Quantitative interpretation of bone marrow biopsies in MPN-What's the point in a molecular age?

The diagnosis of myeloproliferative neoplasms (MPN) requires the integration of clinical, morphological, genetic and immunophenotypic findings. Recently, there has been a transformation in our understanding of the cellular and molecular mechanisms underlying disease initiation and progression in MPN. This has been accompanied by the widespread application of high-resolution quantitative molecular techniques. By contrast, microscopic interpretation of bone marrow biopsies by haematologists/haematopathologists remains subjective and qualitative. However, advances in tissue image analysis and artificial intelligence (AI) promise to transform haematopathology. Pioneering studies in bone marrow image analysis offer to refine our understanding of the boundaries between reactive samples and MPN subtypes and better capture the morphological correlates of high-risk disease. They also demonstrate potential to improve the evaluation of current and novel therapeutics for MPN and other blood cancers. With increased therapeutic targeting of diverse molecular, cellular and extra-cellular components of the marrow, these approaches can address the unmet need for improved objective and quantitative measures of disease modification in the context of clinical trials. This review focuses on the state-of-the-art in image analysis/AI of bone marrow tissue, with an emphasis on its potential to complement and inform future clinical studies and research in MPN.

Oxidation of Protein Kinase A Regulatory Subunit PKARIα Protects Against Myocardial Ischemia-Reperfusion Injury by Inhibiting Lysosomal-Triggered Calcium Release.

BACKGROUND: Kinase oxidation is a critical signaling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, PKARIα (type-1 protein kinase A) can be reversibly oxidized, forming interprotein disulfide bonds in the holoenzyme complex. However, the effect of PKARIα disulfide formation on downstream signaling in the heart, particularly under states of oxidative stress such as ischemia and reperfusion (I/R), remains unexplored. METHODS: Atrial tissue obtained from patients before and after cardiopulmonary bypass and reperfusion and left ventricular (LV) tissue from mice subjected to I/R or sham surgery were used to assess PKARIα disulfide formation by immunoblot. To determine the effect of disulfide formation on PKARIα catalytic activity and subcellular localization, live-cell fluorescence imaging and stimulated emission depletion super-resolution microscopy were performed in prkar1 knock-out mouse embryonic fibroblasts, neonatal myocytes, or adult LV myocytes isolated from "redox dead" (Cys17Ser) PKARIα knock-in mice and their wild-type littermates. Comparison of intracellular calcium dynamics between genotypes was assessed in fura2-loaded LV myocytes, whereas I/R-injury was assessed ex vivo. RESULTS: In both humans and mice, myocardial PKARIα disulfide formation was found to be significantly increased (2-fold in humans, P=0.023; 2.4-fold in mice, P<0.001) in response to I/R in vivo. In mouse LV cardiomyocytes, disulfide-containing PKARIα was not found to impact catalytic activity, but instead led to enhanced AKAP (A-kinase anchoring protein) binding with preferential localization of the holoenzyme to the lysosome. Redox-dependent regulation of lysosomal two-pore channels by PKARIα was sufficient to prevent global calcium release from the sarcoplasmic reticulum in LV myocytes, without affecting intrinsic ryanodine receptor leak or phosphorylation. Absence of I/R-induced PKARIα disulfide formation in "redox dead" knock-in mouse hearts resulted in larger infarcts (2-fold, P<0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, P<0.001), which was prevented by administering the lysosomal two-pore channel inhibitor Ned-19 at the time of reperfusion. CONCLUSIONS: Disulfide modification targets PKARIα to the lysosome, where it acts as a gatekeeper for two-pore channel-mediated triggering of global calcium release. In the postischemic heart, this regulatory mechanism is critical for protection from extensive injury and offers a novel target for the design of cardioprotective therapeutics.

Treatment patterns and outcomes of unfit and elderly patients with Mantle cell lymphoma unfit for standard immunochemotherapy: A UK and Ireland analysis.

