Clinical validation of a tissue-agnostic genome-wide methylome enrichment molecular residual disease assay for head and neck malignancies.

BACKGROUND: Outcomes for patients with locally advanced head and neck cancer (HNC) treated with curative intent remain disappointing, with 5-year survival rates at 50%. Most recurrences occur within the first 2 years after treatment, providing a window of opportunity to identify patients with molecular residual disease (MRD). A tissue-agnostic test for MRD detection in patients with human papillomavirus (HPV) positive and negative HNC, where tissue is often scarce, is needed. PATIENTS AND METHODS: Patients with stage I-IVB HNC, including patients positive and negative for HPV, were enrolled and peripheral blood plasma was collected longitudinally at diagnosis and ∼3, 12, and 24 months after curative intent treatment. The full cohort includes 325 patients with 1155 samples. Samples were split into distinct sets to train and validate a classifier capable of identifying MRD using a tissue-agnostic genome-wide methylome enrichment platform. The primary endpoint was recurrence-free survival (RFS). RESULTS: With a median follow-up of 60 months, patients in the blinded validation set with MRD positivity experienced significantly worse RFS with a hazard ratio (HR) of 35.7 [95% confidence interval (CI) 10.8-117.8; P < 0.0001]. For patients with HPV negativity, HR was 42.3 (95% CI 9.8-182.3; P < 0.0001); for patients with HPV-positive oropharyngeal cancer, HR was 24.1 (95% CI 3.0-196.8; P < 0.0001). Moreover, the lead time between MRD positivity and clinical recurrence was up to 14.9 months, with a mean lead time of 4.1 months. Surveillance sensitivity was 91% (95% CI 77% to 97%) and specificity was 88% (95% CI 80% to 93%). CONCLUSIONS: Here we validate the clinical performance characteristics of a tissue-agnostic genome-wide methylome enrichment assay for MRD detection in patients with HNC. The MRD detection test showed high sensitivity for identifying recurrence at high specificity across different anatomical sites, HPV status, and treatment regimens, highlighting the broad applicability for MRD detection in patients with HNC.

Genome-wide association study of beef bull semen attributes.

BACKGROUND: Cattle production is dependent upon fertility because it results in producing offspring to offset production costs. A number of semen attributes are believed to affect fertility and are frequently measured as part of routine breeding soundness exams or semen collection procedures. The objective of this study was to perform a single-step genome-wide association study (ssGWAS) for beef bull semen attributes. Beef bull fertility phenotypes including volume (VOL), concentration (CONC), number of spermatozoa (NSP), initial motility (IMot), post-thaw motility (PTMot), three-hour post-thaw motility (3HRPTMot), percentage of normal spermatozoa (%NORM), primary abnormalities (PRIM), and secondary abnormalities (SEC) were obtained from two artificial insemination (AI) centers. A total of 1819 Angus bulls with 50,624 collection records were used for ssGWAS. A five-generation pedigree was obtained from the American Angus Association and consisted of 6521 sires and 17,136 dams. Genotypes on 1163 bulls were also obtained from the American Angus Association and utilized in ssGWAS. RESULTS: A multi-trait animal model was used for the estimation of single nucleotide polymorphism (SNP) effects. Significant SNP were those with a -log10 P-value threshold greater than 4.0. Volume, CONC, NSP, IMot, PTMot, 3HRPTMot, %NORM, PRIM, and SEC have five, three, six, seven, two, six, six, and two genome-wide significant SNP, respectively. CONCLUSIONS: Several significant SNP were determined to be near or within quantitative trait loci (QTL) associated with beef bull semen attributes. In addition, genes associated with fertility were found to contain or be near the significant SNP found in the study. The results indicate there are regions of the genome that impact fertility, proving inclusion of genomic information into genetic evaluation should be advantageous for genetic improvement of male fertility traits.

Impact of homologous recombination deficiency biomarkers on outcomes in patients with triple-negative breast cancer treated with adjuvant doxorubicin and cyclophosphamide (SWOG S9313).

