Bone marrow microenvironments that contribute to patient outcomes in newly diagnosed multiple myeloma: A cohort study of patients in the Total Therapy clinical trials.

BACKGROUND: The tumor microenvironment (TME) is increasingly appreciated as an important determinant of cancer outcome, including in multiple myeloma (MM). However, most myeloma microenvironment studies have been based on bone marrow (BM) aspirates, which often do not fully reflect the cellular content of BM tissue itself. To address this limitation in myeloma research, we systematically characterized the whole bone marrow (WBM) microenvironment during premalignant, baseline, on treatment, and post-treatment phases. METHODS AND FINDINGS: Between 2004 and 2019, 998 BM samples were taken from 436 patients with newly diagnosed MM (NDMM) at the University of Arkansas for Medical Sciences in Little Rock, Arkansas, United States of America. These patients were 61% male and 39% female, 89% White, 8% Black, and 3% other/refused, with a mean age of 58 years. Using WBM and matched cluster of differentiation (CD)138-selected tumor gene expression to control for tumor burden, we identified a subgroup of patients with an adverse TME associated with 17 fewer months of progression-free survival (PFS) (95% confidence interval [CI] 5-29, 49-69 versus 70-82 months, χ2 p = 0.001) and 15 fewer months of overall survival (OS; 95% CI -1 to 31, 92-120 versus 113-129 months, χ2 p = 0.036). Using immunohistochemistry-validated computational tools that identify distinct cell types from bulk gene expression, we showed that the adverse outcome was correlated with elevated CD8+ T cell and reduced granulocytic cell proportions. This microenvironment develops during the progression of premalignant to malignant disease and becomes less prevalent after therapy, in which it is associated with improved outcomes. In patients with quantified International Staging System (ISS) stage and 70-gene Prognostic Risk Score (GEP-70) scores, taking the microenvironment into consideration would have identified an additional 40 out of 290 patients (14%, premutation p = 0.001) with significantly worse outcomes (PFS, 95% CI 6-36, 49-73 versus 74-90 months) who were not identified by existing clinical (ISS stage III) and tumor (GEP-70) criteria as high risk. The main limitations of this study are that it relies on computationally identified cell types and that patients were treated with thalidomide rather than current therapies. CONCLUSIONS: In this study, we observe that granulocyte signatures in the MM TME contribute to a more accurate prognosis. This implies that future researchers and clinicians treating patients should quantify TME components, in particular monocytes and granulocytes, which are often ignored in microenvironment studies.

Treatment to suppression of focal lesions on positron emission tomography-computed tomography is a therapeutic goal in newly diagnosed multiple myeloma.

Fluorine-18 fluorodeoxyglucose positron emission tomography with computed tomography attenuation correction (PET-CT) in myeloma can detect and enumerate focal lesions by the quantitative characterization of metabolic activity. The aim of this study was to determine the prognostic significance of the suppression of PET-CT activity at a number of time points post therapy initiation: day 7, post induction, post transplant, and at maintenance therapy. As part of the TT4-6 trial series, 596 patients underwent baseline PET-CT and were evaluated serially during their disease course using peak standardized uptake values above background red marrow signal. We demonstrate that the presence of more than 3 focal lesions at presentation identifies a group of patients with an adverse progression-free survival and overall survival. At day 7 of therapy, patients with complete focal lesion signal suppression revert to the same prognosis as those with no lesions at diagnosis. At later time points, the continued suppression of signal remains prognostically important. We conclude that for newly diagnosed patients with focal lesions, treatment until these lesions are suppressed is an important therapeutic goal as the prognosis of these patients is the same as those without lesions at diagnosis. (clinicaltrials.gov identifiers: 00734877, 02128230, 00869232, 00871013).

Adverse Metaphase Cytogenetics Can Be Overcome by Adding Bortezomib and Thalidomide to Fractionated Melphalan Transplants.

