Pseudomonas aeruginosa community-acquired pneumonia (PA-CAP) were reported in non-immunocompromised patients. We describe a case of Pseudomonas aeruginosa (PA) necrotizing cavitary CAP with a fatal outcome in a 53-year-old man previously infected with SARS-CoV-2, who was admitted for dyspnea, fever, cough, hemoptysis, acute respiratory failure and a right upper lobe opacification. Six hours after admission, despite effective antibiotic therapy, he experienced multi-organ failure and died. Autopsy confirmed necrotizing pneumonia with alveolar hemorrhage. Blood and bronchoalveolar lavage cultures were positive for PA serotype O:9 belonging to ST1184. The strain shares the same virulence factor profile with reference genome PA01. With the aim to better investigate the clinical and molecular characteristics of PA-CAP, we considered the literature of the last 13 years concerning this topic. The prevalence of hospitalized PA-CAP is about 4% and has a mortality rate of 33–66%. Smoking, alcohol abuse and contaminated fluid exposure were the recognized risk factors; most cases presented the same symptoms described above and needed intensive care. Co-infection of PA-influenza A is described, which is possibly caused by influenza-inducing respiratory epithelial cell dysfunction: the same pathophysiological mechanism could be assumed with SARS-CoV-2 infection. Considering the high rate of fatal outcomes, additional studies are needed to identify sources of infections and new risk factors, along with genetic and immunological features. Current CAP guidelines should be revised in light of these results.Rare cases of  Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Moraxella catarrhalis, Chlamydophila pneumoniae, Staphylococcus aureus and Legionella species. Moreover, ATS/IDSA recommend that each acute care center should develop its own validated screening tools for methicillin-resistant S. aureus (MRSA) and Pseudomonas based on local risk factors identified by the stewardship team. Not including the immunocompromised, as per the European guidelines (2011), ATD/IDSA (2019) recommends a non-anti-pseudomonal beta-lactam, plus or minus a macrolide or respiratory fluoroquinolone, as empiric therapy for CAP, not covering MRSA and Pseudomonas aeruginosa (PA) [In the most recent American Thoracic Society and the Infectious Diseases Society of America (ATS/IDSA) guidelines 2019), antibiotic recommendations for the empiric treatment of community-acquired pneumonia (CAP) were based on the following bacterial core respiratory pathogens: 9, antibiosa (PA) ,4,5. In osa (PA) . Howeverosa (PA) . CAP cauPseudomonas aeruginosa and community acquired pneumonia”.We performed a comprehensive literature review, which included publications from 2010 to 2022, via an online literature search using PubMed and the following keywords: “Culture and identification methods. Blood cultures were performed using Plus Aerobic/F, Plus Anaerobic/F culture vials incubated in the automated Bactec FX system . A Direct Gram stain was performed on each positive blood culture bottle, then subcultured on solid agar media. Respiratory sample was analyzed by a semi-quantitative culture, seeded on different media and incubated at 37 °C for up to 48 h. The identification of the isolates was performed using the VITEK MALDI TOF MS (bioMerieux); antimicrobial susceptibility testing of strains was performed using the Vitek 2 automated system . Minimal inhibitory concentrations (MIC) were interpreted using EUCAST clinical breakpoints.Whole Genome Sequencing. Genomic DNA of the PA strain was extracted with DNeasy Blood and Tissue kit , sequencing libraries were prepared using DNA Prep (M) Tagmentation  and run with a Miseq platform (Illumina) producing pair-end reads (2 × 250 bp). Reads were evaluated for quality using FastQC, species and possible contamination were checked through Kraken2 ,9. Raw r2) of 90% at room air, with a slightly increased respiratory rate (20 breaths per minute). Electrocardiogram documented a sinus rhythm. Blood exams reported only hyponatremia , slightly high levels of C-reactive protein , alteration of D-dimer , significant elevation of procalcitonin level  and normal lactates value (1.2 mmol/L) , followed by dyspnea, fever, cough and hemoptysis. His past medical history was unremarkable, except for the paucisymptomatic SARS-CoV-2 infection experienced roughly two months prior. Vital signs at the arrival revealed a temperature of 36 °C, blood pressure of 120/60 mmHg, heart rate of 120 bpm, oxygen saturation (SpO mmol/L) .2 52 mmHg, range 75–100 mmHg, SpO2 90%, range 95–99%), so that an oxygen therapy via a Venturi mask FiO2 35% was implemented, with achievement of SpO2 95%. Chest X-ray displayed a right upper lobe opacification . He was transferred to the Intensive Care Unit where endotracheal intubation was complicated by cardiac arrest. Cardiopulmonary resuscitation was performed, followed by pneumothorax, so that a chest drain was placed. Twenty-four hours after admission, a repeat chest X-ray revealed complete right upper lobe, right lower and left mid-basal opacification, with diffuse thickening of the bilateral lower-middle lung . The whole genome of the isolated PA strain was sequenced: the PA strain belongs to ST1184, a rare ST, not listed among PA high-risk clones ,20,21,22The multinational prevalence of PA-CAP was estimated to be about 4%, specifically 3.8% in Europe , 4.3% in North America, 5.2% in Asia, 4.9% in South America, 5.5% in Africa and 3.1% in Oceania ,25.n = 11) [n = 9) [Rare cases of PA-CAP in healthy patients have been described since the 1960s: Hatchette et al. analyzed all cases indexed in PubMed from 1966 to 2000 (n = 11) , while M [n = 9) . We undeAcinetobacter baumanii and Klebsiella pneumoniae. Most cases started with empirical antibiotic therapy that did not perfectly cover PA and were subsequently modified accordingly with the identification of the pathogen. The duration of therapy varied from 14 to 30 days. Of the reported cases, 75% (9/12) needed intensive care and 33% (4/12) died: three cases died after a few hours from the admission (median time 11.3 h), and another one died after 9 days. Among survivors, 66% (8/12) were discharged after a median recovery of 23,75 days (7–35 days); the median recovery of patients discharged after intensive care  was 30 days.On review of the 12 cases reported, there was male gender predisposition with 4 females and 8 males affected, mean age of 35.33 years (1–67 years). A total of 33% (4/12) were active smokers, and only one case presented alcohol abuse. In total, 42% (5/12) had previous environmental exposure . Upon admission, 58% (7/12) reported pleuritic chest pain, together with right back pain in 71% (5/7). Other symptoms were cough , fever , hemoptysis , dyspnea , diarrhea , weight loss  and diffuse bone pain . Concerning white blood cells (WBC), 42% (5/12) presented leukocytosis, while 42% (5/12) had leucopenia. In relation to radiological features, 83% (10/12) showed right pneumonia, with upper lobe consolidation in 58% (7/12) and evident cavitation in 33% (4/12). The left lung was interested only in 25% (3/12). In addition to the respiratory samples, PA was also isolated from blood in 58% (7/12) and pleural effusion in 16% (2/12). In two cases, co-infection with Influenza A occurred, while in other two cases, other bacteria were isolated from respiratory samples: Pseudomonas aeruginosa, extended-spectrum β-lactamase Enterobacterales and methicillin-resistant Staphylococcus aureus) more promising for detecting PA-CAP, even in the immunocompetent host, independent of the site of pneumonia acquisition [Lung structural abnormalities, immunocompromised condition, chronic heart failure, cerebrovascular disease, burns, malnutrition, prolonged antibacterial therapy, recent hospitalization (in the past 90 days), previous PA infection/colonization and premature birth were reported as risk factors for PA-CAP. In people without these underlying diseases, advanced age, smoking, alcohol use and exposure to contaminated liquids were considered ,44,45,46uisition ,52,53. Iuisition ,55. NevePatients generally have no specific symptoms such as cough, fever and dyspnea; hemoptysis and pleuritic chest pain, particularly in the right back, should induce to consider PA infection, as reported in the cases above ,33,34,35Genomic characterization of PA-CAP strains isolated from patients was only reported in a few cases. Huhulescu et al. 2011)  reported11  reporIn recent years, empirical treatment for patients with CAP has become challenging due to the emerging prevalence of drug-resistant bacteria in community settings ,17. ManyBoth the European guidelines (EG) and ATD/IDSA guidelines recommend empiric therapy with a non-anti-pseudomonal beta-lactam and a macrolide or a quinolone alone in non-severe inpatient pneumonia while recommending a non-anti-pseudomonal beta-lactam, plus either a macrolide or a quinolone, in severe inpatient pneumonia for patients without risk factors for PA or MRSA ,2,3—the The CAP official clinical practice guideline approved by ATS/IDSA and the subsequently published Consensus Statement for treatment of immunocompromised patients with suspected CAP suggested extending empirical therapy beyond core respiratory pathogens when (1) risk factors for drug-resistant organisms and 2) a prior identification of MRSA or PA in the respiratory tract. According to these recommendations, initial empirical therapy should cover PA if there is a history of colonization or infection with a resistant gram-negative bacillus in the prior 12 months, previous hospitalization with exposure to broad-spectrum antibiotics, the presence of a tracheostomy, neutropenia, or a history of pulmonary comorbidity  [ a prior Our case seems to suggest the need for a revision of the post-pandemic SARS-CoV2 epidemiological criteria by including the ‘previous COVID-19 disease’ as a possible risk factor for PA-CAP. Moreover, our fatal outcome underlines the necessity of further study to investigate the effectiveness and safety of using illness severity tools as decision support to guide the intensity of treatment in adults hospitalized for pneumonia.Indeed, the delay in empirical antimicrobial therapy will place the patient at an increased risk of mortality, and patients transferred to an ICU after admission to a hospital ward experience higher mortality than those directly admitted to the ICU from an emergency department ,62,63. TAlthough PA-CAP is rare in immunocompetent patients, clinicians should consider this pathogen in their differential etiological diagnosis for CAP since, after an insidious course, it is characterized by necrotizing pneumonia with high morbidity and mortality. Risk factors identified for PA-CAP in healthy young people, such as smoking, alcohol use and contaminated water exposure, together with atypical clinical signs (hemoptysis and right back pain) and radiological features frequently described (right upper lobe opacification), should guide clinicians to consider PA in patients with CAP. WBC and PCT use are not reliable in the management of localized necrotizing or cavitary pneumonia. The PES score may help to address the use of appropriate empiric therapy in high-risk patients and consider rigorous antibiotic de-escalation whenever possible as part of a responsible antimicrobial stewardship policy. Analysis of this CAP case, together with the comparison of the other PA-CAP case described in the literature, shows that PA isolates with different STs and different virulence profiles, could be responsible for CAP with a rapidly fatal course. Finally, our case underlined that co-infection with a respiratory virus, in particular, with SARS-CoV-2 infection, deserves future attention. Considering the high severity and rapid progression of PA-CAP, further studies are needed to identify sources of infection, unknown risk factors, microbiological features and immunological determinants. These factors could lead to a more selective and earlier identification of patients at risk for PA-CAP in order to better define a target treatment algorithm.