Mantle cell lymphoma (MCL) presenting in elderly, unfit patients represents a clinical challenge. Front-line 'attenuated' or low-intensity immunochemotherapy is often employed, although outcomes are relatively unexplored. We report outcomes of attenuated immunochemotherapy in 95 patients with MCL across 19 centres in the UK and Ireland considered unfit for full-dose rituximab-bendamustine or rituximab-cyclophosphamide, doxorubicin, vincristine, prednisolone (R-CHOP). Regimens examined were rituximab-cyclophosphamide, vincristine, prednisolone (R-CVP) (n = 19), dose-attenuated R-CHOP (n = 22), dose attenuated rituximab-bendamustine (n = 24) and rituximab-chlorambucil (n = 30). The primary outcome was progression-free survival (PFS). The secondary outcomes included overall response, overall survival (OS) and toxicity. The median (range) age was 79 (58-89) years and 50% were aged ≥80 years. The median (range) Cumulative Illness Rating Scale-Geriatric score was 6 (0-24). The median PFS for all patients was 15 months [95% confidence interval (CI) 8·7-21·2) and median OS was 31·4 months (95% CI 19·7-43·2). By multivariable analysis (MVA), the only clinical factor associated with an inferior PFS was blastoid morphology [hazard ratio (HR) 2·90, P = 0·01). Notably, higher treatment intensity (R-CHOP/R-bendamustine composite) provided an independently superior PFS compared with R-CVP/R-chlorambucil (MVA HR 0·49, P = 0·02). Factors associated with inferior OS by MVA were Eastern Cooperative Oncology Group Performance Status (HR 2·14, P = 0·04), blastoid morphology (HR 4·08, P = 0·001) and progression of disease at <24 months status (HR 5·68, P < 0·001). Overall, survival after front-line dose-attenuated immunochemotherapy is unsatisfactory. Clinical trials investigating novel agents such as Bruton tyrosine kinase and B-cell lymphoma 2 inhibitors in this specific clinical setting are warranted.

The microenvironment in myeloma.

PURPOSE OF REVIEW: The aim of the review is to describe recent advances in our understanding of how multiple myeloma interacts with its cellular and molecular neighbours in the bone marrow microenvironment, and how this may provide targets for prognostication and prevention. RECENT FINDINGS: The bone marrow microenvironment in myeloma is beginning to yield targets that are amenable to therapy. A number of trials demonstrate some clinical efficacy in heavily pretreated disease. The challenge remains for how and when these therapeutic interventions are of particular benefit early in disease progression. SUMMARY: Multiple myeloma is rarely curable and its interactions with the bone marrow microenvironment are evident. However, separating cause from effect remains a challenge. We propose that targeting specific niches within the bone marrow will yield therapies that have the potential for significant benefit in myeloma and may facilitate earlier intervention to disrupt an environment that is permissive for myeloma progression.

Phosphodiesterases maintain signaling fidelity via compartmentalization of cyclic nucleotides.

Novel technological advances have improved our understanding of how cyclic nucleotides are able to convey signals faithfully between cellular compartments. Phosphodiesterases play a crucial role in shaping these signals in health and disease. The concept of compartmentalization is guiding the search for therapies that have the potential to offer greater efficacy and tolerability compared with current treatments.

A population-scale temporal case-control evaluation of COVID-19 disease phenotype and related outcome rates in patients with cancer in England (UKCCP).

Patients with cancer are at increased risk of hospitalisation and mortality following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the SARS-CoV-2 phenotype evolution in patients with cancer since 2020 has not previously been described. We therefore evaluated SARS-CoV-2 on a UK populationscale from 01/11/2020-31/08/2022, assessing case-outcome rates of hospital assessment(s), intensive care admission and mortality. We observed that the SARS-CoV-2 disease phenotype has become less severe in patients with cancer and the non-cancer population. Case-hospitalisation rates for patients with cancer dropped from 30.58% in early 2021 to 7.45% in 2022 while case-mortality rates decreased from 20.53% to 3.25%. However, the risk of hospitalisation and mortality remains 2.10x and 2.54x higher in patients with cancer, respectively. Overall, the SARS-CoV-2 disease phenotype is less severe in 2022 compared to 2020 but patients with cancer remain at higher risk than the non-cancer population. Patients with cancer must therefore be empowered to live more normal lives, to see loved ones and families, while also being safeguarded with expanded measures to reduce the risk of transmission.

COVID-19: Third dose booster vaccine effectiveness against breakthrough coronavirus infection, hospitalisations and death in patients with cancer: A population-based study.