Purpose: To determine whether a reduction in the intensity of Total Therapy (TT) reduces toxicity and maintains efficacy.Experimental Design: A total of 289 patients with gene expression profiling (GEP70)-defined low-risk multiple myeloma were randomized between a standard arm (TT4-S) and a light arm (TT4-L). TT4-L employed one instead of two inductions and consolidations. To compensate for potential loss of efficacy of TT4-L, bortezomib and thalidomide were added to fractionated melphalan 50 mg/m2/d for 4 days.Results: Grade ≥3 toxicities and treatment-related mortalities were not reduced in TT4-L. Complete response (CR) rates were virtually identical (P = 0.2; TT4-S, 59%; TT4-L, 61% at 2 years), although CR duration was superior with TT4-S (P = 0.05; TT4-S, 87%; TT4-L, 81% at 2 years). With a median follow-up of 4.5 years, there was no difference in overall survival (OS) and progression-free survival (PFS). Whereas metaphase cytogenetic abnormalities (CAs) tended to be an adverse feature in TT4-S, as with predecessor TT trials, the reverse applied to TT4-L. Employing historical TT3a as training and TT3b as test set, 51 gene probes (GEP51) significantly differentiated the presence and absence of CA (q < 0.0001), seven of which function in DNA replication, recombination, and repair. Applying the GEP51 model to clinical outcomes, OS and PFS were significantly inferior with GEP51/CA in TT4-S; such a difference was not observed in TT4-L.Conclusions: We identified a prognostic CA-linked GEP51 signature, the adversity of which could be overcome by potentially synergizing anti-multiple myeloma effects of melphalan and bortezomib. These exploratory findings require confirmation in a prospective randomized trial. Clin Cancer Res; 23(11); 2665-72. ©2016 AACR.

Assessment of Total Lesion Glycolysis by 18F FDG PET/CT Significantly Improves Prognostic Value of GEP and ISS in Myeloma.

Purpose: Fluorine-18 fluorodeoxyglucose positron emission tomography with CT attenuation correction (18F-FDG PET/CT) is useful in the detection and enumeration of focal lesions and in semiquantitative characterization of metabolic activity (glycolytic phenotype) by calculation of glucose uptake. Total lesion glycolysis (TLG) and metabolic tumor volume (MTV) have the potential to improve the value of this approach and enhance the prognostic value of disease burden measures. This study aims to determine whether TLG and MTV are associated with progression-free survival (PFS) and overall survival (OS), and whether they improve risk assessments such as International Staging System (ISS) stage and GEP70 risk.Experimental Design: 192 patients underwent whole body PET/CT in the Total Therapy 3A (TT3A) trial and were evaluated using three-dimensional region-of-interest analysis with TLG, MTV, and standard measurement parameters derived for all focal lesions with peak SUV above the background red marrow signal.Results: In multivariate analysis, baseline TLG > 620 g and MTV > 210 cm3 remained a significant factor of poor PFS and OS after adjusting for baseline myeloma variables. Combined with the GEP70 risk score, TLG > 205 g identifies a high-risk-behaving subgroup with poor expected survival. In addition, TLG > 205 g accurately divides ISS stage II patients into two subgroups with similar outcomes to ISS stage I and ISS stage III, respectively.Conclusions: TLG and MTV have significant survival implications at baseline and offer a more precise quantitation of the glycolytic phenotype of active disease. These measures can be assessed more readily than before using FDA-approved software and should be standardized and incorporated into clinical trials moving forward. Clin Cancer Res; 23(8); 1981-7. ©2016 AACR.

Four genes predict high risk of progression from smoldering to symptomatic multiple myeloma (SWOG S0120).