Breast cancer is a heterogeneous disease characterized by distinct molecular subtypes, varied prognoses, and differential treatment responses. Understanding the molecular landscape and identifying therapeutic targets, such as trophoblast cell-surface antigen 2 (TROP2), is vital. TROP2 is notably overexpressed in breast cancer, playing a significant role in tumor growth, invasion, metastasis, and treatment resistance. While significant progress has been made in targeting TROP2 in breast cancer, several challenges and knowledge gaps remain. These challenges include the heterogeneity of TROP2 expression within breast cancer subtypes, resistance to its targeted therapies, potential off-target effects, limited therapeutic agents, and identifying optimal combination treatments. Integrating findings from clinical trials into clinical practice further complicates the landscape. This review article delves deep into TROP2 in breast cancer, highlighting its expression patterns, clinical implications, and therapeutic advancements. By understanding the role of TROP2, we can pave the way for personalized treatments, and transform the landscape of breast cancer care. These statistics underlie the ongoing efforts to evolve and refine breast cancer treatment strategies.Breast cancer remains the most commonly diagnosed cancer among women globally, accounting for approximately 30% of all new cancer diagnoses in women annually. Predictions for 2023 estimate 297,790 new invasive breast cancer cases, 55,720 new cases of ductal carcinoma Notably, breast cancer is a heterogeneous disease  with disAlthough progress has been made in breast cancer management, obstacles like treatment resistance and paucity of therapeutic options persist –9. WithiHowever, translating the potential of TROP2 into effective therapeutic strategies are challenging. Heterogeneity in TROP2 expression within breast cancer subtypes can affect treatment response and clinical outcomes , 18. OthIt is pivotal to decode the complexities of TROP2’s role in breast cancer for progress in personalized treatment and overcoming resistance. Understanding the expression patterns of TROP2, prognostic relevance, and therapeutic innovations offers avenues for better-targeted therapies, optimizing therapeutic response, and enhancing breast cancer patient outcomes , 21, 22.Despite accumulating evidence on TROP2’s therapeutic potential in breast cancer, a significant knowledge gap regarding its precise role and therapeutic application challenges . AddressIn this review, we endeavor to shed light on TROP2 in breast cancer, focusing on its expression patterns, clinical implications, and therapeutic progress. We explore the variability of TROP2 expression among different breast cancer subtypes and its correlation with clinicopathological factors. Additionally, we discuss the prognostic value of TROP2 expression, its association with treatment response, and its potential as a predictive biomarker. The latest therapeutic innovations targeting TROP2, including monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), and immunotherapeutics, are also examined. Finally, our focus shifts to prospective avenues and challenges in harnessing TROP2 therapeutically. Emphasizing the necessity for more in-depth research to elucidate TROP2’s molecular mechanisms and navigate the obstacles in developing effective TROP2-targeted therapies, we aim to uplift patient outcomes and reshape breast cancer treatment paradigms.22.1The heterogeneity of breast cancer manifests as distinct molecular subtypes, each with specific molecular characteristics and clinical behaviors. These subtypes significantly influence treatment approaches and outcomes. The major subtypes include HR+, HER2+, and TNBC, each has its own therapeutic considerations , 24.In a recent groundbreaking multi-omics study conducted by Jin et al. , the intTherapeutic considerations are different for each subtype. HR+ breast cancers, primarily driven by estrogen receptor (ER) and/or progesterone receptor (PR) are amenable to endocrine therapies. These receptors serve as therapeutic targets, and as such, endocrine therapies are highly effective in this subtype . HormoneIn summary, the treatment landscape for breast cancer is significantly influenced by the specific molecular subtype, with each subtype requiring distinct therapeutic strategies. Understanding the molecular intricacies of breast cancer, particularly the HR+/HER2- subtype, is crucial for optimizing therapeutic responses and outcomes, including the potential benefits of immunotherapy, as emphasized in the recent multi-omics study by Jin et al. .2.2Though traditional treatments such as surgery, chemotherapy, radiation therapy, and hormonal therapy have undoubtedly improved outcomes for many breast cancer patients. However, the persisting challenges such as treatment resistance –30, and 2.3TROP2, a 323 amino acids transmembrane glycoprotein , is prom2.4TROP2’s involvement in breast cancer progression spans various facets, from promoting cell proliferation to resisting therapies . CrucialThis diagram illustrates the multifaceted involvement of TROP2 in various oncogenic signaling cascades. TROP 2 activates the ERK1/2-MAPK axis, promoting malignant transformation and driving tumorigenesis. Additionally, it modulates the Notch pathway, influencing stem cell functions and potential tumor differentiation and hierarchy . TROP2 a33.1TROP2 exhibits notable overexpression in TNBC, a subtype distinguished by its aggressive phenotype, establishing it as a pivotal therapeutic target and prognosis biomarker , 36. Con3.2Elevated TROP2 expression levels in breast cancer correlate with unfavorable prognostic markers, including larger tumor size, and increased risk of recurrence , 44. Spe3.3Studies on the associations between TROP2 expression and clinical-pathological characteristics in TNBC present mixed findings. While some studies found no substantial correlation between TROP2 levels and clinicopathological factors in breast cancer, like age, histologic subtype, tumor grade, stage, lymphovascular invasion, or tumor-infiltrating lymphocytes (TILs) levels , the sig3.4TROP2 stands out as a promising predictive biomarker in breast cancer, with its expression levels informing on treatment response. Elevated TROP2 expression levels in tumors have been associated with resistance to specific therapies, such as chemotherapy and endocrine treatments . NotablyIn summary, TROP2’s expression patterns and implications in breast cancer solidify its stature as both a prognostic and predictive biomarker. As we advance our understanding, the insights gathered can guide clinical decisions, promote personalized treatments, and improve patient outcomes.4Numerous clinical trials are currently investigating TROP2-targeted therapy for breast cancer, including mAbs, ADCs, and CAR T-cell therapies. These therapies hold great promise for advancing breast cancer treatment by targeting TROP2 specifically and effectively.4.1Several TROP2-targeted mAbs are under evaluation, with a focus on improving therapeutic efficiency while sparing healthy cells , 50. NotPromising ADCs under investigation include PF-06664178 , 52, IMM4.1.1Developed by Pfizer, PF-06664178 represents a cutting-edge ADC drug that utilizes a humanized IgG1 mAb targeting TROP2, a prominent antigen on breast cancer cells. The mechanism of this ADC is intriguing: once it binds to TROP 2 and is internalized by the cancer cell, it is directed to the lysosomes, it is within these cellular compartments that the ADC releases its cytotoxic payload, the auristatin-based compound known as Aur0101 . Prelimi4.1.2Sacituzumab Govitecan (Trodelvy or IMMU-132) is an FDA-approved ADC tailed for TROP2-positive cancers, particularly TNBC. Its mAb component specifically targets TROP2, delivering the cytotoxic agent SN-38 directly to the tumor cells. Clinical trials have demonstrated its effectiveness for metastatic TNBC patients, positioning it as a promising frontline treatment –58.4.1.3Dato-DXd, or DS-1062a, represents a promising addition to the landscape of TROP2-targeted therapies in breast cancer. This innovative ADC pairs a TROP2-targeting antibody with topoisomerase I inhibitor payload, creating a highly specific and effective therapeutic approach . The rat4.1.4SKB264, also known as AKB264, represents another noteworthy member of the TROP2-targeted ADC family. It shares the same mAb as IMMU-132, which targets the TROP2 receptor in breast cancer cells, is being investigated for its potential therapeutic effects. Current clinical trials aim to ascertain its effectiveness in various breast cancer stages, particularly in advanced forms . The key4.2in vitro. Moreover, these T2-CAR T cells produced a plethora of effector cytokines upon antigen stimulation  with different co-stimulatory intercellular domains and found that T2-CAR T cells exhibited robust cytotoxic activity against Trop2-positive cells mulation . Interesin vitro and in vivo settings  targeting TROP2 effectively killed TROP2-positive pancreatic cancer cells and inhibited tumor growth in xenograft models. These findings suggest that TROP2-CAR T-cells, including breast cancer, can be a potent therapeutic strategy for TROP2-positive cancer types. In another study, Zhao et al. developed bi-specific CAR T-cells targeting TROP2 and PD-L1 and showcased their superior tumoricidal activity in both settings . CollectIn conclusion, TROP2-targeted therapies are gaining momentum as potential treatments for breast cancer. Ongoing clinical trials will continue to define the role of these therapies in the treatment landscape. For a comprehensive list of ongoing clinical trials focusing on TROP2 inhibitors in TNBC, please refer to 5The future of breast cancer treatment sees promise in advanced interventions such as ADCs, next-generation CAR T-cell therapies, and novel immunotherapeutic interventions. Integrating TROP2-targeted therapies with conventional treatments could enhance efficacy and patient outcomes. Crucial steps ahead include biomarker validation, understanding resistance mechanisms, and refining therapeutic avenues through rigorous clinical trials and translational research. Integrating TROP2-targeted therapy into personalized medicine and ensuring equitable access are important objectives to enhance patient care.Nevertheless, targeting TROP2 in breast cancer treatment is not devoid of challenges. Ensuring therapy specificity is vital to minimize toxicity in normal tissues. The varied nature of breast cancer subtypes necessitates strategies capable of addressing each subtype effectively. Key challenges lie in surmounting treatment resistance, pinpointing predictive biomarkers, and gaining an in-depth understanding of TROP2 signaling dynamics. Addressing these aspects will pave the way for fully harnessing the potential of TROP2-targeted therapy in breast cancer treatment.6TROP2’s role in breast cancer, highlighted by its pronounced overexpression and pivotal function in tumor dynamics, establishes it as a compelling therapeutic target. With an array of promising TROP2-centric interventions, from mAbs, ADCs, to CAR T-cell therapy, the landscape of treatment, especially for TNBC, is evolving. The endorsement of Sacituzumab Govitecan by the FDA for metastatic TNBC highlights this potential. However, the journey is not without obstacles, with resistance emergence, of breast cancer subtypes variation, and the need for reliable biomarkers being foremost. To truly harness the promise of TROP2-based interventions, these challenges mandate focused research. The horizon of TROP2-targeted strategies shines brightly with promise for advancing breast cancer therapeutics.LY: Conceptualization, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing, Methodology, Writing – original draft. JC: Data curation, Formal Analysis, Investigation, Methodology, Resources, Validation, Writing – original draft, Writing – review & editing. WM: Conceptualization, Formal Analysis, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing.


