivity analysis through leave-one-out method and found that these individual results were consistent with the conclusion of meta-analysis. In addition, We constructed a funnel diagram and used the Begg-Mazumdar’s rank test and the Egger’s regression test to assess publication bias, defining significant publication bias as a p value < 0.05. Since the numbers of studies included in other subgroups were less than 5, we only assessed the publication bias of the effect of anti-VEGF monotherapy vs combined therapy on BCVA at 6-month follow-up. The P value was less than 0.05, indicating that no significant publication bias was detected  with 95% confidence interval (CI). Continuous data were provided as mean and SD. We used odds ratio (OR) with 95% CI in the assessment of adverse events such as cataracts and ocular hypertension. All the data synthesis were based on random effects models. We used the Cochran Q test to evaluate the magnitude of heterogeneity among the included studies. When the value of p was less than 0.1, we regarded it as evidence of heterogeneity. IWe utilized Review Manager  and STATA  for all statistical analyses.A total of 366 records were identified, of which 10 studies were remained in our analysis after screening  in BCVA compared with anti-VEGF monotherapy in month 1 , month 2 , month 3, month 4, month 5 , month 6 , month 12 , month 24   in BCVA in week 1 , month 1 , month 3 , month 6 , month 12 , month 24   at improving on CMT in month 1, month 2 , month 3 , month 4 , month 5 , month 6   . The results were consistent at all follow-up time points of week 1 , month 1 , month 3 , or month 6    Figs.  and 9.FiP = 0.003)  showed recurrent ME associated with decreased visual acuity. Compared to control group treated with anti-VEGF alone, the combination group was associated with a longer average time to first anti-VEGF reinjection  Fig. . The resOur results of analysis showed that patients receiving the combination of anti-VEGF and steroids therapy were more likely to achieve the target of improving BCVA compared with patients receiving anti-VEGF or steroids therapy alone. Compared to anti-VEGF therapy alone, the combination of anti-VEGF and steroids therapy offers a greater reduction of CMT and SCS, and this combination can significantly reduce IOP compared to steroids therapy alone, thereby reducing ocular hypertension complications after treatment.Combination therapy of steroids with anti-VEGF has a better safety profile than steroids therapy alone, a longer duration of therapeutic effect than anti-VEGF therapy alone and a better cost effectiveness than DEX implants. Besides, the greatest advantage of combination therapy is that once the number of injections is reduced, the time from the first injection to the next injection is longer than that of monotherapy. Compared to traditional monotherapy with a high injection frequency, the convenience of treatment and lack of identifiable complications for patients can relieve the pressure of receiving therapy and improve the quality of life of patients. What’s more, patients receiving combined treatment required fewer anti-VEGF reinjections compared to those receiving monotherapy, according to data from several trials , 16, 18.Studies have found that retinal pigment epithelial cells, as an important component of the blood-retina barrier, which play an important role in regulating ocular inflammation, are able to interact with leukocytes extensively infiltrating in the choroidal circulation and respond to IL-1β and TNF-α by secreting chemokines including IL-8 and MCP-1, while ICAM-1 mediates VEGF-induced retinal vascular permeability. DEX can inhibit IL-1β induction of MCP-1 and IL-8 by human retinal pigment epithelial cells (HRPE) , while TTaken together, our results were robust and consistent, independent of the length of follow-up, indicating that anti-VEGF with steroids combination therapy can be used as a therapeutic strategy to improve ME secondary to RVO, showing a likehood of solving the problem that laser photocoagulation is ineffective for ME secondary to CRVO. Combination therapy is more cost-effective and can reduce the dose and injection frequency to some extent, lower the risk of complications and achieve better outcomes of visual acuity compared with monotherapy.The limitation of our study is that the number of samples that can be included in the study is small for analysis of each outcome indicator. For example, it showed no significant difference in the efficacy of two treatments on VCT, SCT and TCT, likely to be a result of only one trial included in this analysis group. Endophthalmitis is probably the most visually destructive complication of intraocular injection, despite its low incidence . More trIn this study, the analysis was not performed according to the detailed classification of RVO. Based on the anatomical location of the occlusion, RVO can be divided into CRVO and BRVO, and BRVO can be further divided into major BRVO and macular BRVO . PreviouAlthough further trials and studies needs to be conducted to establish the optimal range of application in detail of anti-VEGF with steroids combination therapy in the clinical treatment of ME secondary to RVO, evidence of our findings suggest that this combination is superior to monotherapy in improving clinical indicators and reducing adverse events, especially refractory and recurrent ME secondary to RVO.Additional file 1:Supplement Figure 1. The publication bias of the effect of anti-VEGF monotherapy and vs combined therapy on BCVA at 6-month follow-up.Additional file 2:Supplement Table 1. The publication bias of the effect of anti-VEGF monotherapy and vs combined therapy on BCVA at 6-month follow-up. The P value was less than 0.05, indicating that no significant publication bias was detected.Additional file 3: Supplement Table 2. Bias risk of non-RCT according to MINORS.