Multiple myeloma is preceded by an asymptomatic phase, comprising monoclonal gammopathy of uncertain significance and smoldering myeloma. Compared to the former, smoldering myeloma has a higher and non-uniform rate of progression to clinical myeloma, reflecting a subset of patients with higher risk. We evaluated the gene expression profile of smoldering myeloma plasma cells among 105 patients enrolled in a prospective observational trial at our institution, with a view to identifying a high-risk signature. Baseline clinical, bone marrow, cytogenetic and radiologic data were evaluated for their potential to predict time to therapy for symptomatic myeloma. A gene signature derived from four genes, at an optimal binary cut-point of 9.28, identified 14 patients (13%) with a 2-year therapy risk of 85.7%. Conversely, a low four-gene score (< 9.28) combined with baseline monoclonal protein < 3 g/dL and albumin ≥ 3.5 g/dL identified 61 patients with low-risk smoldering myeloma with a 5.0% chance of progression at 2 years. The top 40 probe sets showed concordance with indices of chromosome instability. These data demonstrate high discriminatory power of a gene-based assay and suggest a role for dysregulation of mitotic checkpoints in the context of genomic instability as a hallmark of high-risk smoldering myeloma.

Fulminant onset of acute leukemia from normal hematopoiesis within 3 months of follow up for multiple myeloma treated with total therapy protocols.

Assiduous surveillance for genetic aberrations is necessary in patients on cytotoxic therapies to detect therapy-related myeloid neoplasms (t-MN). Current modalities include metaphase cytogenetics and FISH. Since t-MN may develop abruptly in cytogenetically normal patients, a discussion exploring additional methods such as SNP-array and targeted-deep-sequencing to detect subchromosomal abnormalities is needed.

Clinical, genomic, and imaging predictors of myeloma progression from asymptomatic monoclonal gammopathies (SWOG S0120).

All cases of clinical myeloma (CMM) are preceded by an asymptomatic monoclonal gammopathy (AMG), classified as either monoclonal gammopathy of undetermined significance (MGUS) or asymptomatic multiple myeloma (AMM). We analyzed data from AMG patients (n = 331) enrolled in a prospective, observational clinical trial (S0120). Baseline data from clinical variables, gene expression profiles (GEP) of purified tumor cells, and findings of magnetic resonance imaging (MRI) were correlated with the risk of progression to CMM requiring therapy. GEP of purified tumor cells revealed that all molecular subtypes of CMM are also represented in the AMG phase. An increased risk score (>-0.26) (based on a 70-gene signature, GEP70) was an independent predictor of the risk of progression to CMM. Combination of elevated serum free light chain, M-spike, and GEP70 risk score identified a subset with high risk (67% at 2 years) of progression to CMM requiring therapy. Importantly, absence of these factors in AMM patients predicted low risk similar to MGUS. Detection of multiple (>1) focal lesions by MRI also conferred an increased risk of progression. These data demonstrate that signatures associated with high-risk CMM impact disease risk and support inclusion of genomic analysis in the clinical management of AMGs.

Metronomic therapy is an effective salvage treatment for heavily pre-treated relapsed/refractory multiple myeloma.

Relapsed/refractory multiple myeloma represents a major challenge in multiple myeloma therapy. For patients with relapsed/refractory multiple myeloma, we developed a treatment schema of metronomically scheduled drug therapy. We identified 186 patients who had been treated with metronomic therapy between March 2004 and January 2012 with a median follow up of 24.2 months. Median age was 61 years (range 36-83). Median number of prior therapies was 14 (range 1-51). Median number of completed metronomic therapy cycles was 1 (range 1-5), while 45 of 186 (25%) received 2 or more cycles. Responses included complete remission in 11 of 186 patients (6%), very good partial remission in 12 of 186 (7%), partial remission in 65 of 179 (36%), and minimal response in 29 of 186 (16%), for an overall response rate of 63% (117 of 186). Median overall survival and progression-free survival were 11.2 and 3.6 months, respectively. Hematologic toxicity grading was problematic as 146 of 186 (78%) of patients presented with at least grade 2 thrombocytopenia within 90 days prior to starting metronomic therapy. Grade 4 leukopenia, anemia, and/or thrombocytopenia following metronomic therapy occurred in 108 of 186 (58%), 12 of 186 (6%), and 147 of 186 (79%) patients, respectively. Incidence of grade 3-4 neutropenic fever was 4 of 186 (2%). Most patients (177 of 186, 95%) were treated in an outpatient unit and secondary admissions due to regimen-related toxicity occurred in 37 of 186 (20%). Treatment-related mortality was evident in 2 of 186 (1%). In conclusion, metronomic therapy is an effective late salvage treatment in relapsed/refractory multiple myeloma, with a high overall response rate and a favorable toxicity profile.