Alzheimer disease (AD) models are based on the notion that abnormal protein aggregation is the primary event in AD, which begins a decade or longer prior to symptom onset, and culminates in neurodegeneration; however, emerging evidence from animal and clinical studies suggests that reduced blood flow due to capillary loss and endothelial dysfunction are early and primary events in AD pathogenesis, which may precede amyloid and tau aggregation, and contribute to neuronal and synaptic injury via direct and indirect mechanisms. Recent data from clinical studies suggests that endothelial dysfunction is closely associated with cognitive outcomes in AD and that therapeutic strategies which promote endothelial repair in early AD may offer a potential opportunity to prevent or slow disease progression. This review examines evidence from clinical, imaging, neuropathological, and animal studies supporting vascular contributions to the onset and progression of AD pathology. Together, these observations support the notion that the onset of AD may be primarily influenced by vascular, rather than neurodegenerative, mechanisms and emphasize the importance of further investigations into the vascular hypothesis of AD. Alzheimer disease (AD) is the most common cause of dementia, accounting for over 70% of dementia cases in individuals above the age of 65 years . The twoVascular disease, including arteriolosclerosis, atherosclerosis, microinfarcts, and cerebral amyloid angiopathy (CAA), is a common co-pathology which is observed in 20–80% of AD brains at autopsy ,5,6,7. FThis review will examine evidence from clinical, imaging, neuropathological, and animal studies supporting vascular contributions to the onset and progression of AD pathology, including amyloid and tau aggregation, as well as direct vascular contributions to synaptic injury, impaired axonal repair, and neurodegeneration. Importantly, the present review discusses evidence supporting the role of specific vascular constituents  and vascular abnormalities at the capillary level  in mediating AD onset and progression. Together, these observations support the notion that the onset of AD may be primarily influenced by vascular, rather than neurodegenerative, mechanisms and emphasize the importance of further investigations into the vascular hypothesis of AD.Several lines of evidence support the presence of vascular contributions to AD pathogenesis including associations of vascular pathology  with mechanisms involved in AD pathogenesis, including amyloid and tau aggregation, oxidative stress, mitochondrial dysfunction, and vascular inflammation, which cumulatively contribute to synaptic loss and neurodegeneration ,15,19,20The vascular hypothesis of AD, which was first proposed over 30 years ago, is based on the notion that the first inciting event in AD pathogenesis is vascular dysregulation, which then initiates a cascade of molecular and neuropathological changes leading to neuronal dysfunction and the onset of AD pathologies, such as amyloid and tau aggregation ,24,25,26This review summarizes the literature supporting this hypothesis and presents new data suggesting additional direct links between specific vascular components and cognition. Importantly, this current review expands the vascular hypothesis beyond associations of macrovascular pathology with AD to highlight the importance of “micro”-vascular constituents at the cellular and molecular level in AD onset and progression.APOE4) genotype, the most significant genetic risk factor for AD [E2, E3, and E4. The presence of 1 or 2 APOE4 alleles increases AD risk by 2–3-fold and 10–15-fold, respectively, due to increased amyloid and tau aggregation, dysregulated cholesterol metabolism, and impaired synaptic plasticity [APOE4 allele have a higher risk for both late-onset AD and cardiovascular disease, further supporting an important link between vascular disease and AD pathogenesis [The association of vascular risk factors, including hypertension, hyperlipidemia, diabetes mellitus (DM), obesity, and metabolic syndrome, with AD has been well documented in several clinical and epidemiological studies ,31,32,33r for AD ,38. Apolasticity ,40,41. Cogenesis .APOE4 [APOB, which codes for apolipoprotein B, an important component of LDL [Other studies have shown associations of AD with midlife hypertension, while associations of AD with late-life hypertension have been inconsistent. In the longitudinal Honolulu-Asia Aging Study, midlife hypertension was associated with a higher risk for AD in middle-aged Japanese American men followed over 25 years, who demonstrated neuropathological evidence of increased hippocampal tau pathology and brain atrophy over follow-up, compared to normotensive controls . InteresAPOE4 ,45,46. AAPOE4 ,48. Furtt of LDL . High LDt of LDL ,51,52.The Rotterdam study, which examined over 7000 older adults, reported a higher risk for AD in association with vascular risk factors, such as DM, thrombotic episodes, high fibrinogen or homocysteine levels, atrial fibrillation, smoking, alcoholism, and atherosclerosis ,53. PartFurthermore, vascular risk factors appear to have additive or synergistic effects on AD risk, with a higher risk being reported in the presence of multiple vascular risk factors ,58. One Vascular disease, including subcortical white matter lesions or lacunes, is a common co-pathology in AD, being reported in 18–84% of AD brains ,7; howevTogether, these findings suggest that vascular risk and Aβ likely have independent, yet overlapping , effectsStructural distortions to brain capillaries, including increased tortuosity, looping or kinking, basement membrane thickening, and luminal buckling or narrowing, are consistently observed in AD brains ,16,17,18Endothelial dysfunction is observed in almost all AD brains at autopsy , includiEndothelial alterations have previously been considered secondary to amyloid or tau toxicity on cerebral vasculature; however, emerging evidence from animal studies suggests that endothelial dysfunction is an early and primary process in AD that may precede amyloid and tau pathologies and promotes abnormal protein aggregation and synaptic loss ,15,69. RAPOE-isoform-specific manner [Pericytes are specialized cells located in the capillary basement membrane, which have contractile properties, and are uniquely positioned to interact closely with other cellular components of the neurovascular unit, such as endothelium, neurons, and astrocytes ,82. Peric manner ; howeverc manner ,85,86,87APOE4-mediated disruption of the neurovascular unit in clinical and animal studies. APOE4 expression results in upregulation of the LRP-1-dependent proinflammatory cyclophilin A (CypA)–nuclear factor κB (NF-κB)– MMP-9 pathway in pericytes and brain endothelium. Activated MMP-9 from pericytes leads to endothelial toxicity, BBB breakdown, and neuronal injury [APOE4-carriers years prior to the onset of cognitive decline and is associated with increased CSF levels of MMP-9 derived from pericytes [Pericyte dysfunction has been associated with l injury . An age-ericytes .+/−Pdgfrβ) mice were crossed with mice overexpressing the Swedish mutation of the human amyloid precursor protein  [+/−Pdgfrβ nor sw/0APP mice demonstrated tau pathology, sw/0APP/Pdgfrβ+/− mice had evidence of tau pathology as early as 9 months. Neuronal loss and impaired hippocampal functions were observed in pericyte-deficient mice, although these changes appeared to be more severe when excess Aβ was also present [Pericytes also appear to contribute to tau aggregation in AD; pericyte loss is associated with neuronal accumulation of hyperphosphorylated tau, caspase-cleaved tau, and tau aggregation in animal studies ,91. Inte present ,91. Othe present . While i present .Astrocytes are specialized glia that interact with other components of the neurovascular unit through long cellular processes  . SeveralAPOE4-dependent and APOE4-independent pathways [Reactive astrocytes in AD may contribute to amyloid pathology through both pathways . Under ppathways ,96. Astrpathways . Astrocypathways .APOE4 expression by astrocytes also stimulates their transformation into the pro-inflammatory phenotype, which further contributes to Aβ aggregation [Conversely, inflammatory conditions with high TNF-α and interferon-γ (INF-γ) levels are associated with increased Aβ synthesis and release by astrocytes ,100. Excregation .APOE4-dependent tau hyperphosphorylation [APOE4, WWOX, CLU, CDK2AP1, MEF2C, and IQCK) and are linked to both amyloid and tau metabolism [Astrocytes have been linked to increased tau phosphorylation and tau-mediated neurodegeneration in AD, including tau accumulation within astrocytes and the release of tau oligomers through exosome-dependent pathways . Astrocyrylation . Severaltabolism ,103.APP knock-in mice at an early age and that CSF decorin levels correlate with Aβ plaque load and decorin levels in the choroid plexus [Other vascular constituents, such as the extracellular matrix (ECM) and basement membrane proteins, may have a role in AD pathogenesis . It has d plexus . Consistd plexus . Elevated plexus .Impaired CBF, measured by arterial spin labeling magnetic resonance imaging (MRI), is well documented early in AD, including preclinical stages, as well as in animal studies ,108,109.APP processing that sequentially cleave APP to produce the Aβ42 peptide), and impaired Aβ clearance due to reduced LRP-1 and increased RAGE expression in the brain endothelium [Consistent with these findings, animal models of chronic brain hypoperfusion  demonstrate AD pathology, including amyloid and tau aggregation and capillary changes in the CA1 region of the hippocampus that are almost identical to those seen in human AD brains . In thesothelium .While neuronal dysfunction and subsequent hypometabolism may contribute to reduced CBF in AD, several observations suggest that hypoperfusion in AD cannot be fully attributed to hypometabolism ; CBF altAPP gene. Capillary degeneration [Additional support for the importance of vascular contributions to AD pathogenesis comes from studies of individuals with trisomy 21 (Down syndrome) who have an additional copy of the neration  and reduneration ,138. Oxineration , a findiThe brain endothelium is a highly active interface that regulates the neuronal milieu via diffusible mediators and, as a part of the neurovascular unit, controls blood flow and neuronal metabolism ,140. BraSeveral studies suggest that hypoxia causes structural and functional alterations in the brain endothelium via different mechanisms, including rearrangement of the endothelial cytoskeleton, altering intercellular adhesions of endothelial cells, and promoting endothelial constriction, apoptosis, and degeneration . StudiesBACE1) enzyme involved in Aβ synthesis [BACE1 levels are increased in human AD brains, including those in the preclinical or early symptomatic stages , and may correlate with Aβ plaque burden and cognitive decline [BACE1 expression in AD include a hypoxia-inducible factor (HIF)-1α-mediated increase in BACE1 mRNA expression and enzymatic activity [BACE1 gene promoter contains a functional hypoxia-response element (HRE), and HIF-1α can effectively bind to the BACE1 promoter [BACE1 levels are observed in HIF-1α knockout mice. BACE1 upregulation due to hypoxia is also mediated by caspase-3, which cleaves GGA3  involved in BACE1 trafficking, thereby reducing BACE1 degradation [APP cleavage by γ-secretase, stimulates the transcription of the HIF-1α gene, potentially creating a positive forward loop between HIF-1α and γ-secretase [APP, Presenilin-1 (PS1), Presenilin-2 (PS2), and BACE1 [APP processing, which reduces the production of the extracellular sAPPα fragment, and results in subsequent loss of its neuroprotective and neurotrophic effects [PSEN2 (aka PS2) that lacks exon 5, referred to as PSEN2V [PSEN2V variant is abundantly expressed in the hippocampus of AD brains, promotes Aβ synthesis, and increases the vulnerability of the endoplasmic reticulum (ER) to stress [Data from animal and human studies suggests that capillary constriction in AD is sufficient to reduce CBF by up to 50%, leading to upregulation of the β-secretase1  expression and γ-secretase activity, contributing to increased amyloid pathology [PSEN1 gene promoter and induces its transcription [BACE1 expression [ER stress and impaired autophagy also occur due to hypoxia and are associated with increased athology , and m-cathology . Two majathology , are actcription , and PERpression . ER strepression ,167.Endothelial dysfunction also contributes to