The landscape of cytogenetic and molecular genetic methods in diagnostics for hematologic neoplasia.

Improvements made during the last decades in the management of patients with hematologic neoplasia have resulted in increase of overall survival. These advancements have become possible through progress in our understanding of genetic basis of different hematologic malignancies and their role in the current risk-adapted treatment protocols. In this review, we provide an overview of current cytogenetic and molecular genetic methods, commonly used in the genetic characterization of hematologic malignancies, describe the current developments in the cytogenetic and molecular diagnostics, and give an outlook into their future development. Furthermore, we give a brief overview of the most important public databases and guidelines for sequence variant interpretation.

Chemotherapy-Free Targeted Anti-BCR-ABL+ Acute Lymphoblastic Leukemia Therapy May Benefit the Heart.

Targeted therapies are currently considered the best cost-benefit anti-cancer treatment. In hematological malignancies, however, relapse rates and non-hematopoietic side effects including cardiotoxicity remain high. Here, we describe significant heart damage due to advanced acute lymphoblastic leukemia (ALL) with t(9;22) encoding the bcr-abl oncogene (BCR-ABL+ ALL) in murine xenotransplantation models. Echocardiography reveals severe cardiac dysfunction with impaired left ventricular function and reduced heart and cardiomyocyte dimensions associated with increased apoptosis. This cardiac damage is fully reversible, but cardiac recovery depends on the therapy used to induce ALL remission. Chemotherapy-free combination therapy with dasatinib (DAS), venetoclax (VEN) (targeting the BCR-ABL oncoprotein and mitochondrial B-cell CLL/Lymphoma 2 (BCL2), respectively), and dexamethasone (DEX) can fully revert cardiac defects, whereas the depletion of otherwise identical ALL in a genetic model using herpes simplex virus type 1 thymidine kinase (HSV-TK) cannot. Mechanistically, dexamethasone induces a pro-apoptotic BCL2-interacting mediator of cell death (BIM) expression and apoptosis in ALL cells but enhances pro-survival B-cell lymphoma extra-large (BCLXL) expression in cardiomyocytes and clinical recovery with the reversion of cardiac atrophy. These data demonstrate that therapies designed to optimize apoptosis induction in ALL may circumvent cardiac on-target side effects and may even activate cardiac recovery. In the future, combining the careful clinical monitoring of cardiotoxicity in leukemic patients with the further characterization of organ-specific side effects and signaling pathways activated by malignancy and/or anti-tumor therapies seems reasonable.

Anthracycline-free tumor elimination in mice leads to functional and molecular cardiac recovery from cancer-induced alterations in contrast to long-lasting doxorubicin treatment effects.

Systemic effects of advanced cancer impact on the heart leading to cardiac atrophy and functional impairment. Using a murine melanoma cancer model (B16F10 melanoma cells stably transduced with a Ganciclovir (GCV)-inducible suicide gene), the present study analysed the recovery potential of cancer-induced cardiomyopathy with or without use of doxorubicin (Dox). After Dox-free tumor elimination and recovery for 70 ± 5 days, cancer-induced morphologic, functional, metabolic and molecular changes were largely reversible in mice previously bearing tumors. Moreover, grip strength and cardiac response to angiotensin II-induced high blood pressure were comparable with healthy control mice. In turn, addition of Dox (12 mg/kg BW) to melanoma-bearing mice reduced survival in the acute phase compared to GCV-alone induced recovery, while long-term effects on cardiac morphologic and functional recovery were similar. However, Dox treatment was associated with permanent changes in the cardiac gene expression pattern, especially the circadian rhythm pathway associated with the DNA damage repair system. Thus, the heart can recover from cancer-induced damage after chemotherapy-free tumor elimination. In contrast, treatment with the cardiotoxic drug Dox induces, besides well-known adverse acute effects, long-term subclinical changes in the heart, especially of circadian clock genes. Since the circadian clock is known to impact on cardiac repair mechanisms, these changes may render the heart more sensitive to additional stress during lifetime, which, at least in part, could contribute to late cardiac toxicity.