PURPOSE: People living with cancer and haematological malignancies are at an increased risk of hospitalisation and death following infection with acute respiratory syndrome coronavirus 2. Coronavirus third dose vaccine boosters are proposed to boost waning immune responses in immunocompromised individuals and increase coronavirus protection; however, their effectiveness has not yet been systematically evaluated. METHODS: This study is a population-scale real-world evaluation of the United Kingdom's third dose vaccine booster programme for cancer patients from 8th December 2020 to 7th December 2021. The cancer cohort comprises individuals from Public Health England's national cancer dataset, excluding individuals less than 18 years. A test-negative case-control design was used to assess the third dose booster vaccine effectiveness. Multivariable logistic regression models were fitted to compare risk in the cancer cohort relative to the general population. RESULTS: The cancer cohort comprised of 2,258,553 tests from 361,098 individuals. Third dose boosters were evaluated by reference to 87,039,743 polymerase chain reaction coronavirus tests. Vaccine effectiveness against breakthrough infections, symptomatic infections, coronavirus hospitalisation and death in cancer patients were 59.1%, 62.8%, 80.5% and 94.5%, respectively. Lower vaccine effectiveness was associated with a cancer diagnosis within 12 months, lymphoma, recent systemic anti-cancer therapy (SACT) or radiotherapy. Patients with lymphoma had low levels of protection from symptomatic disease. In spite of third dose boosters, following multivariable adjustment, individuals with cancer remain at an increased risk of coronavirus hospitalisation and death compared to the population control (OR 3.38, 3.01, respectively. p < 0.001 for both). CONCLUSIONS: Third dose boosters are effective for most individuals with cancer, increasing protection from coronavirus. However, their effectiveness is heterogenous and lower than the general population. Many patients with cancer will remain at the increased risk of coronavirus infections even after 3 doses. In the case of patients with lymphoma, there is a particularly strong disparity of vaccine effectiveness against breakthrough infection and severe disease. Breakthrough infections will disrupt cancer care and treatment with potentially adverse consequences on survival outcomes. The data support the role of vaccine boosters in preventing severe disease, and further pharmacological intervention to prevent transmission and aid viral clearance to limit the disruption of cancer care as the delivery of care continues to evolve during the coronavirus pandemic.

Complicated forms of tachycardia-mediated cardiomyopathy associated with idiopathic left ventricular tachycardia.

Idiopathic left ventricular tachycardia is an infrequent form of ventricular tachycardia associated with a structurally normal heart. The prognosis is usually benign; however, sustained cases have been reported. In this report, we describe two cases of persistent idiopathic left ventricular tachycardia complicated by tachycardia-mediated cardiomyopathy. In the first case, the patient developed a right ventricular thrombus with subsequent pulmonary embolism. In the second case, the patient developed acute pulmonary edema. Both cases were cured by catheter ablation.

Validation of clinical-grade whole genome sequencing reproduces cytogenetic analysis and identifies mutational landscape in newly-diagnosed multiple myeloma patients: A pilot study from the 100,000 Genomes Project.

Multiple myeloma is characterized by chromosomal abnormalities and genetic variation, which may inform prognosis and guide treatment. This pilot study sought to examine the feasibility of incorporating Whole Genome Sequencing (WGS) alongside the routine laboratory evaluation of 14 patients with newly diagnosed multiple myeloma who had enrolled in the 100,000 Genomes Project. In all 14 cases, WGS data could be obtained in a timely fashion within existing clinical frameworks in a tertiary hospital setting. The data not only replicated standard-of-care FISH analysis of chromosomal abnormalities but also provided further chromosomal and molecular genetic insights that may influence patient management.

Minimal Residual Disease in Myeloma: Application for Clinical Care and New Drug Registration.

The development of novel agents has transformed the treatment paradigm for multiple myeloma, with minimal residual disease (MRD) negativity now achievable across the entire disease spectrum. Bone marrow-based technologies to assess MRD, including approaches using next-generation flow and next-generation sequencing, have provided real-time clinical tools for the sensitive detection and monitoring of MRD in patients with multiple myeloma. Complementary liquid biopsy-based assays are now quickly progressing with some, such as mass spectrometry methods, being very close to clinical use, while others utilizing nucleic acid-based technologies are still developing and will prove important to further our understanding of the biology of MRD. On the regulatory front, multiple retrospective individual patient and clinical trial level meta-analyses have already shown and will continue to assess the potential of MRD as a surrogate for patient outcome. Given all this progress, it is not surprising that a number of clinicians are now considering using MRD to inform real-world clinical care of patients across the spectrum from smoldering myeloma to relapsed refractory multiple myeloma, with each disease setting presenting key challenges and questions that will need to be addressed through clinical trials. The pace of advances in targeted and immune therapies in multiple myeloma is unprecedented, and novel MRD-driven biomarker strategies are essential to accelerate innovative clinical trials leading to regulatory approval of novel treatments and continued improvement in patient outcomes.

Clinical Controversies in the Management of Smoldering Multiple Myeloma.