increased amyloid and tau aggregation via different mechanisms, including ineffective Aβ and tau clearance, dysregulated lipid raft endocytosis, and reduced endothelial nitric oxide synthase (eNOS), which are described below.APOE4-containing lipoproteins [Two neuronal cell surface receptors involved in Aβ clearance are impaired in AD: the downregulation of LRP-1, which transports free parenchymal Aβ into the systemic circulation, and the upregulation of RAGE, which transports Aβ across the BBB into the brain . Severalproteins .eNOS knockout (eNOS−/−) mice with a mouse model of amyloid pathology (APP/PS1 mice) was associated with increased phosphorylated tau (p-tau), which was attributed to an increase in Cdk5 activation by p25, while other kinases, such as GSK-3β, were unchanged [eNOS−/− and the APP/PS1/eNOS−/− mice, supporting the notion that the brain endothelium can influence neuronal mechanisms via soluble mediators [eNOS knockout alone was not sufficient to induce tau pathology, as increased p-tau was only observed in eNOS knockout mice that also had amyloid pathology  [Endothelial NOS (eNOS) plays an essential role in tau homeostasis and regulating tau phosphorylation by balancing the activity of tau kinases and phosphatases . Crossinnchanged ,177. Intediators ,177. Ano/− mice) ,177. Thi/− mice) ; however/− mice) . These fAPP processing within the lipid rafts [PS1 and NOTCH3 into lipid drafts due to oxidative stress [APOE4 and p-tau to the membrane in an age-dependent manner [Altered lipid raft endocytosis has been implicated in abnormal protein aggregation in AD and other neurodegenerative disorders and has been proposed as an additional mechanism by which hypoxia or endothelial dysfunction contribute to AD pathogenesis ,181. Lipid rafts ,184, ande stress . Aβ in lt manner . Interest manner , and p-tt manner .Lipid raft clusters are also present in endothelial cells and contribute to vascular inflammation . FurtherAngiogenesis and vascular remodeling are impaired in AD as evidenced by altered expression profiles of several angiogenic factors, such as VEGF, vascular-restricted mesenchyme homeobox-2 (MEOX2), MMP-9, angiopoietin-2, integrins (αvβ3 and αvβ5), and the transferrin receptor, leading to vascular regression and abnormal vessel sprouting ,192. SevAlthough several angiogenic factors are upregulated in AD, Aβ toxicity and low MEOX-2 levels in the brain endothelium, which impair angiogenesis, are also present. Hypoxia suppresses MEOX-2 expression in brain endothelium, and MEOX-2-deficient mice demonstrate vascular regression . TherefoVEGF is an important pro-angiogenic factor that is present in the vessel wall, reactive astrocytes, and perivascular amyloid deposits . While aThrombin is an endothelium-derived factor with important roles in angiogenesis and inflammation and contributes to tau pathology and neurodegeneration in AD. High levels of thrombin and the thrombin receptor, protease-activated receptor-1 (PAR-1) ,204, andRecent studies have shown that endothelial cells and pericytes demonstrate immunoregulatory functions and influence the neuronal milieu through the release of inflammatory and immune mediators. The brain endothelium in AD expresses high levels of inflammatory mediators such as MCP-1, intercellular adhesion molecule-1 , and cationic antimicrobial protein 37 kDa (CAP37) ,218,219.Endothelial dysfunction indirectly contributes to neuronal and synaptic loss via increased protein aggregation, vascular inflammation, impaired angiogenesis, and dysregulated thrombosis; however, it has become increasingly recognized that endothelial injury can also cause neuronal and synaptic loss via other mechanisms, including loss of endothelial GLUT-1 (glucose transporter protein type 1) which leads to impaired neuronal glucose uptake and metabolism, impaired neurovascular coupling, and potential direct endothelial interactions with pathways involved in synaptic plasticity and axonal growth and repair ,229.There is growing evidence from animal and translational AD studies that endothelial dysfunction exerts direct effects on synaptic and neuronal functions that are independent of amyloid or tau . For exaRecent studies highlight several pathways by which endothelial dysfunction influences axonal repair, neuronal integrity, and synaptic plasticity independently of amyloid and tau, including interactions of endothelial proteins with semaphorin-3A involved in memory or learning functions ,22. EndoWhile the mechanisms that underly neurovascular uncoupling in AD are complex, there is ample evidence to suggest that brain endothelial dysfunction plays an important role in this process ,236. NADIn addition to neurons, brain endothelial cells also produce brain-derived neurotrophic factor (BDNF), which plays an important role in neurogenesis and neuroplasticity . One mecProposed mechanisms by which endothelial BDNF promotes neuroplasticity include direct binding to neuronal tyrosine receptor kinase B (TrKB) receptors or sustained activation of endothelial TrKB receptors, which results in increased NO production and induces long-term potentiation, an essential process for memory and learning ,248. TheTogether, these findings support the role of dysfunctional endothelium in mediating neurodegeneration in AD. Further support for this comes from studies that show that the direct co-culture of neurons with microvessels from AD brains, or the exposure of cultured neurons to conditioned mediums from AD microvessels but not microvessels from normal aging brains, is associated with neurotoxicity , and thaFinally, improving brain endothelial health has the potential to support neuronal regeneration and repair. Endothelial progenitor cells (EPCs), which express the surface markers CD34, VEGFR2, and CD133, are bone-marrow derived cells that can differentiate into mature endothelial cells and produce angiogenic growth factors to support endothelial repair . ReducedAlthough the current hypothesis of neurodegeneration in AD is centered around abnormal amyloid and tau aggregation, emerging evidence suggests that endothelial dysfunction is an early and primary event in AD pathogenesis that may precede abnormal protein aggregation and directly contribute to neurodegeneration and synaptic injury. Together, studies examining endothelial contributions to neuronal and synaptic dysfunction in AD suggest it may be a potential therapeutic target in AD. Brain endothelial health has both direct and indirect effects on neuronal and axonal repair mechanisms and synaptic plasticity via endothelial–neuronal interactions as well as diffusible endothelial mediators that regulate neuronal functions. Therapeutic interventions, which improve endothelial health and promote endothelial repair, have the potential to improve neurovascular coupling, reduce vascular inflammation and thrombosis, and decrease amyloid and tau aggregation, ultimately leading to restoring CBF and improving cognition. Specifically, the implementation of EPCs may offer an exciting opportunity for endothelial repair and mitigating the deleterious effects of endothelial dysfunction early in the disease process. Further investigations into the mechanisms by which endothelial repair may improve neuronal and synaptic plasticity, and the development of novel AD therapies targeting endothelial injury in AD, are needed.


IGH::CCND1 rearrangement was 26% in this cohort, but with no apparent differences across races, ages, or disease groups. Almost all cases with abnormal karyotypes presented a complex or composite karyotype, with most featuring five or more chromosomal abnormalities, and chromosome 1 structural abnormalities were the most prevalent (65%). The genomic complexity escalated from MGUS to SMM and further to NDMM and RRMM. Elevated frequencies of high-risk cytogenetics (59%) and the presence of subclones (48%) were particularly notable in RRMM cases.In this study we examined the cytogenetic profile of 1087 patients with plasma cell neoplasms (PCNs) at different stages, including monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), newly diagnosed multiple myeloma (NDMM), and refractory/relapsed multiple myeloma (RRMM). Our study demonstrated that >95% of patients exhibited at least one cytogenetic abnormality detected by FISH tests and/or chromosomal analysis. The frequency of MYC-rearrangement (MYC-R), del(1p), and tetraploidy. Elevated frequencies of high-risk cytogenetics (59%), 1q gain (44%), and del(17p) (23%), as well as the presence of subclones (48%), were particularly notable in RRMM cases. IGH::CCND1 was observed in 26% of the cases, with no apparent variations across races, ages, or disease groups. Concurrent chromosomal analysis with FISH revealed that the incidence of abnormal karyotypes was strongly correlated with the extent of neoplastic plasma cell infiltration, genomic complexity, and the presence of specific abnormalities like del(17p) and MYC-R. Approximately 98% of the cases with abnormal karyotypes were complex, with most featuring five or more abnormalities. Chromosome 1 structural abnormalities were the most prevalent, found in 65% of cases. The frequent presence of subclones and composite karyotypes underscored the genomic heterogeneity and instability in this cohort.The aim of this study was to examine the cytogenetic profiles of plasma cell neoplasms (PCNs) at various disease stages, encompassing 1087 patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), newly diagnosed multiple myeloma (NDMM), and refractory/relapsed multiple myeloma (RRMM). Fluorescence in situ hybridization (FISH) analyses were conducted on highly purified plasma cell samples, revealing that 96% of patients exhibited at least one cytogenetic abnormality. The genomic complexity escalated from MGUS to SMM and further to NDMM and RRMM, largely driven by 1q gain, del(17p),  Almost all patients with multiple myeloma (MM) evolve from a premalignant stage termed monoclonal gammopathy of undetermined significance (MGUS) ,2. MGUS www.msmart.org (lastly accessed on 30 August 2023)) are the most widely applied in clinical practice. The ISS, developed in 2005, ref. [IGH translocations t, t, and t; 1q+; del(17p); ISS stage III; and elevated serum LDH are identified as independent high-risk factors associated with decreased overall survival [Multiple factors contribute to patient prognosis and survival, including host characteristics, tumor burden (stage), cytogenetic abnormalities, and response to therapy. Among several risk stratification systems, the International Staging System (ISS)  and the 05, ref. , is base05, ref. , is deri05, ref. ,13,14,1505, ref. , 1q+ has 1q+; delp; ISS stIGH rearrangement (IGH-R) [IGH-R in t, t, and t are considered to be primary cytogenetic abnormalities and occur at the time of the establishment of MGUS. Other cytogenetic changes, termed secondary cytogenetic abnormalities, arise along the disease course and/or during progression, including 1q+, del(1p), del(17p), and MYC rearrangement (MYC-R) [More than 90% of plasma cell neoplasms (PCNs) harbor at least one chromosomal abnormality detected via fluorescence in situ hybridization (FISH) and/or conventional chromosomal analysis (karyotyping). The cytogenetic abnormalities feature trisomies of odd-numbered chromosomes  and/or  (IGH-R) ,19. Tris (MYC-R) ,19. BothIn this study, we analyzed the cytogenetic profiles of 1087 patients with MGUS, SMM, newly diagnosed MM (NDMM), and refractory/relapsed MM (RRMM) via karyotyping and FISH studies. About 91% of the specimens underwent the enrichment of plasma cells (EPC) prior to FISH analysis, while the remaining 9% of cases all had PC >20% in the bone marrow (BM).Patients with MGUS, SMM, and MM who were diagnosed and managed at our institution from 2 January 2021 through 31 May 2023 formed the study cohort. The diagnosis of MGUS, SMM, and MM were based on the World Health Organization Classification of Tumors of Hematopoietic and Lymphoid Tissues [The degree of plasma cells’ (PCs) involvement was evaluated via: (1) differential count (diff) on BM aspirate smears; (2) immunohistochemistry for CD138 on BM core biopsy and/or clot sections; and (3) flow cytometric immunophenotypic analysis that included markers specific to CD19, CD27, CD38, CD45, CD56, CD81, CD117, CD138, cytoplasmic (cyto) kappa, and lambda immunoglobin light chain as described previously . Aberrang for 10 min and the supernatant was carefully removed. The cell pellet was then resuspended in 2 mL D-PBS. After adding 2 mL red blood cell lysis buffer to the suspension, the specimen tube was placed