Risk factors for MDS and acute leukemia following total therapy 2 and 3 for multiple myeloma.

Lenalidomide has been linked to myelodysplastic syndrome (MDS) after autotransplants for myeloma. Total therapy trials (TT; TT2(-/+) thalidomide) and TT3 (TT3a with bortezomib, thalidomide; TT3b with additional lenalidomide) offered the opportunity to examine the contribution of these immune-modulatory agents to MDS-associated cytogenetic abnormalities (MDS-CA) and clinical MDS or acute leukemia ("clinical MDS/AL"). Of 1080 patients with serial cytogenetic studies, MDS-CA occurred in 11% and clinical MDS/AL in 3%. Risk features of MDS-CA included TT3b, age ≥65 years, male gender, levels of ß-2-microglobulin >5.5 mg/L, and multiple myeloma relapse. Clinical MDS/AL occurred less frequently in the control arm of TT2 and more often with TT3a and TT3b. Since MDS-CA often antedated clinical disease, periodic cytogenetic monitoring is recommended. Larger CD34 quantities should be collected upfront as the risk of MDS could be reduced by applying higher CD34 doses with transplant. Thus, treatment, host, and myeloma features could be linked to MDS development after therapy for this malignancy. This trial was registered at www.clinicaltrials.gov: TT3A: NCT00081939, TT3B: NCT00572169.

Prognostic implications of serial 18-fluoro-deoxyglucose emission tomography in multiple myeloma treated with total therapy 3.

Prognostic implications of 3 imaging tools, metastatic bone survey, magnetic resonance imaging, and positron emission tomography (PET), were evaluated in 2 consecutive Total Therapy 3 trials for newly diagnosed myeloma. Data including PET at baseline and on day 7 of induction as well as standard prognostic factors were available in 302 patients of whom 277 also had gene expression profiling (GEP)-derived risk information. According to multivariate analysis, more than 3 focal lesions on day 7 imparted inferior overall survival and progression-free survival, overall and in the subset with GEP-risk data. GEP high-risk designation retained independent significance for all 3 end points examined. Thus, the presence of > 3 focal lesions on day 7 PET follow-up may be exploited toward early therapy change, especially for the 15% of patients with GEP-defined high-risk disease with a median overall survival expectation of 2 years. This trial was registered at www.clinicaltrials.gov as #NCT00081939 and # NCT00572169.

Thalidomide in total therapy 2 overcomes inferior prognosis of myeloma with low expression of the glucocorticoid receptor gene NR3C1.

PURPOSE: Because dexamethasone remains a key component of myeloma therapy, we wished to examine the impact of baseline and relapse expression levels of the glucocorticoid receptor gene NR3C1 on survival outcomes in the context of treatment with or without thalidomide. EXPERIMENTAL DESIGN: We investigated the clinical impact of gene expression profiling (GEP)-derived expression levels of NR3C1 in 351 patients with GEP data available at baseline and in 130 with data available at relapse, among 668 subjects accrued to total therapy 2 (TT2). RESULTS: Low NR3C1 expression levels had a negative impact on progression-free survival (PFS; HR, 1.47; P = 0.030) and overall survival (OS; HR, 1.90; P = 0.002) in the no-thalidomide arm. Conversely, there was a significant clinical benefit of thalidomide for patients with low receptor levels (OS: HR, 0.54; P = 0.015; PFS: HR, 0.54; P = 0.004), mediated most likely by thalidomide's upregulation of NR3C1. In the context of both baseline and relapse parameters, post-relapse survival (PRS) was adversely affected by low NR3C1 levels at relapse in a multivariate analysis (HR, 2.61; P = 0.012). CONCLUSION: These findings justify the inclusion of NR3C1 expression data in the work-up of patients with myeloma as it can significantly influence the choice of therapy and, ultimately, OS. The identification of an interaction term between thalidomide and NR3C1 underscores the importance of pharmacogenomic studies in the systematic study of new drugs.