Forty years ago this year, smoldering multiple myeloma was defined as a clinical entity that identifies a group of patients with a substantial burden of disease but with a relatively indolent natural history compared with symptomatic disease. Since then, there has been a revolution in the therapeutic options for multiple myeloma. The aim of this article is to describe recent advances in the identification of those patients who are at the highest risk of progression and whether they may benefit from therapy. Treatment of smoldering myeloma is an area of active debate and we present contrasting interpretations of the available trial data. We conclude by identifying the priorities for research that will help to clarify the management of this condition, which can be challenging for physicians and patients alike.

Monoclonal Gammopathy of Undetermined Significance (MGUS)-Not So Asymptomatic after All.

Monoclonal Gammopathy of Undetermined Significance (MGUS) is considered to be a benign precursor condition that may progress to a lymphoproliferative disease or multiple myeloma. Most patients do not progress to an overt condition, but nevertheless, MGUS is associated with a shortened life expectancy and, in a minority of cases, a number of co-morbid conditions that include an increased fracture risk, renal impairment, peripheral neuropathy, secondary immunodeficiency, and cardiovascular disease. This review aims to consolidate current evidence for the significance of these co-morbidities before considering how best to approach these symptoms and signs, which are often encountered in primary care or within a number of specialties in secondary care.

Genome instability in multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by clonal proliferation of plasma cells and a heterogenous genomic landscape. Copy number and structural changes due to chromosomal instability (CIN) are common features of MM. In this review, we describe how primary and secondary genetic events caused by CIN can contribute to increased instability across the genome of malignant plasma cells; with a focus on specific driver genomic events, and how they interfere with cell-cycle checkpoints, to prompt accelerated proliferation. We also provide insight into other forms of CIN, such as chromothripsis and chromoplexy. We evaluate how the tumor microenvironment can contribute to a further increase in chromosomal instability in myeloma cells. Lastly, we highlight the role of certain mutational signatures in leading to high mutation rate and genome instability in certain MM patients. We suggest that assessing CIN in MM and its precursors states may help improve predicting the risk of progression to symptomatic disease and relapse and identifying future therapeutic targets.

Usefulness of C-reactive protein in predicting early and late recurrences after atrial fibrillation ablation.

We studied the C-reactive protein level and its predictive value for early and late arrhythmia recurrences after atrial fibrillation (AF) ablation. We analysed data from 125 consecutive patients (52% with paroxysmal AF) who underwent a first AF ablation. C-reactive protein level was determined at baseline and at Days 1, 2, and 3 post-AF ablation. An early recurrence (ER) was defined as any arrhythmia occurring within the first month following the index ablation. Sixty-eight patients (54%) experienced ER. Twenty-nine patients were re-ablated for ER within the first month and were not included for the long-term follow-up analysis. Of the remaining 96 patients, 59 (61%) experienced late recurrences. C-reactive protein level increased significantly after AF ablation with a peak (40 +/- 32 mg/L) occurring 2 days after the index procedure. Patients without ER had a significant higher C-reactive protein level (49 +/- 52 vs. 32 +/- 22 mg/L, P=0.02) compared with patients without ER. However, the C-reactive protein level was similar in patients with or without late recurrences. C-reactive protein level was the only independent predictor of ERs (P = 0.03). C-reactive protein level increased significantly after AF ablation. High C-reactive protein level was associated with fewer early arrhythmia recurrences. No relationship was found between C-reactive protein level and late arrhythmia recurrences.

Impact of a patent foramen ovale on paroxysmal atrial fibrillation ablation.

INTRODUCTION: A patent foramen ovale (PFO) is located at the anterior and superior part of the anatomical interatrial septum, the area that is targeted during transseptal puncture. This study sought to investigate the impact of accessing the left atrium via a PFO on paroxysmal AF ablation. METHODS: From March 2004, 203 patients (55 +/- 11 years) underwent catheter ablation for paroxysmal AF (80 +/- 71 months), with the endpoint being electrical isolation of all pulmonary veins (PV) and AF noninducibility. The presence of a PFO was determined by both transesophageal echocardiography and catheter probing. Procedural difficulty was evaluated by radiofrequency (RF), procedural, and fluoroscopic durations. Clinical follow-up was also investigated. RESULTS: A PFO was detected in 27 patients (13%) by transesophageal echocardiography and in 22 additional patients (total 49 patients, 24%), by catheter probing (P < 0.001). A PFO was associated with longer total RF applications (57 +/- 19 vs 51 +/- 18 min, P = 0.04) and RF applications to isolate the PVs (42 +/- 16 vs 35 +/- 12 min, P = 0.001). Procedural and fluoroscopic times were unaffected. Seventy-three patients (36%) required a second procedure; there was no difference in the number of PV reconnections (1.3 vs 1.8 veins, P = NS). After a mean follow-up of 19 +/- 9 months, 194/203 patients (96%) were free of AF, with no difference in patients in whom a PFO had been used. CONCLUSION: Although isolation of PVs is longer, overall procedural duration and success is not affected when using a PFO compared with a transseptal puncture. The presence of a PFO is underestimated by transesophageal echocardiography with brachial injection when compared with catheter probing.

Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes.

AIMS: 3',5'-Cyclic adenosine monophosphate (cAMP) signals in the heart are often confined to concentration microdomains shaped by cAMP diffusion and enzymatic degradation. While the importance of phosphodiesterases (degradative enzymes) in sculpting cAMP microdomains is well established in cardiomyocytes, less is known about cAMP diffusivity (DcAMP) and factors affecting it. Many earlier studies have reported fast diffusivity, which argues against sharply defined microdomains. METHODS AND RESULTS: [cAMP] dynamics in the cytoplasm of adult rat ventricular myocytes were imaged using a fourth generation genetically encoded FRET-based sensor. The [cAMP]-response to the addition and removal of isoproterenol (ß-adrenoceptor agonist) quantified the rates of cAMP synthesis and degradation. To obtain a read out of DcAMP, a stable [cAMP] gradient was generated using a microfluidic device which delivered agonist to one half of the myocyte only. After accounting for phosphodiesterase activity, DcAMP was calculated to be 32 µm(2)/s; an order of magnitude lower than in water. Diffusivity was independent of the amount of cAMP produced. Saturating cAMP-binding sites with the analogue 6-Bnz-cAMP did not accelerate DcAMP, arguing against a role of buffering in restricting cAMP mobility. cAMP diffused at a comparable rate to chemically unrelated but similar sized molecules, arguing for a common physical cause of restricted diffusivity. Lower mitochondrial density and order in neonatal cardiac myocytes allowed for faster diffusion, demonstrating the importance of mitochondria as physical barriers to cAMP mobility. CONCLUSION: In adult cardiac myocytes, tortuosity due to physical barriers, notably mitochondria, restricts cAMP diffusion to levels that are more compatible with microdomain signalling.

Up-regulation of miR-31 in human atrial fibrillation begets the arrhythmia by depleting dystrophin and neuronal nitric oxide synthase.

Atrial fibrillation (AF) is a growing public health burden, and its treatment remains a challenge. AF leads to electrical remodeling of the atria, which in turn promotes AF maintenance and resistance to treatment. Although remodeling has long been a therapeutic target in AF, its causes remain poorly understood. We show that atrial-specific up-regulation of microRNA-31 (miR-31) in goat and human AF depletes neuronal nitric oxide synthase (nNOS) by accelerating mRNA decay and alters nNOS subcellular localization by repressing dystrophin translation. By shortening action potential duration and abolishing rate-dependent adaptation of the action potential duration, miR-31 overexpression and/or disruption of nNOS signaling recapitulates features of AF-induced remodeling and significantly increases AF inducibility in mice in vivo. By contrast, silencing miR-31 in atrial myocytes from patients with AF restores dystrophin and nNOS and normalizes action potential duration and its rate dependency. These findings identify atrial-specific up-regulation of miR-31 in human AF as a key mechanism causing atrial dystrophin and nNOS depletion, which in turn contributes to the atrial phenotype begetting this arrhythmia. miR-31 may therefore represent a potential therapeutic target in AF.

Adenoviral transduction of FRET-based biosensors for cAMP in primary adult mouse cardiomyocytes.

Genetically encoded biosensors that make use of fluorescence resonance energy transfer (FRET) are important tools for the study of compartmentalized cyclic nucleotide signaling in living cells. With the advent of germ line and tissue-specific transgenic technologies, the adult mouse represents a useful tool for the study of cardiovascular pathophysiology. The use of FRET-based genetically encoded biosensors coupled with this animal model represents a powerful combination for the study of cAMP signaling in live primary cardiomyocytes. In this chapter, we describe the steps required during the isolation, viral transduction, and culture of cardiomyocytes from an adult mouse to obtain reliable expression of genetically encoded FRET biosensors for the study of cAMP signaling in living cells.