Increased prostaglandin-D2 in male STAT3-deficient hearts shifts cardiac progenitor cells from endothelial to white adipocyte differentiation.

Cardiac levels of the signal transducer and activator of transcription factor-3 (STAT3) decline with age, and male but not female mice with a cardiomyocyte-specific STAT3 deficiency conditional knockout (CKO) display premature age-related heart failure associated with reduced cardiac capillary density. In the present study, isolated male and female CKO-cardiomyocytes exhibit increased prostaglandin (PG)-generating cyclooxygenase-2 (COX-2) expression. The PG-degrading hydroxyprostaglandin-dehydrogenase-15 (HPGD) expression is only reduced in male cardiomyocytes, which is associated with increased prostaglandin D2 (PGD2) secretion from isolated male but not female CKO-cardiomyocytes. Reduced HPGD expression in male cardiomyocytes derive from impaired androgen receptor (AR)-signaling due to loss of its cofactor STAT3. Elevated PGD2 secretion in males is associated with increased white adipocyte accumulation in aged male but not female hearts. Adipocyte differentiation is enhanced in isolated stem cell antigen-1 (SCA-1)+ cardiac progenitor cells (CPC) from young male CKO-mice compared with the adipocyte differentiation of male wild-type (WT)-CPC and CPC isolated from female mice. Epigenetic analysis in freshly isolated male CKO-CPC display hypermethylation in pro-angiogenic genes (Fgfr2, Epas1) and hypomethylation in the white adipocyte differentiation gene Zfp423 associated with up-regulated ZFP423 expression and a shift from endothelial to white adipocyte differentiation compared with WT-CPC. The expression of the histone-methyltransferase EZH2 is reduced in male CKO-CPC compared with male WT-CPC, whereas no differences in the EZH2 expression in female CPC were observed. Clonally expanded CPC can differentiate into endothelial cells or into adipocytes depending on the differentiation conditions. ZFP423 overexpression is sufficient to induce white adipocyte differentiation of clonal CPC. In isolated WT-CPC, PGD2 stimulation reduces the expression of EZH2, thereby up-regulating ZFP423 expression and promoting white adipocyte differentiation. The treatment of young male CKO mice with the COX inhibitor Ibuprofen or the PGD2 receptor (DP)2 receptor antagonist BAY-u 3405 in vivo increased EZH2 expression and reduced ZFP423 expression and adipocyte differentiation in CKO-CPC. Thus, cardiomyocyte STAT3 deficiency leads to age-related and sex-specific cardiac remodeling and failure in part due to sex-specific alterations in PGD2 secretion and subsequent epigenetic impairment of the differentiation potential of CPC. Causally involved is the impaired AR signaling in absence of STAT3, which reduces the expression of the PG-degrading enzyme HPGD.

Common genetic predisposition for heart failure and cancer.

Cardiovascular diseases and cancer are major causes of mortality in industrialized societies. They share common risk factors (e.g., genetics, lifestyle, age, infection, toxins, and pollution) and might also mutually promote the onset of the respective other disease. Cancer can affect cardiac function directly while antitumor therapies may have acute- and/or late-onset cardiotoxic effects. Recent studies suggest that heart failure might promote tumorigenesis and tumor progression. In both cancer and cardiovascular diseases, genetic predisposition is implicated in the disease onset and development. In this regard, genetic variants classically associated with cardiomyopathies increase the risk for toxic side effects on the cardiovascular system. Genetic variants associated with increased cancer risk are frequent in patients with peripartum cardiomyopathy complicated by cancer, pointing to a common genetic predisposition for both diseases. Common risk factors, cardiotoxic antitumor treatment, genetic variants (associated with cardiomyopathies and/or cancer), and increased cardiac stress lead us to propose the "multi-hit hypothesis" linking cancer and cardiovascular diseases. In the present review, we summarize the current knowledge on potential connecting factors between cancer and cardiovascular diseases with a major focus on the role of genetic predisposition and its implication for individual therapeutic strategies and risk assessment in the novel field of oncocardiology.