Second malignancies in total therapy 2 and 3 for newly diagnosed multiple myeloma: influence of thalidomide and lenalidomide during maintenance.

Thalidomide and lenalidomide constitute an important part of effective myeloma therapy. Recent data from the Intergroup Francophone du Myélome, Cancer and Leukemia Group B, and Gruppo Italiano Malattie Ematologiche dell Adulto MM-015 trials suggest that lenalidomide maintenance therapy is associated with a higher incidence of second primary malignancies (SPMs), including both hematologic and solid malignancies. In the present study, we analyzed data from the Total Therapy 2 (TT2) trial, along with the 2 Total Therapy 3 (TT3) trials. TT2 patients were assigned randomly to either a control group (no thalidomide) or to the experimental group (thalidomide during induction, between transplantations, and during consolidation and maintenance). The 2 TT3 trials used thalidomide and bortezomib during induction, before and in consolidation after tandem melphalan-based transplantation; TT3A applied VTD (bortezomib, thalidomide, dexamethasone) in the first year of maintenance and TD for 2 more years, whereas TT3B used VRD (bortezomib, lenalidomide, dexamethasone) maintenance for 3 years. The cumulative incidence of SPMs did not differ significantly among the TT trial components when measured from enrollment (P = .78) or from initiation of maintenance (P = .82). However, a pairwise comparison of the TT2 arms suggested a lower incidence of hematologic SPMs in the thalidomide maintenance arm (hazard ratio = 0.38; P = .09). These trials are registered at www.clinicaltrials.gov as NCT00573391 (TT2), NCT00081939 (TT3A), and NCT00572169 (TT3B).

Total Therapy 3 for multiple myeloma: prognostic implications of cumulative dosing and premature discontinuation of VTD maintenance components, bortezomib, thalidomide, and dexamethasone, relevant to all phases of therapy.

The impact of cumulative dosing and premature drug discontinuation (PMDD) of bortezomib (V), thalidomide (T), and dexamethasone (D) on overall survival (OS), event-free survival (EFS), time to next therapy, and post-relapse survival in Total Therapy 3 were examined, using time-dependent methodology, relevant to induction, peritransplantation, consolidation, and maintenance phases. Univariately, OS and EFS were longer in case higher doses were used of all agents during induction, consolidation (except T), and maintenance (except V and T). The favorable OS and EFS impact of D induction dosing provided the rationale for examining the expression of glucocorticoid receptor NR3C1, top-tertile levels of which significantly prolonged OS and EFS and rendered outcomes independent of D and T dosing, whereas T and D, but not V, dosing was critical to outcome improvement in the bottom-tertile NR3C1 setting. PMDD of V was an independent highly adverse feature for OS (hazard ratio = 6.44; P < .001), whereas PMDD of both T and D independently imparted shorter time to next therapy. The absence of adverse effects on postrelapse survival of dosing of any VTD components and indeed a benefit from V supports the use up-front of all active agents in a dose-dense and dose-intense fashion, as practiced in Total Therapy 3, toward maximizing myeloma survival.

Reiterative survival analyses of total therapy 2 for multiple myeloma elucidate follow-up time dependency of prognostic variables and treatment arms.