in the RoboSep machine and the procedure was run accordingly. The positive selected cells (PCs) were collected in 1 mL D-PBS. To estimate the purity of plasma cells in the post-enrichment samples, we prepared cytospin slide by using 100 uL post-enrichment collection. The cytospin slide then underwent Wright-Giemsa stain, and the purity of plasma cells was estimated via morphological examination . Overall, the purity of plasma cells in post-enrichment specimens ranged from 60% to nearly 100%. The left-over (~900 uL) post-enrichment solution was spun down , and the cell pellet was then resuspended in fixation buffer (3:1 methanol: acetic acid) for FISH analysis. Based on our laboratory algorithm, if BM aspirate is insufficient for both karyotyping and EPC/FISH, the latter takes priority unless a diagnosis of a myelodysplastic syndrome is suspected.Enrichment of plasma cells (EPC) was conducted on BM aspirates when PCs were ≤20%. Meanwhile, the aberrant (neoplastic) PCs comprised at least 0.05% of the total analyzed cells, and these aberrant PCs made up 25% or more of the total plasma cell population (estimated via flow cytometry). Enrichment was achieved by using RoboSep  and anti-CD138 magnetic microbeads according to the manufacturer’s instructions. Briefly, 2 mL BM aspirate was mixed with 8 mL D-PBS then filtered through 70 um strainer. The filtered specimen was spun down at 300× CDKN2C)/1q21 (CKS1B), 9p21 (CDKN2A)/CEP9, 13q14 (RB1)/13q34, and 17p13 (TP53)/CEP17; and four for gene rearrangements , including t/IGH::FGFR3 ; t/IGH::CCND1 ; t/IGH::MAF ; and 8q24/MYC (break-apart). IGH (break-apart) FISH testing was reflexed when IGH exhibited three signals but was not fused with CCND1, FGFR3, or MAF. At least 100 cells were counted for each FISH probe set. The cut-off values for common abnormal patterns established in our Clinical Cytogenetics Laboratory are summarized in CKS1B, and 1q amplification (amp) was defined as 5 or more copies of CKS1B (lumped as 1q+ if not otherwise specified in this study). The presence of subclone(s) was defined by the following criteria: (1) co-existence of two or more abnormalities in the same gene/locus, e.g., different copies of CKS1B are detected in one specimen; (2) simultaneous presence of both diploid and aneuploid populations; and (3) the difference in clonal size exceeds 30%.FISH analysis was either performed on cultured cells when PCs were >20% or on EPC following manufacturer’s instructions . Eight dual-color FISH probe sets were included in our myeloma FISH panel as recommended by the International Myeloma Working Group (IMWG) : four prChromosomal analysis was performed on metaphase cells prepared from BM aspirates cultured for 72 h with lipopolysaccharide (LPS) and/or 24 h without mitogens using standard techniques. Twenty Giemsa-banded metaphases were analyzed, and the results were reported using the International System for Human Cytogenetic Nomenclature (ISCN 2020). Cases with 3 or more unrelated chromosomal abnormalities  were considered to have a complex karyotype. Structural abnormalities included deletion (del), duplication (dup), additional material of unknown origin (add), translocation (t), isochromosome, etc. The karyotype was arbitrarily divided into hypodiploidy ; diploidy ; hyperdiploidy ; and aneuploidy (mainly tetraploidy) in this study.This study cohort included 1087 patients: 137 with MGUS, 205 with SMM, 252 with NDMM, and 493 with RRMM. The demographic information  is summarized in All patient specimens were assessed using a MM FISH panel: 986 (91%) on EPC and 101 (9%) on cultured cells. A total of 802 patients had concurrent karyotyping, while 285 other cases had insufficient BM specimen and it was not possible to do karyotyping. The percentage of plasma cells in bone marrow aspirate , the corresponding FISH analysis and karyotyping, and the cases with abnormal results are summarized in Number of cytogenetic abnormalities. Overall, 1043 patients (96%) showed at least one PCN-related cytogenetic abnormality: 93% in MGUS patients, 98% in SMM, and 96% in NDMM and RRMM. Most patients in the MGUS group (65%) showed two or fewer cytogenetic abnormalities, whereas most of the MM patients (68%) showed three or more abnormalities. Furthermore, 34~35% of patients with MM showed five or more abnormalities, versus only 15% in SMM and 7% in MGUS patients. The average number of cytogenetic abnormalities per case was 1.8 for MGUS, 3 for SMM, and 4.2 for MM. The cytogenetic complexity was similar between RRMM and NDMM cases , del(13q)/−13, and tri/tetra11, present in ≥40% of patients and at a similar frequency among MGUS, SMM, NDMM, and RRMM. The other two cytogenetic abnormalities present at a similar frequency with MGUS, SMM, and MM were t and t, present in ~26% and ~5% of patients, respectively. On the other hand, the frequency of 1q+/amp increased from 14% for MGUS to 44% for RRMM. A similar pattern was also observed in del(17p)/−17 (from 1% for MGUS to 23% for RRMM) and del(1p) (from 1% in MGUS to 12% in RRMM). Other cytogenetic abnormalities, including t, MYC-R, and tetraploidy, were rare or absent with MGUS but detected in 8% to 12% of RRMM cases , t, and del(17p), were detected in 10% of MGUS, 19% of SMM, 27% of NDMM, and 33% of RRMM patients. The most common high-risk cytogenetic abnormalities were t in MGUS, t in SMM, and del(17p) in NDMM and RRMM patients , aneusomies of chr9, chr13, and chr17, del(17p), MYC-R, losses of CDKN2C, IGH, FGFR3, MAF, and aneuploidy. Sixty cases with RRMM (12%) showed a spectrum of 1q copies (up to 12 copies) within one specimen.Cytogenetic abnormalities among demographic groups . There was no apparent difference between Caucasian and African American in aspects of disease stages, frequencies of t; t, t, and del(17p), and high-risk cytogenetics. Other races were not compared due to the limited number of patients included in this cohort.Cases with one or two cytogenetic abnormalities. A single cytogenetic abnormality was detected in 158 patients (15%). The most common single abnormality was t , followed by trisomy 11 (n = 15), trisomy 9 (n = 10), and del(13q) (n = 6). Two abnormalities were detected in 210 patients (19%), with the most common being trisomy 11 plus trisomy 9 (n = 45), t plus del(13q) (n = 43), t plus 1q+ (n = 16), and t plus del(13q) (n = 13).Frequency of abnormal karyotype. Karyotyping was performed in 802 patients. Of these, 623 patients showed a normal karyotype (including 18 cases with minus Y only); 164 (21%) showed a PCN-related abnormal karyotype, of which 161 (98.2%) had a complex karyotype, 2 had hyperdiploidy (only chromosome gains), and 1 had 2 abnormalities , diploid (8%), hyperdiploid (61%), and aneuploid (4%). Most of the cases showed five or more abnormalities. The most involved chromosomes were chr1 (67%), chr11 (67%), chr9 (66%), chr15 (58%), chr5 (52%), and chr14 (50%). Structural abnormalities were most frequently detected in chr1 (65%), followed by chr14 (41%) and chr11. Among trisomies, +15 was the most common (46%), followed by +9, +11, and other odd number chromosomes, except for chr17 and chr13 (no cases showed +13). On the other hand, monosomy 13 was the most common aneusomy resulting from loss (30%) (ss (30%) . Marker Correlation of FISH results with karyotype. The yield of an abnormal karyotype was closely associated with the number of cytogenetic abnormalities detected via FISH; the greater number of FISH abnormalities, the higher thr yield of an abnormal karyotype (MYC-R (detected via FISH) were more likely to have an abnormal karyotype: 44% of cases with del(17p) and 43% of cases with MYC-R showed an abnormal karyotype. In contrast, cases with t (detected via FISH) were the least likely to have an abnormal karyotype, occurring only in 13% of cases , t, +9, +11, and del(13q) detected in patients with MGUS, SMM, and MM supports the theory that these cytogenetic abnormalities are primary events, occurring during an early stage of PCN, and are unlikely to play roles in disease progression. We observed a slightly increased frequency of tetrasomy 9 and −13 with SMM and MM, which was likely associated with clonal evolution. Conversely, t was more commonly detected with SMM and MM, indicating that t as a primary event poses a higher risk of progression to SMM and MM ,24. We dCKS1B; in RRMM, ~40% of the cases with 1q+ showed four or more CKS1B copies, including 15% of the cases that showed CKS1B amplification (five or more copies), and 12% of the cases that showed subclones with different CKS1B copies, which is consistent with clonal evolution and disease progression [CKS1B leads to CKS1B overexpression [STAT3 and MEK/ERK pathways [The frequency of 1q+ and the copy number of 1q increased steadily from MGUS to SMM, NDMM, and RRMM. The frequency of 1q+ detected in MM in our cohort was similar to what has been reported by other studies ,26. For gression ,28,29. Pgression ,28, causpression , which hpression . Subsequpathways . Despitepathways ,15,16, 1TP53 deletion with a complex karyotype and genomic instability. We also observed a high frequency of abnormal karyotypes in the presence of del(17p), suggesting that del(17p) may confer proliferative advantages to neoplastic PCs. Del(17p) is consistently recognized as a high-risk factor across all guidelines [Chromosome 17p deletion is another cytogenetic abnormality that was more common in RRDD, from 1% in MUGS to 23% in RRMM. All cases with del(17p) showed three or more abnormalities detected via FISH, supporting the association of del(17p)/idelines .MYC-R was not detected in any case with MGUS but was present in 10% of SMM and 15% of MM cases. Other studies have shown a frequency of MYC-R in MM varying from 8% to 50%, depending on the methods used for detection [MYC-R had immunoglobulin gene partners [MYC detected in MM included: 1p12 (FAM46C), 6p24.3 (TXNDC5), 6q21 , 8q24.13/NSMCE2, 11q13.3 (CCND1), and 7p21.3 (GLCCI1). Most of these partner genes are also super-enhancers [MYC partners included IGH , IGL, IGK, NSMCE2, TXNDC5, and CCND1, but the partner was uncertain for most cases. The breakpoints showed a much wider range around the MYC locus during MYC-R, and the breakpoint clusters were not as distinct in MM as the clusters in lymphomas [MYC-R in MM via FISH [MYC-R is associated with elevated serum levels of beta-2-microglobin and a high disease burden and is an independent adverse prognostic factor in patients with NDMM [etection ,32,33,34partners ,32, whicpartners . The recymphomas , which mvia FISH . MYC-R iAneuploid (mainly near-tetraploidy in our cohort) clones were not detected in MGUS, were rare (~3%) in SMM, and were found in about 10% of MM cases in this cohort. Tetraploidy was reported in 6% to 10% of MM patients in other studies ,36. MostMYC-R, and the complexity of the genome. Almost all cases with an abnormal karyotype showed a complex karyotype, and most showed abnormalities on chr1 , chr11 and chr9 , and +15. A marker chromosome and composite karyotype were detected in more than 60% of the patients, corresponding to the high frequency of subclones detected via FISH. These findings highly suggest clonal heterogeneity and genome instability in PCNs showing an abnormal karyotype.The poor growth of PCs under in vitro culture conditions leads to difficulty in obtaining metaphase cells for karyotyping, especially when the BM involvement by PCs is low. This fact likely explains why the karyotypes of MGUS and SMM have been rarely reported. In our cohort, no case with MGUS and only 11% of cases with SMM showed an abnormal karyotype, with chromosomal gains as the major abnormalities. The likelihood of producing metaphase cells from PCN was highly associated with high-level BM involvement, the presence of del(17p) and MYC-R, and high-copy number of 1q which enable proliferative advantages in neoplastic plasma cells. In the cases where a PCN-related karyotype was produced, the karyotype was often highly complex, with structural abnormalities of chr1 being the most common. To enhance our understanding of the whole genomic profile of PCNs, future studies could incorporate a DNA-based assay that doesn’t require metaphase cells but covers the entire genome, such as optical genome mapping.In summary, our FISH panel identified cytogenetic abnormalities in 96% of PCNs. We observed low genomic complexity in MGUS patients, with an increasing degree of complexity for SMM and MM, and the later-acquired del(17p),