Background: Homologous recombination deficiency (HRD)-causing alterations have been reported in triple-negative breast cancer (TNBC). We hypothesized that TNBCs with HRD alterations might be more sensitive to anthracycline plus cyclophosphamide-based chemotherapy and report on HRD status and BRCA1 promoter methylation (PM) as prognostic markers in TNBC patients treated with adjuvant doxorubicin (A) and cyclophosphamide (C) in SWOG9313. Patients and methods: In total, 425 TNBC patients were identified from S9313. HRD score, tumor BRCA1/2 sequencing, and BRCA1 PM were carried out on DNA isolated from formalin-fixed paraffin-embedded tissue. Positive HRD status was defined as either a deleterious tumor BRCA1/2 (tBRCA) mutation or a pre-defined HRD score ≥42. Markers were tested for prognostic value on disease-free survival (DFS) and overall survival (OS) using Cox regression models adjusted for treatment assignment and nodal status. Results: HRD status was determined in 89% (379/425) of cases. Of these, 67% were HRD positive (27% with tBRCA mutation, 40% tBRCA-negative but HRD score ≥42). HRD-positive status was associated with a better DFS [hazard ratio (HR) 0.72; 95% confidence interval (CI) 0.51-1.00; P = 0.049] and non-significant trend toward better OS (HR = 0.71; 95% CI 0.48-1.03; P = 0.073). High HRD score (≥42) in tBRCA-negative patients (n = 274) was also associated with better DFS (HR = 0.64; 95% CI 0.43-0.94; P = 0.023) and OS (HR = 0.65; 95% CI 0.42-1.00; P = 0.049). BRCA1 PM was evaluated successfully in 82% (348/425) and detected in 32% of cases. The DFS HR for BRCA1 PM was similar to that for HRD but did not reach statistical significance (HR = 0.79; 95% CI 0.54-1.17; P = 0.25). Conclusions: HRD positivity was observed in two-thirds of TNBC patients receiving adjuvant AC and was associated with better DFS. HRD status may identify TNBC patients who receive greater benefit from AC-based chemotherapy and should be evaluated further in prospective studies. Clinical Trials Number: Int0137 (The trial pre-dates Clinicaltrial.Gov website establishment).

Survival analysis of carboplatin added to an anthracycline/taxane-based neoadjuvant chemotherapy and HRD score as predictor of response-final results from GeparSixto.

Background: In the neoadjuvant GeparSixto study, adding carboplatin to taxane- and anthracycline-based chemotherapy improved pathological complete response (pCR) rates in patients with triple-negative breast cancer (TNBC). Here, we present survival data and the potential prognostic and predictive role of homologous recombination deficiency (HRD). Patients and methods: Patients were randomized to paclitaxel plus nonpegylated liposomal doxorubicin (Myocet®) (PM) or PM plus carboplatin (PMCb). The secondary study end points disease-free survival (DFS) and overall survival (OS) were analyzed. Median follow-up was 47.3 months. HRD was among the exploratory analyses in GeparSixto and was successfully measured in formalin-fixed, paraffin-embedded tumor samples of 193/315 (61.3%) participants with TNBC. Homologous recombination (HR) deficiency was defined as HRD score ≥42 and/or presence of tumor BRCA mutations (tmBRCA). Results: A significantly better DFS (hazard ratio 0.56, 95% CI 0.34-0.93; P = 0.022) was observed in patients with TNBC when treated with PMCb. The improvement of OS with PMCb was not statistically significant. Additional carboplatin did not improve DFS or OS in patients with HER2-positive tumors. HR deficiency was detected in 136 (70.5%) of 193 triple-negative tumors, of which 82 (60.3%) showed high HRD score without tmBRCA. HR deficiency independently predicted pCR (ypT0 ypN0) [odds ratio (OR) 2.60, 95% CI 1.26-5.37, P = 0.008]. Adding carboplatin to PM significantly increased the pCR rate from 33.9% to 63.5% in HR deficient tumors (P = 0.001), but only marginally in HR nondeficient tumors (from 20.0% to 29.6%, P = 0.540; test for interaction P = 0.327). pCR rates with carboplatin were also higher (63.2%) than without carboplatin (31.7%; OR 3.69, 1.46-9.37, P = 0.005) in patients with high HRD score but no tmBRCA. DFS rates were improved with addition of carboplatin, both in HR nondeficient (hazard ratio 0.44, 0.17-1.17, P = 0.086) and HR deficient tumors (hazard ratio 0.49, 0.23-1.04, P = 0.059). Conclusions: The addition of carboplatin to neoadjuvant PM improved DFS significantly in TNBC. Long-term survival analyses support the neoadjuvant use of carboplatin in TNBC. HR deficiency in TNBC and HRD score in non-tmBRCA TNBC are predictors of response. HRD does not predict for carboplatin benefit.

Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation.

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

Cell line dependence of Bcl-2-induced alteration of glutathione handling.

Bcl-2 has been associated with both oxidative and antioxidative effects in vivo. Moreover, despite evidence that Bcl-2 is antiapoptotic by virtue of its effect on reactive oxygen species and their scavengers, Bcl-2 exerts its antiapoptotic effects even under anaerobic conditions. The reasons for the variable relationship between Bcl-2 and reactive oxygen species are not clear. The present studies demonstrate that the impact of Bcl-2 on glutathione (GSH) metabolism is cell line-dependent. Bcl-2 overproduction in PC12 cells is associated with increased functional thiol reserves, increased reductive activation of chemotherapeutic prodrugs, and GSH accumulation after treatment with N-acetylcysteine. In contrast, Bcl-2-overproducing MCF-7 breast cancer cells demonstrate neither altered GSH handling nor potentiation of chemotherapeutic prodrug reduction. These findings indicate that the effects of Bcl-2 on GSH handling are millieu-dependent. This could account for the variable effects of Bcl-2 in in vivo systems. Furthermore, since our previous studies have demonstrated that reduction-dependent prodrugs may be useful chemotherapeutic agents against tumors that demonstrate altered GSH handling, screening in vitro for alteration of GSH handling may predict responsiveness of such tumors to these reduction-dependent agents.

Opioid and opiate immunoregulatory processes.

The discovery of the ability of the nervous system to communicate through "public" circuits with other systems of the body is attributed to Ernst and Berta Scharrer, who described the neurosecretory process in 1928. Indeed, the immune system has been identified as another important neuroendocrine target tissue. Opioid peptides are involved in this communication (i.e., neuroimmune) and with that of autoimmunoregulation (communication between immunocytes). The significance of opioid neuropeptide involvement with the immune system is ascertained from the presence of novel delta, mu, and kappa receptors on inflammatory cells that result in modulation of cellular activity after activation, as well as the presence of specific enzymatic degradation and regulation processes. In contrast to the relatively uniform antinociceptive action of opiate and opioid signal molecules in neural tissues, the presence of naturally occurring morphine in plasma and a novel mu3, opiate-specific receptor on inflammatory cells adds to the growing knowledge that opioid and opiate signal molecules may have antagonistic actions in select tissues. In examining various disorders (e.g., human immunodeficiency virus, substance abuse, parasitism, and the diffuse inflammatory response associated with surgery) evidence has also been found for the involvement of opiate/opioid signaling in prominent mechanisms. In addition, the presence of similar mechanisms in man and organisms 500 million years divergent in evolution bespeaks the importance of this family of signal molecules. The present review provides an overview of recent advances in the field of opiate and opioid immunoregulatory processes and speculates as to their significance in diverse biological systems.

Survival, disease-free survival and adverse effects of conditioning for allogeneic bone marrow transplantation with busulfan/cyclophosphamide vs total body irradiation: a meta-analysis.