PURPOSE: In Total Therapy 2, after randomly assigning 323 patients with myeloma to thalidomide and 345 to a control arm, no difference was observed in overall survival, with a median follow-up of 42 months, although at 72 months, survival was superior on the thalidomide arm in the one third exhibiting cytogenetic abnormalities (CA). After further follow-up of 87 months, we examined, in reiterative analyses, the effect of increasing time intervals on clinical outcomes relevant to baseline prognostic variables and treatment randomization. PATIENTS AND METHODS: We investigated clinical trial end points as a function of increasing time intervals from protocol enrollment to determine consistencies of results by treatment and prognostic variables. RESULTS: The complete congruence of serial survival plots for both study arms combined attested to stable patient characteristics over the time of accrual and the quality of follow-up management. Presence of CA was associated with consistently inferior survival curves from year 3 onward. Although 80% of patients randomly assigned to thalidomide discontinued study drug after 2 years because of toxicity, its clinical benefit did not reach statistical significance until year 10. The relative ranking order in multivariate models of prognostic factors remained stable over time. Decline in initially high hazard ratio values of gene array-defined high risk is consistent with an initial crisis phase that is time limited. CONCLUSION: Reporting potentially time-sensitive features as a part of clinical trial results will enable the critical reader to judge the robustness of prognostic factors and the time sensitivity of outcome predictors, with important implications for future trial designs.

Gene expression profiling of plasma cells at myeloma relapse from tandem transplantation trial Total Therapy 2 predicts subsequent survival.

We report on prognostic implications for post-relapse survival (PRS) of a gene expression profiling (GEP)-defined risk score at relapse available in 120 myeloma patients previously enrolled in tandem transplantation trial Total Therapy 2. Among the 71 patients with additional GEP baseline information, 3-year PRS was 71% in 40 patients with low risk present both at baseline and relapse contrasting with only 17% in 28 patients with high risk at relapse, 12 of whom with baseline low-risk status fared better than the remainder (P = .08). On multivariate analysis of relapse parameters available in 104 patients, high risk conferred short PRS (hazard ratio = 4.00, P < .001, R(2) = 33%), whereas relapse hyperdiploidy predicted long PRS (hazard ratio = 0.37, P = .022, cumulative R(2) = 41%). In case the initial partial response lasted less than 2 years, relapse low-risk identified 26 patients with superior 3-year PRS of 61% versus 9% among 32 with relapse high-risk (P < .001). Based on its PRS predictive power, GEP analysis should be an integral part of new agent trials in search of better therapy for high-risk myeloma.

Seven-year median time to progression with thalidomide for smoldering myeloma: partial response identifies subset requiring earlier salvage therapy for symptomatic disease.

Smoldering multiple myeloma (SMM) is usually followed expectantly without therapy. We conducted a phase 2 trial in 76 eligible patients with SMM, combining thalidomide (THAL, 200 mg/d) with monthly pamidronate. In the first 2 years, THAL dose reduction was required in 86% and drug was discontinued in 50%. Within 4 years, 63% improved, including 25% qualifying for partial response (PR); by then, 34 patients had progressed and 17 required salvage therapy. Unexpectedly, attaining PR status was associated with a shorter time to salvage therapy for disease progression (P < .001), perhaps reflecting greater drug sensitivity of more aggressive disease. Low beta-2-microglobulin levels less than 2 mg/L were independently associated with superior overall and event-free survival. Four-year survival and event-free survival estimates of 91% and 60%, respectively, together with a median postsalvage therapy survival of more than 5 years justify the conduct of a prospective randomized clinical trial to determine the clinical value of preemptive therapy in SMM. Trial registered at http://www.clinicaltrials.gov under identifier NCT00083382.

Thalidomide arm of Total Therapy 2 improves complete remission duration and survival in myeloma patients with metaphase cytogenetic abnormalities.