Proteome analyses in patients with newly diagnosed, untreated giant cell arteritis (GCA) have not been reported previously, nor are changes of protein expression upon treatment with glucocorticoids (GC) and/or tocilizumab (TCZ) known. The GUSTO trial allows to address these questions, provides the opportunity to learn about the differential effects of GC and TCZ on proteomics and may help to identify serum proteins to monitor disease activity.Serum samples obtained from 16 patients with new-onset GCA at different time points  during the GUSTO trial (NCT03745586) were examined for 1436 differentially expressed proteins (DEPs) based on proximity extension assay technology. The patients received 500 mg methylprednisolone intravenously for 3 consecutive days followed by TCZ monotherapy.When comparing day 0 (before the first GC infusion) with week 52 (lasting remission), 434 DEPs  were identified. In response to treatment, the majority of changes occurred within 10 days. GC inversely regulated 25 proteins compared to remission. No difference was observed between weeks 24 and 52 during established remission and ongoing TCZ treatment. Expression of CCL7, MMP12, and CXCL9 was not regulated by IL6.Disease-regulated serum proteins improved within 10 days and were normalized within 24 weeks, showing a kinetic corresponding to the gradual achievement of clinical remission. The proteins inversely regulated by GC and TCZ shed light on the differential effects of the two drugs. CCL7, CXCL9, and MMP12 are biomarkers that reflect disease activity despite normalized C-reactive protein levels. Giant cell arteritis (GCA) is the most common systemic vasculitis in elderly adults. It is characterized by invading of immune cells through the adventitial vasa vasorum network in the immune-privileged three-layered vessel wall . Upon acThe current definition of disease activity in GCA is based on symptoms, findings in imaging, as well as the inflammatory markers C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) . TocilizUntil now, most studies on serum proteins in GCA were hypothesis-driven and used pre-specified panels of proteins, e.g. cellular markers, cytokines and effector molecules. Several proteins, like ANGPT2, CD163, CHI3L1, CXCL13, CXCL9, CXCL10, IL1B, IL18, IL2RA, IL6, IL6R, MMP2, MMP3, PTX3, S100A9, SPP1, TEK, TIMP1, TNC, TNFRSF1A, and VEGFA, were described as potential markers of active GCA. Furthermore, most data were generated using sera of patients pre-treated with glucocorticoids (GC) –12. ThisA multiplex analysis bears the advantage of identifying previously unrecognized molecules, while offering the opportunity to confirm or reject hypotheses.The goals of our study were (i) to describe the profile of serum proteins in active, newly diagnosed naïve GCA patients compared to patients in lasting remission at week 52, (ii) to investigate and compare the effects of pulsed GC treatment and ensuing long-term TCZ monotherapy on changes in serum protein expression pattern, and (iii) to identify serum proteins which are not under the control of IL6 and, thus, may qualify as biomarkers of disease activity in patients with GCA, especially in patients treated with TCZ.Patients with newly diagnosed GCA were enrolled in the GCA treatment with ultra-short GCs and TCZ (GUSTO) trial and received 500 mg/d methylprednisolone intravenously for 3 consecutive days (NCT03745586) . ThereafA total of 18 patients were included in the GUSTO study and 13/18 patients completed the 52-week treatment course. 16/18 patients contributed samples for this study. In 13/16 patients, the first sample was obtained prior to any GC treatment, 3/16 patients had a median of 3 days  of prior oral GC treatment (daily dose: 20-60 mg prednisolone). Samples from all 13 patients completing the study were analyzed (6 time points/patient). Additionally, two samples  from one patient who discontinued the study due to diverticulitis after achieving remission, and six samples  from two patients who discontinued the study after day 10 due to persistent cranial symptoms were included. In total, 86 samples were assessed. Baseline characteristics are displayed in the Serum samples were analyzed on Olink Explore 1536 , a proximity extension assays (PEA) multiplex immunoassay platform capturing 1463 proteins  were used for analysis. Principal component analysis (PCA) and sample-wise NPX-distribution was evaluated for outliers. Samples identified as outliers were excluded from downstream analysis along with those with quality control (QC)-warning status.Protein-wise t-tests compared NPX-values between the two groups  and results were adjusted for multiple testing (Benjamini-Hochberg) to identify differentially expressed proteins (DEPs). In the first step, we investigated whether there was a difference in day 0 samples of those patients with as compared to without prior GC treatment. In case of a difference due to previous GC treatment, we intended to analyze both groups separately. In a second step, we compared samples between day 0 versus day 3 (referred to as GC treatment), week 4 versus week 52 (referred to as TCZ treatment), day 0 versus week 52 (referred to as remission), day 3 versus week 4 (weaning effect of GC and evolving effect of TCZ treatment), and week 4 (comparison based on remission status).post-hoc analysis (Tukey’s test).Protein-wise ANOVA compared NPX-values between groups  and results were adjusted for multiple testing using the Benjamini-Hochberg method. Significant differences were determined by Analyses were performed on Olink Insights Stat Analysis. Venn diagrams were created using the online tool InteractiVenn . CorrespCompared to week 52 (stable remission for more than six months), 213 proteins were up-regulated, and 221 proteins were down-regulated at day 0 (active disease prior to administration of GC pulse therapy).The ten most significant DEPs included: IL6R, PRTG, ITGAV , LBP, ITIH3, PLA2G2A, LRIG1, TNFSF14, TNC, and FURIN  were compared to day 0 samples from patients without prior GC treatment (n=13). Only one protein, namely CLPP (down-regulated upon GC treatment), exhibited differential expression between the two groups  as compared to patients without prior oral GC treatment revealed 36 DEPs. The ten most significant included MMP12, TNC, ANGPTL1, CHRDL1, GALNT3, CLPP, IL20RA, PREB (down-regulated upon GC treatment), and MMP3, CTSF (up-regulated upon GC treatment).In response to GC-pulse therapy for 3 days, the NPX-value of 208 proteins changed. In total, 94 proteins were up- and 114 down-regulated. The ten most significant DEPs included ANGPTL7, AREG, IL1RL1, MATN3, MMP3, SPARCL1 (up-regulated) and IL12A, IL12B, KLK10, SIGLEC6 (down-regulated) Figure 2Twenty-five proteins were down- and 24 up-regulated with GC treatment and upon remission. On the other hand, we identified 19 DEPs that were down-regulated at day 3 and up-regulated at week 52 as compared to day 0, and 6 proteins  that were up-regulated at day 3 and down-regulated at week 52 compared to day 0.Upon GC-pulse therapy, many proteins showed impressive changes, which  returned to pre-treatment levels, allowing an estimation of the kinetics and lasting effect of GC-pulse therapy. Further, we investigated the kinetics after day 3 of the 159/208 DEPs related to GC treatment that did not mirror disease activity to estimate the duration of the GC effect on serum proteins. We assessed the time to return to the day 0 levels for those proteins. 70/159 were up-regulated, and 89/159 were down-regulated upon day 3. After removal of those proteins  that displayed an opposite regulation upon GC treatment and remission, 64/70 proteins were up-, and 70/89 down-regulated on day 3. Most of those proteins returned to the day 0 level at day 10 (46/64 and 64/70). However, some proteins changed later, namely at week 4 (11/64 and 1/70), week 24 (4/64 and 4/70), and even at week 52 (3/64 and 1/70).The interval between day 3 and week 4 reflects the weaning GC effect and the evolving TCZ effect. We identified a total of 497 DEPs between day 3 and week 4. The ten most significant DEPs included IL6R, CDON, IL12A, IL12B, CD83, TNFRSF4, ADAM23, IL6, FLT3LG, and SIGLEC6 . IL6R, CDON, CD83, ADAM23, IL6, and FLT3LG displayed the same trend upon remission (higher level at week 52 as compared to day 0). IL12A, IL12B, TNFRSF4, and SIGLEC6 displayed no significant changes upon remission (week 52 as compared to day 0). In total, 303/497 proteins were up- and 194/497 down-regulated.Based on the results above the changes measured between week 4 and week 52 are to an increasing extent induced by TCZ. Eleven DEPs  were identified. All eleven DEPs were down-regulated at week 52 as compared to week 4. Five/11 DEPs displayed the same trend upon remission . TNC, however, was up-regulated upon GC treatment (day 0 versus day 3), and CLEC5A displayed a slow kinetic – yet mirroring disease activity – with no changes between day 0 versus day 3, day 10, and week 4 but with down-regulation on day 0 versus week 24 and 52, respectively. On the other hand, 2/11 DEPs  displayed the opposite trend upon TCZ treatment (down-regulated) and remission (up-regulated).Results of concomitant ultrasound analysis have been reported previously . There wAt week 4 there was only one protein, IL4R, that was different between patients in remission (n=4) and patients not in remission (n=10). Patients in remission displayed lower levels of IL4R as compared to patients not in remission.Boxplots of selected proteins of interest  are displayed in Compared to patients in remission, newly diagnosed, active GCA patients showed 434/1463 differentially regulated proteins. 208/1463 proteins were regulated by pulsed GC treatment and 11/1463 by subsequent TCZ monotherapy. To our knowledge, this is the first study to report an in-depth analysis of serum proteins in new-onset GCA. The study design allowed for the comparative analysis of the effects of GC and TCZ treatment on the protein serum signature in GCA.versus off GC treatment): 3550 genes were differentially expressed in GC-treated patients . The high number reflects the combined genomic and non-genomic effects . In addipatients . RemarkaRemission had a broader impact on serum protein expression in patients with GCA compared to pulse GC treatment and changed every third protein . This profound impact of disease activity on serum proteomics in GCA is a novel finding. Longitudinally monitoring of protein expression revealed that these changes occurred early on. They were already seen within 10 days after initiation of treatment and concluded by week 24. Data published in 2018 on rheumatoid arthritis demonstrated a 24-week treatment with TCZ to result in broader proteomic changes toward healthy controls compared to infliximab or methotrexate. Of note, most of these changes occurred within 4 weeks . This isBased on the kinetics of GC-induced changes, any regulation of protein expression after week 4 is likely related to TCZ treatment. Between weeks 4 and 52, 11 DEPs were identified, and 5/11  proteins displayed the same trend upon TCZ treatment and during remission. Of these, TNC, associated with active AAV, was found to be up-regulated upon GC treatment and, therefore, might not be a reliable marker in conventional co-medication of GC plus TCZ . The serDue to their potential deleterious role in the destruction of the arterial vessel wall and formation of aneurysm, MMPs merit a special attention. In line with a previous study on RNA microarray analysis in temporal artery biopsies of GCA patients, we identified MMP12 and MMP9, which enable the trafficking of inflammatory cells to the vessel wall, as markers of disease activity in GCA . Of the As expected, there was an increase of the soluble IL6R related to TCZ treatment over time, achieving stable values after week 4. IL6, biologically not active under TCZ treatment, displayed peak levels at day 10 and a decline from day 10 to week 24, with stable values between week 24 and 52. This decline was not expected as IL6 levels did not decrease during TCZ treatment in the first RCT about TCZ to treat GCA . In a re+ macrophage subset produces CHI3L1, which may facilitate tissue destruction and angiogenesis . The patients/participants provided their written informed consent to participate in this study.LC and PV initiated the study. AG, FB, FK, LC, PV, SR, and TG were involved in the study conception, design and implementation and data interpretation. AG, LC and PV drafted the manuscript. It was finalized by AG, FB, FK, LC, PV, SR, and TG. AG, LC, and PV had full access to all the data in the study, and accessed and verified the data. All authors contributed to the article and approved the submitted version.