Outcome in German and South African peripartum cardiomyopathy cohorts associates with medical therapy and fibrosis markers.

AIMS: This study aims to compare the clinical course of peripartum cardiomyopathy (PPCM) cohorts from Germany (G-PPCM) and South Africa (SA-PPCM) with fibrosis-related markers to get insights into novel pathomechanisms of PPCM. METHODS AND RESULTS: G-PPCM (n = 79) and SA-PPCM (n = 72) patients and healthy pregnancy-matched women from Germany (n = 56) and South Africa (n = 40) were enrolled. Circulating levels of procollagen type-I (PINP) and type-III (PIIINP) N-terminal propeptides, soluble ST2, galectin-3, and full-length and cleaved osteopontin (OPN) were measured at diagnosis (baseline) and 6 months of follow-up. Both cohorts received standard heart failure therapy while anticoagulation therapy was applied in 100% of G-PPCM but only in 7% of SA-PPCM patients. In G-PPCM patients, baseline left ventricular ejection fraction (LVEF) was lower, and outcome was better (baseline LVEF, 24 ± 8%, full recovery: 52%, mortality: 0%) compared with SA-PPCM patients (baseline LVEF: 30 ± 9%, full recovery: 32%, mortality: 11%; P < 0.05). At baseline, PINP/PIIINP ratio was lower in SA-PPCM and higher in G-PPCM compared with respective controls, whereas total OPN was elevated in both collectives. Cleaved OPN, which increases PIIINP levels, is generated by thrombin and was reduced in patients receiving anticoagulation therapy. High baseline galectin-3, soluble ST2, and OPN levels were associated with poor outcome in all PPCM patients. CONCLUSIONS: SA-PPCM patients displayed a more profibrotic biomarker profile, which was associated with a less favourable outcome despite better cardiac function at baseline, compared with G-PPCM patients. Use of bromocriptine and anticoagulation therapy in G-PPCM may counteract fibrosis and may in part be responsible for their better outcome.

Modulation of cardiac AKT and STAT3 signalling in preclinical cancer models and their impact on the heart.

BACKGROUND: Advanced cancer induces fundamental cardiac changes and promotes body wasting and heart failure. We evaluated the impact of cancer on major cardiac signalling pathways, and resulting consequences for the heart. METHODS AND RESULTS: Metastatic melanoma disease was induced in male C57BL/6 N mice by intraperitoneal injection of the melanoma cell line B16F10 and lead to cardiac atrophy and heart failure. Analyses of key cardiac signalling pathways in left ventricular tissue revealed increased activation of STAT3 and reduced activation of AKT, p38 and ERK1/2. Markers of the ubiquitin proteasomal system (UPS: Atrogin-1) and of mitophagy/autophagy (LC3b, BNIP3) were upregulated. Tumour-bearing C57BL/6 N mice with a cardiomyocyte-specific overexpression of a constitutively active AKT transgene (AKTtg) displayed less cardiac atrophy and dysfunction and normalized Atrogin-1, LC3b and BNIP3 expression while the cardiomyocyte-specific knockout of STAT3 (CKO) had no major effect on these parameters compared to WT. CONCLUSION: Cancer alters major cardiac signalling pathways and subsequently the UPS, mitophagy and autophagy. The present study suggests that cancer-induced reduction of cardiomyocyte AKT contributes to these alterations as they were attenuated in tumour-bearing AKTtg mice. In turn, increased cardiomyocyte STAT3 activation appears less relevant, as tumour-induced impairment on the heart was largely similar in CKO and WT mice. Since oncologic therapies frequently target AKT and/or STAT3, their impact on the heart might be different in tumour-bearing mice compared to healthy mice, a feature suggesting to test tumour therapies also in tumour disease models and not only under healthy conditions. This article is part of a Special Issue entitled: Cardiomyocyte biology: new pathways of differentiation and regeneration edited by Marijke Brink, Marcus C. Schaub, and Christian Zuppinger.