Total Therapy 2 examined the clinical benefit of adding thalidomide up-front to a tandem transplant regimen for newly diagnosed patients with multiple myeloma. When initially reported with a median follow-up of 42 months, complete response rate and event-free survival were superior among the 323 patients randomized to thalidomide, whereas overall survival was indistinguishable from that of the 345 patients treated on the control arm. With further follow-up currently at a median of 72 months, survival plots segregated 5 years after initiation of therapy in favor of thalidomide (P = .09), reaching statistical significance for the one third of patients exhibiting cytogenetic abnormalities (CAs; P = .02), a well-recognized adverse prognostic feature. The duration of complete remission was also superior in the cohort presenting with CAs such that, at 7 years from onset of complete remission, 45% remained relapse-free as opposed to 20% on the control arm (P = .05). These observations were confirmed when examined by multivariate analysis demonstrating that thalidomide reduced the hazard of death by 41% among patients with CA-positive disease (P = .008). Because two thirds of patients without CAs have remained alive at 7 years, the presently emerging separation in favor of thalidomide may eventually reach statistical significance as well.

Incorporating bortezomib into upfront treatment for multiple myeloma: early results of total therapy 3.

Total therapy 3 incorporated bortezomib into a melphalan-based tandem transplant regimen for 303 newly diagnosed patients with myeloma. Induction chemotherapy prior to and consolidation chemotherapy after transplants each consisted of two cycles of VTD-PACE (bortezomib, thalidomide, dexamethasone and 4-d continuous infusions of cis-platin, doxorubicin, cyclophosphamide, etoposide); 3-year maintenance comprised monthly cycles of VTD in the first and TD in the remaining years. The median age was 59 years (age >64 years, 28%). A minimum of 20 x 10(6) CD34 cells/kg was collected in 87% of patients; 83% completed both transplants, and only 5% suffered a treatment-related death. At 24 months, 83% had achieved near-complete remission, which was sustained in 88% at 2 years from its onset. With a median follow-up of 20 months, 2-year estimates of event-free and overall survival were 84% and 86% respectively. The 44 patients who experienced an event more often had a high-risk gene array profile, cytogenetic abnormalities and indicators of high lactate dehydrogenase, beta-2-microglobulin, creatinine and International Staging System stage. Toxicities of grade > 2 included thrombo-embolic events in 27% and peripheral neuropathy in 12%. Results of this phase-2 study demonstrated that bortezomib could be safely combined with multi-agent chemotherapy, effecting near-complete remission status and 2-year survival rates in more than 80% of patients.

Linoleic acid metabolites act to increase contractility in isolated rat heart.

Previous in vivo studies in dogs suggest that the 9,10-monoepoxide of linoleic acid (9,10-cis-epoxyoctadecenoic acid [9,10-EOA]) has toxic cardiovascular effects that result in death at higher doses. More recent work with rabbit renal proximal tubule cells suggests that the 12,13-metabolites of linoleic acid are more toxic than the 9,10-isomers. Thus, in the current study, we tested the hypothesis that 12,13-EOA and 12,13-dihydroxyoctadecadienoic acid (12,13-DHOA) have direct adverse effects on the heart. Langendorff-perfused rat hearts were exposed to 30 microM linoleic acid, 30 microM 12,13-EOA, or 30 microM 12,13-DHOA for 60 min followed by a 30-min recovery period. As indicated by peak left intraventricular pressure and/or +dP/dt(max), all three of the agents elicited moderate increases in contractile function that peaked within 10 20 min. The effects of linoleic acid and 12,13-EOA returned to control values during the remainder of the 60-min exposure, whereas the positive inotropic response to 12,13-DHOA was maintained until washout. Sustained arrhythmias and negative inotropic actions were not observed with any of the three compounds. Subsequently, the monoepoxides were infused into conscious rats (35 mg/kg/h) while blood pressure, heart rate, and EKG were monitored for 24 h using biotelemetry techniques. The only effect observed was a slight decline in blood pressure. Thus, current data suggest that linoleic acid and its oxidative metabolites do not have direct cardiotoxic effects during acute exposure.