GATA-4 gene, located on chromosome 8p23.1, encodes GATA-binding protein 4 (GATA-4), a transcription factor that is essential for cardiac and gonadal development and sexual differentiation. Case Description: A child with a history of micropenis and cryptorchidism. At 8 years of age, he came under our observation for an increase in sexual pubic hair (pubarche). The laboratory parameters and the GnRH test suggested a central precocious puberty (CPP). Treatment with GnRH analogs was started, and we decided to perform genetic tests for DSD. The NGS genetic investigation showed a novel and heterozygous variant in the GATA-4 gene. Discussion: In the literature, 26 cases with 46,XY DSD due to the GATA4 gene were reported. Conclusion: The novel variant in the GATA-4 gene of our patient was not previously associated with DSD. This is the first case of a DSD due to a GATA-4 mutation that develops precocious puberty. Precocious puberty could be associated with DSD and considered a prelude to hypogonadism in some cases.Background: Disorders/Differences of sex development (DSD) are often due to disruptions of the genetic programs that regulate gonad development. The  Disorders/Differences of sex development (DSD) represent a heterogenous group of congenital conditions with abnormal development of internal and/or external genitalia . AnormalDSDs are estimated to affect 1:4500 newborns and include different abnormalities of urogenital differentiation divided into several categories, according to the 2006 Chicago Consensus classification: chromosomal DSD, 46,XX DSD, and 46,XY DSD .DSD related to sex chromosomes are the most frequent class of DSD, and they include syndromic conditions such as Turner Syndrome (TS), Klinefelter Syndrome (KS) and mixed gonadal dysgenesis. KS is characterized by the 47,XXY karyotype and its incidence is about 1 in 500 males . During The 46,XX DSDs are caused by an excess of androgens that lead to precocious virilization. This group presents ovotesticular and testicular cases. Classical congenital adrenal hyperplasia (CAH) is the most common disease of 46,XX DSD, and it is due to a significantly reduced activity of 21-hydroxylase that determines two subtypes: the simple virilizing and the salt-wasting forms. Clinical manifestations of this condition are already present at birth, with ambiguous genitalia and electrolyte alterations appearing after a few weeks when there is no residual enzyme activity  ,4. 46,XXGATA4 haploinsufficiency, a rare cause of 46,XY DSD, could cause a wide spectrum of genital abnormalities, including micropenis, cryptorchidism, and hypospadias, as reported in the 26 patients with DSD due to the GATA4 mutations that are included in previous studies [Complete Androgen Insensitivity Syndrome (CAIS) and Persistent Mullerian ducts syndrome (PMDS) belong to the 46,XY DSD family. The incidence of 46,XY DSD can vary considerably, from rare disorders, such as complete gonadal dysgenesis affecting 1/20,000 births, to common conditions, such as hypospadias affecting 1/250 male births . The com studies ,17,18,19GATA4 gene, located on chromosome 8p23.1, is essential for cardiac and gonadal development. GATA-binding protein 4 (GATA4) regulates the expression of multiple genes’ coding for hormones or components of the steroidogenic pathway during testis development and function. GATA4 is expressed in the gonads together with GATA1 and GATA6, but only GATA4 is involved in early gonad development [SRY), SRY-box transcription factor 9 (SOX-9), and anti-Mullerian hormone (AMH) genes, GATA4 initiates testis differentiation [GATA4 appears as an amplifier of AMH transcription. In Leydig cells, GATA4 modulates a couple of steroidogenic genes. A blockade of GATA4 expression in mice Leydig cell lines leads to the suppression of the steroidogenic program and a decrease in hormone production [GATA-binding proteins (GATA), a group of six transcriptional regulator factors, interact with specific nucleotide sequences thanks to zinc finger DNA-binding domains. The elopment . Regulatntiation . In Sertoduction .Within the pituitary gland, GATA transcription factors have been shown to be critical for pituitary cell differentiation and function: GATA2 regulates gonadotropins gene expression and GATA2-GATA4 stimulates basal and GnRH-induced ADCYAP1, a polypeptide able to stimulate LH and FSH secretion by primary pituitary cells and the gonadotrope cell line ,24.GATA4 has an important role in the development of congenital heart disease (CHD), DSD and CHD can be isolated or combined in patients with GATA4 mutations [GATA4 mutations have been identified in more than 140 patients with ventricular septal defect, tetralogy of Fallot, or other cardiac malformations [Even if utations . Heterozrmations .GATA4 mutation without CHD. In addition, we aimed to review the characteristics of DSD cases related to GATA-4 alterations to understand if additional clinical findings could link the GATA-4 pathway with precocious puberty.The aim of this study was to present the peculiar case of an 8-year-old child with 46,XY DSD, central precocious puberty (CPP), and a novel ERBB4 gene, previously never described in association with DSD and precocious puberty. A Next Generation Sequencing (NGS) panel of genes related to gonadal dysgenesis and precocious puberty was adopted. The gene panel included AMH, AMHR2, AR, CBX2, CYPIIAI, CYP11AI, DHH, DMRTI, GATA4, HSDI7B3, MAP3KI, NROBI, NR5A1, SOX9, SRD5A2, SRY, WNT4, WTI, WWOX, KISS1, KISSIR, and MKRN3. The NGS analysis identified a heterozygous and missense variant c.671CG (p.Ser224Cys) in the GATA-4 gene.The child is a second-born of healthy non-consanguineous parents, born at 40 gestational weeks by cesarean section after a normal pregnancy. At birth, he had mild respiratory distress: his Apgar scores were 6 and 8 at 1′ and 5′, respectively. His birth weight and length were 3900 gr and 54 cm, respectively. The family history was negative for endocrinological pathologies. Genitalia examination at birth showed a micropenis (length 1.5 cm) and scrota without gonads palpable. Due to the presence of micropenis (1.5 × 1.0 cm), in the second month of life, a treatment of intramuscular testosterone at a dose of 25 mg/month was administered for 3 months. When testosterone therapy was started, the patient had a length of 58.3 cm (+2.1 SDS) and bilateral undescended testis. Then, the patient was lost at follow-up, and when he was 2 years old, in a different hospital, orchidopexy was performed for bilateral cryptorchidism. After 6 years, when he was 8 years old, the patient was referred again to our endocrinology clinic for a precocious pubarche. Height was 147.1 cm (+2.95 SDS), weight was 42.0 kg (+2.21 SDS), and bone age was significantly advanced . Physical examination showed hypertelorism, a singular 5 cm wide cafe-au-lait spot in the abdomen, curly pubic hair, testes palpable in the scrota with a volume of 3 mL, and a penis length of 4 cm. For suspected precocious puberty, appropriate investigations were performed during hospitalization, such as the GnRH test, and are reported in Segregation analysis in the parents is still being carried out. Furthermore, genetic investigations have ruled out other conditions such as McCune–Albright syndrome.AR and NR5A1 genes. A 3-year-old patient assigned at birth as a female, with typical female genitalia and dwarfism, was the only 46,XY DSD case with the GATA4 mutation (p.G12R) [AR and NR5A1 genes were seen more frequently in 46,XY DSD patients and only 1–2% of cases were related to the GATA4 gene [GATA4 haploinsufficiency is therefore a rare cause of DSD in genetic males.The genetic causes of 46,XY DSD are determined in almost half of the cases, although the latest genetic techniques  can identify new genes and novel mutations . In a la(p.G12R) . In anotTA4 gene ,13. GATAGATA4 gene have been reported until now (GATA4 variants were p.P407Q (31%) and p.G221R (12%). The P.407Q variant was found in a female 46,XY with complete female genitalia, in four males with cryptorchidism and micropenis, and in three males with hypospadias and cryptorchidism. In experimental studies [AMH and SRY genes. Instead, two patients presented the same variant, p.W228C, and similar clinical phenotype characterized micropenis and cryptorchidism [GATA4 interaction partners. In fact, p.P226L and p.T228C variants act on the N-terminal region without changing the overall structure of GATA4 [In the literature, 26 cases with 46,XY DSD due to the ntil now  [9,10,11 studies ,15 this rchidism . Accordi p.G221R %. The P.of GATA4 .GATA4 activity is mostly deficient in patients with both cardiac defects and DSD than that observed in patients with only CHD [GATA4 in five different patients with CHD of which only one also presented a DSD. In this way, they assumed that normal development of the heart required stricter regulation of GATA4 transcription than gonadal development [CHD was found in five patients, and three cases of them had atrial septal defects. Yui Shichiri et al. suggested that only CHD . This coelopment .GATA4 gene are responsible for 46,XY DSD but the phenotypes, ranging from a mild under-virilization to complete female external genitalia, are different in patients with the same mutation, and it could be linked to a variable expressivity and penetrance of the GATA4 gene [GATA gene. In fact, clinical presentations of our case were micropenis ad cryptorchidism without a family history of DSD. NGS investigation of our patient, for DSD panel genes, showed heterozygous and missense variant, c.671CG, in the GATA-4 gene. This variant was classified as likely pathogenic according to The American College of Medical Genetics and Genomics (ACMG), and it causes punctiform mutation, p.Ser224Cys, in the specific aminoacidic sequence. The C.671CG variant was not previously associated with DSD, described in scientific literature, nor noted in reference databases. A particular additional finding in our case was the precocious pubarche that led to a diagnosis of precocious puberty. Other peculiar elements were the elevated basal levels of testosterone together with a significant elevation of LH and FSH, which could suggest a hypergonadotropic hypogonadism. None of the 26 cases with 46XY DSD due to the GATA4 gene already reported presented precocious puberty, while some cases have been documented as physiological pubertal development. Wagner-Mahler K et al. described a follow-up of over almost 15 years of a child born with perineal hypospadias, micropenis, cryptorchidism, and 8p23 deletion [GATA4 missense mutation (p.G221R) in three 46,XY DSD patients from one family [GATA4 mutation (p.G221R) was due to a loss-of-function effect on GATA4 in the developing gonad, interfering with its ability to interact with FOG2, and disturbing activation of the AMH promoter together with NR5A1 [Different variants of the TA4 gene . Analyzideletion . Subseque family . The indth NR5A1 . This coth NR5A1 , to undeLHCGR mutation (2p16.3) had presented precocious puberty with 46,XY DSD, altered gonadal differentiation, and Leydig cell hypoplasia. Lele