Data on left ventricular expression of STAT3 and AKT in transgenic mouse models with B16F10 melanoma.

The dataset describes protein expression of phosphorylated and total signal transducer and activator of transcription 3 (STAT3), protein kinase B (AKT) and suppressor of cytokine signalling 3 (SOCS3) in left ventricular tissue (LV) from healthy and B16F10 melanoma tumour-bearing (B16F10-TM) wildtype (WT) mice, mice with cardiomyocyte-specific constitutively active AKT transgene (AKTtg) and mice with cardiomyocyte-restricted deletion of STAT3 (CKO) analysed in Western blot and/or fluorescence microscopy experiments. The data presented in this article are related to the research paper entitled "Modulation of cardiac AKT and STAT3 signalling in preclinical cancer models and their impact on the heart", available in Biochim. Biophys. Acta Mol. Cell Res. (1).

Increased Cancer Prevalence in Peripartum Cardiomyopathy.

OBJECTIVES: This study was designed to analyze the prevalence and potential genetic basis of cancer and heart failure in peripartum cardiomyopathy (PPCM). BACKGROUND: PPCM manifests as heart failure late in pregnancy or postpartum in women without previous heart disease. METHODS: Clinical history and cancer prevalence were evaluated in a cohort of 236 PPCM patients from Germany and Sweden. Exome sequencing assessed variants in 133 genes associated with cancer predisposition syndromes (CPS) and in 115 genes associated with dilated/hypertrophic cardiomyopathy (DCM/HCM) in 14 PPCM patients with a history of cancer, and in 6 PPCM patients without a history of cancer. RESULTS: The prevalence of cancer was 16-fold higher (8.9%, 21 of 236 patients) in PPCM patients compared to age-matched women (German cancer registry, Robert-Koch-Institute: 0.59%; p < 0.001). Cancer before PPCM occurred in 12 of 21 patients of whom 11 obtained cardiotoxic cancer therapies. Of those, 17% fully recovered cardiac function by 7 ± 2 months of follow-up compared to 55% of PPCM patients without cancer (p = 0.015). Cancer occurred after PPCM in 10 of 21 patients; 80% had left ventricular ejection fraction of ≥50% after cancer therapy. Whole-exome sequencing in 14 PPCM patients with cancer revealed that 43% (6 of 14 patients) carried likely pathogenic (Class IV) or pathogenic (Class V) gene variants associated with DCM/HCM in CPT2, DSP, MYH7, TTN, and/or with CPS in ATM, ERCC5, NBN, RECQL4, and SLX4. All CPS variants affected DNA damage response genes. CONCLUSIONS: Cardiotoxic cancer therapy before PPCM is associated with delayed full recovery. The high cancer prevalence in PPCM is linked to likely pathogenic/pathogenic gene variants associated with DCM/HCM and/or CPS/DNA damage response-related cancer risk. This may warrant genetic testing and screening for heart failure in pregnant women with a cancer history and screening for cancer in PPCM patients.

Insulin supplementation attenuates cancer-induced cardiomyopathy and slows tumor disease progression.