Li et al. [MAMLD1 variant. Hypospadias was the most prevalent phenotype among 10 cases, but it was not present in a male patient with normal external genitalia at birth. The latter case developed premature secondary sexual characteristics  at the age of 1 year and in addition, he showed elevated LH, FSH, and T-levels which indicated precocious puberty. His testosterone level later decreased, and so he displayed hypergonadotropic hypogonadism. The patient’s genotype was a missense variant of MAMLD1, p.A421P, classified as VUS according to ACMG criteria [GATA4, the MAMLD1 gene plays a molecular role in the pituitary gland level and its mutations could be a rare cause of DSD.We analyzed the literature to find cases of precocious puberty in patients with 46,XY DSD caused by monogenic alterations. This research allows us to identify two cases. Eggers et al.  analyzedi et al.  evaluatecriteria . Like GAMoreover, CPP could be a clinical element in patients with DSD. In fact, some patients with chromosomal DSD also presented precocious puberty. Chunxiu Gong et al.  reportedGATA4 mutation is a rare cause of 46,XY DSD with variable expressivity and genetic penetrance that can lead to different phenotypes. We encountered precocious puberty as a new clinical element in a patient with 46,XY DSD and testicular dysgenesis, probably secondary to a novel GATA4 mutation. The combination of CPP and DSD manifested in our patient with a pathologic baseline increase in FSH and LH, secondary testosterone, and the clinical signs of virilization, accelerated growth, and bone maturation. Precocious puberty could be considered an additional finding in the clinical phenotype of DSD patients, although pathophysiological processes have yet to be understood. To the best of our knowledge, the novel missense variant of our case, c.671CG, was not already described, and this is the first patient in which precocious puberty is associated with a DSD due to a GATA4 mutation.The


The identification of proteins in the cellular membrane of the tumoral cell is a key to the design and guidance of therapeutic agents. Recently, a bi-specific antibody termed amivantamab, targeting epidermal growth factor receptor (EGFR) and mesenchymal–epithelial transition factor (MET), which are oncogenic membrane proteins, received regulatory approval for the treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC). In this study, the authors explored tumor types with high levels of expression of EGFR and MET and focused on prostate adenocarcinoma and pancreatic adenocarcinoma, where anti-PD(L)1 agents alone have not shown relevant signs of activity. In addition, the authors confirmed that high expression of either receptor is associated with poor response to anti-PD(L)1 therapy, independently of the expression of PD-L1, suggesting that blocking these receptors with bi-specific EGFR/MET antibodies could augment the efficacy of anti-PD(L)1 inhibitors.Background: The identification of proteins in the cellular membrane of the tumoral cell is a key to the design of therapeutic agents. Recently, the bi-specific antibody amivantamab, targeting the oncogenic membrane proteins EGFR and MET, received regulatory approval for the treatment of adult patients with locally advanced or metastatic NSCLC. Methods: The authors interrogated several publicly available genomic datasets to evaluate the expression of both receptors and PD-L1 in most of the solid and hematologic malignancies and focused on prostate adenocarcinoma (PRAD) and pancreatic adenocarcinoma (PAAD). Results: In PAAD, EGFR highly correlated with PD-L1 and MET, and MET showed a moderate correlation with PD-L1, while in PRAD, EGFR, MET and PD-L1 showed a strong correlation. In addition, in tumors treated with immune checkpoint inhibitors, including anti-PD(L)1 and anti-CTLA4, a high expression of EGFR and MET predicted detrimental survival. When exploring the relationship of immune populations with these receptors, the authors observed that in PAAD and PRAD, EGFR moderately correlated with CD8+ T cells. Furthermore, EGFR and MET correlated with neutrophils in PRAD. Conclusions: The authors identified tumor types where EGFR and MET were highly expressed and correlated with a high expression of PD-L1, opening the door for the future combination of bi-specific EGFR/MET antibodies with anti-PD(L)1 inhibitors. The identification of genomic alterations is a main task in cancer research . ProteinMore recently, novel antibody formats have been developed with the aim to improve clinical efficiency. Bi-specific antibodies were designed to bind two proteins through the Fab fraction, therefore, acting on both targets at the same time ,13. ThisIn solid tumors, only one antibody, amivantamab, that targets EGFR and MET, has received regulatory approval for the treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) with EGFR exon 20 insertion mutations ,15. OtheA further step to boost the efficacy of this family of agents is to exploit combinations of these compounds with antibodies that act on the immune response . Immune Here, the authors interrogated genomic data to identify indications where both EGFR and MET receptors were up-regulated and, therefore, could be susceptible to be targetable with antibodies. In addition, the authors explored indications where these antibodies could be combined with immunotherapies, through the evaluation of the presence of immunomodulatory molecules or PD-L1 positivity within the selected tumors.http://gepia2.cancer-pku.cn/#index; last accessed on 6 February 2023) [https://www.cbioportal.org/; last accessed on 20 February 2023) [RNA-sequencing expression data to evaluate the transcriptomic levels of EGFR and MET in tumor and normal tissue were obtained from the GTEx (Genotype-Tissue Expression) and TCGA (The Cancer Genome Atlas) databases , using try 2023) ,22 (httpry 2023) ), CHOL (ry 2023) ), PAAD (ry 2023) ), SARC (ry 2023) ) and LUAry 2023) ).https://timer.cistrome.org/; last accessed on 8 March 2023) [p-values were obtained from Spearman’s correlation test.Tumor Immune Estimation Resource (TIMER 2.0) web server  (https:/ch 2023)  was emplch 2023) ,32. The https://kmplot.com/analysis/; last accessed on 22 March 2023) [p-value (p), the hazard ratio (HR) and the false-discovery rate (FDR). Genes with an HR < 1 and p < 0.05 predict a favorable clinical outcome.The Kaplan–Meier Plotter Online Tool  (https:/ch 2023)  was utilThe authors first evaluated the expression of EGFR and MET across a wide number of solid tumors using publicly available human genomic datasets, as described in the Materials and Methods section. EGFR was highly expressed with ≥32 transcripts per million (TPM) in glioblastoma (GBM), low-grade glioma (LGG), kidney renal clear cell carcinoma (KIRC), esophageal carcinoma (ESCA) and head and neck squamous cell carcinoma (HNSC) A. MET waIndications with the highest fold change between tumor and non-transformed tissue for EGFR included thymoma (THYM) (>600-fold change), GBM (>20-fold change) and LGG (≥5-fold change) C,E, follThe authors next explored the correlation between the expression of EGFR and MET individually with the expression of PD-L1 to identify indications where combinations of both therapeutic strategies could be of interest. As can be seen in p = 7.4 × 10−11; FDR = 1% and MET: HR = 1.42 CI = 1.2–1.69, p = 4.5 × 10−5; FDR = 2%). When selecting patients treated only with anti-PD1, the authors also observed a detrimental outcome: HR = 1.76 CI = 1.33–2.32, p = 5.9 × 10−5; FDR = 1% and HR = 1.36 CI = 1.02–1.82, p = 0.035; FDR 50% for EGFR and MET, respectively , BRCA-LumA , PAAD , BRCA , THCA , HNSC-HPV+ , KIRP ; and with less strength PRAD , HNSC , HNSC-HPV- , READ , BLCA , KICH , MESO , KIRC  and BRCA-basal   A.p = 1.79 × 10−19) and a moderate correlation between MET and PD-L1  (p = 2.31 × 10−26). As in p = 1.47 × 10−54), as well as between MET and PD-L1  and EGFR and MET . We also noted that EGFR, MET and PD-L1 correlated among them in THCA and THYM, as shown in Taking into consideration the data described before, the authors observed some potential opportunities for clinical development in indications where anti-PD(L)1 agents alone have not shown relevant signs of activity. In this context, we selected the tumor types that also showed a moderate or high correlation between EGFR, MET and PD-L1. Among them, PAAD displayed a high correlation of EGFR and PD-L1  B. In addp = 4.57 × 10−8), neutrophils  and myeloid dendritic cells (DCs)  (p = 8.19 × 10−23)  and myeloid dendritic cells  (p = 6.42 × 10−21)  A,B. No a × 10−9) A,B. The × 10−23) A. In addctively) B. In thi× 10−21) A. No cor× 10−21) A,B.p = 3.21 × 10−23) between MET and HAVCR2 in PRAD  and OV for MET (more than 5%).When exploring the contribution to the anti-PD (L) 1 response of EGFR and MET receptors, the authors confirmed a clear detrimental effect. A high expression of EGFR and MET in a dataset of patients treated with ICIs was associated with poor survival. These data verified the authors’ previous observations describing the lack of activity of anti-PD (L) 1 antibodies in lung cancer patients with activation of EGFR due to kinase mutations ,36,37,38A clear example that was not evaluated previously includes PAAD, where EGFR and MET correlated with each other and with PD-L1. This tumor also had a mild correlation between EGFR and CD8+ T cells and dendritic cells. Likewise, in PRAD, EGFR and MET correlated with the presence of PD-L1, and in addition to that, EGFR had a mild correlation with CD8+ T cells, neutrophils and DCs, while MET correlated strongly with neutrophils.In these two indications, PAAD and PRAD, the activity of ICI was limited. For instance, in PRAD, anti-PD1 antibodies have shown a lack of efficacy in monotherapy or in combination with hormonotherapy, suggesting the necessity for the evaluation in combination with other immune-modulating agents ,45. In aA combination of antibodies with anti-PD-(L)1 inhibitors can potentiate the activity of each agent alone through the activation of ADCC and CDC immunologic mechanisms . AntibodThe authors’ study has limitations. First, this is a bioinformatic analysis using publicly available genomic datasets. Therefore, the authors may expect that in the near future, the findings described here could be confirmed with immunohistochemistry studies using human samples. Secondly, the combinations suggested here should also be evaluated preclinically. In addition, we acknowledge that genetic variations, including single-nucleotide polymorphisms (SNPs) or EGFRex20 insertions, which can lead to differences in ethnicities or therapeutic efficacies, could not be explored due to the lack of available datasets.In summary, the authors have described tumor types, where the presence of EGFR/MET is associated with detrimental response to anti-PD(L)1 therapies and with an immune-suppressive environment. Therefore, the authors suggest that co-targeting EGFR/MET and PD(L)1 with antibodies could enhance treatment efficacy, particularly in those tumors with high correlation among receptors, like PAAD and PRAD.