Advanced cancer induces fundamental changes in metabolism and promotes cardiac atrophy and heart failure. We discovered systemic insulin deficiency in cachectic cancer patients. Similarly, mice with advanced B16F10 melanoma (B16F10-TM) or colon 26 carcinoma (C26-TM) displayed decreased systemic insulin associated with marked cardiac atrophy, metabolic impairment, and function. B16F10 and C26 tumors decrease systemic insulin via high glucose consumption, lowering pancreatic insulin production and producing insulin-degrading enzyme. As tumor cells consume glucose in an insulin-independent manner, they shift glucose away from cardiomyocytes. Since cardiomyocytes in both tumor models remained insulin responsive, low-dose insulin supplementation by subcutaneous implantation of insulin-releasing pellets improved cardiac glucose uptake, atrophy, and function, with no adverse side effects. In addition, by redirecting glucose to the heart in addition to other organs, the systemic insulin treatment lowered glucose usage by the tumor and thereby decreased tumor growth and volume. Insulin corrected the cancer-induced reduction in cardiac Akt activation and the subsequent overactivation of the proteasome and autophagy. Thus, cancer-induced systemic insulin depletion contributes to cardiac wasting and failure and may promote tumor growth. Low-dose insulin supplementation attenuates these processes and may be supportive in cardio-oncologic treatment concepts.

Low STAT3 expression sensitizes to toxic effects of ß-adrenergic receptor stimulation in peripartum cardiomyopathy.

Aims: The benefit of the ß1-adrenergic receptor (ß1-AR) agonist dobutamine for treatment of acute heart failure in peripartum cardiomyopathy (PPCM) is controversial. Cardiac STAT3 expression is reduced in PPCM patients. Mice carrying a cardiomyocyte-restricted deletion of STAT3 (CKO) develop PPCM. We hypothesized that STAT3-dependent signalling networks may influence the response to ß-AR agonist treatment in PPCM patients and analysed this hypothesis in CKO mice. Methods and Results: Follow-up analyses in 27 patients with severe PPCM (left ventricular ejection fraction ≤25%) revealed that 19 of 20 patients not obtaining dobutamine improved cardiac function. All seven patients obtaining dobutamine received heart transplantation (n = 4) or left ventricular assist devices (n = 3). They displayed diminished myocardial triglyceride, pyruvate, and lactate content compared with non-failing controls. The ß-AR agonist isoproterenol (Iso) induced heart failure with high mortality in postpartum female, in non-pregnant female and in male CKO, but not in wild-type mice. Iso induced heart failure and high mortality in CKO mice by impairing fatty acid and glucose uptake, thereby generating a metabolic deficit. The latter was governed by disturbed STAT3-dependent signalling networks, microRNA-199a-5p, microRNA-7a-5p, insulin/glucose transporter-4, and neuregulin/ErbB signalling. The resulting cardiac energy depletion and oxidative stress promoted dysfunction and cardiomyocyte loss inducing irreversible heart failure, which could be attenuated by the ß1-AR blocker metoprolol or glucose-uptake-promoting drugs perhexiline and etomoxir. Conclusions: Iso impairs glucose uptake, induces energy depletion, oxidative stress, dysfunction, and death in STAT3-deficient cardiomyocytes mainly via ß1-AR stimulation. These cellular alterations may underlie the dobutamine-induced irreversible heart failure progression in PPCM patients who frequently display reduced cardiac STAT3 expression.

Cardiomyocytes display low mitochondrial priming and are highly resistant toward cytotoxic T-cell killing.

Following heart transplantation, alloimmune responses can cause graft rejection by damaging donor vascular and parenchymal cells. However, it remains unclear whether cardiomyocytes are also directly killed by immune cells. Here, we used two-photon microscopy to investigate how graft-specific effector CD8(+) T cells interact with cardiomyocytes in a mouse heart transplantation model. Surprisingly, we observed that CD8(+) T cells are completely impaired in killing cardiomyocytes. Even after virus-mediated preactivation, antigen-specific CD8(+) T cells largely fail to lyse these cells although both cell types engage in dynamic interactions. Furthermore, we established a two-photon microscopy-based assay using intact myocardium to determine the susceptibility of cardiomyocytes to undergo apoptosis. This feature, also known as mitochondrial priming reveals an unexpected weak predisposition of cardiomyocytes to undergo apoptosis in situ. These observations together with the early exhaustion phenotype of graft-infiltrating specific T cells provide an explanation why cardiomyocytes are largely protected from direct CD8(+) T-cell-mediated killing.