Randomized, prospective studies comparing BUCY to TBI conditioning regimens for allogeneic bone marrow transplantation have yielded conflicting results. We investigated the overall survival, the disease-free survival and the toxicities of BUCY vs TBI-based regimens by conducting a meta-analysis of all published, randomized, prospective trials comparing these regimens. Five studies were analyzed. We evaluated six endpoints: survival, disease-free survival, veno-occlusive disease (VOD) of the liver, acute GVHD, chronic GVHD, and interstitial pneumonitis. We combined individual study results using a random effects model. Survival and disease-free survival were better with TBI-based regimens than with BUCY, but these differences were not statistically significant (survival odds ratio 1.4, 95% confidence interval 0.9-2.2, P = 0.09; disease-free survival odds ratio 1.2, 95% confidence interval 0.7-2.1, P = 0.44). A power analysis indicated that BUCY was unlikely to have a clinically relevant survival or disease-free survival advantage. The power analysis could not exclude the possibility of such an advantage for TBI-based regimens. A significantly greater incidence of VOD occurred with BUCY (odds ratio 2.5, 95% confidence interval 1.2-5.2, P = 0.02). For the other side-effects, there were no significant differences. We concluded that TBI-based regimens cause less VOD than BUCY and are at least as good for survival and disease-free survival.

Meta-analysis of adjuvant cyclophosphamide/methotrexate/5-fluorouracil chemotherapy in postmenopausal women with estrogen receptor-positive, node-positive breast cancer.

Conflicting results have been published regarding the efficacy of adjuvant cyclophosphamide/methotrexate/5-fluorouracil (CMF)-type chemotherapy in postmenopausal, estrogen receptor (ER)-positive women. The Oxford overview suggests real but limited benefit of any chemotherapy in this group of patients but avoids analyzing smaller subsets. We wished to better quantitate the benefit of adding CMF to tamoxifen in postmenopausal ER-positive women with tumor involvement of axillary lymph nodes. Six randomized studies comparing CMF plus tamoxifen to tomoxifen alone in postmenopausal, ER-positive, node-positive women have been published since 1992. They include 2368 patients. We performed a meta-analysis of 6 endpoints: survival, disease-free survival, locoregional recurrence, distant recurrence, contralateral breast recurrence, and thromboembolic complications. There was a statistically significant increase in disease-free survival from the addition of CMF-type chemotherapy to tamoxifen in this population; the absolute risk of relapse was reduced by 5.5% at 5 years. Effects of locoregional recurrence were greater than those on overall recurrence. No significant survival benefit was observed.

Fiberoptic examination of the inferior vena cava during circulatory arrest for complete removal of renal cell carcinoma thrombus.

Renal cell carcinoma is known to invade the inferior vena cava and may extend its entire length. Profound hypothermic circulatory arrest has been demonstrated to be a very effective technique to facilitate removal of tumor thrombus from the cava while limiting the amount of blood loss. We describe an innovative method of ensuring complete removal of tumor thrombus from the retrohepatic cava with a fiberoptic bronchoscope introduced through the right atrium during profound hypothermic circulatory arrest. Fiberoptic examination of the cava and hepatic vein orifices under these circumstances will prevent incomplete removal of tumor.

Autologous whole plasma fibrin gel. Intraoperative procurement.

Fibrin glue is a relatively recent addition to the armamentarium of hemostatic agents for surgical use. Its efficacy has been repeatedly demonstrated in almost all surgical disciplines and subspecialties. Its use in the United States has been limited because of the risk of viral transmission associated with the use of human plasma. Previous authors have described techniques that limit this risk, but they are frequently impractical, expensive, or cumbersome. We describe the use of patients' own fresh plasma to make fibrin gel at the operative field. It provided hemostasis at least as good as that from heterologous plasma glue in 40 cardiac surgical patients. Autologous whole plasma fibrin gel is inexpensive and safe and eliminates the risk of viral transmission associated with glue derived from heterologous donor plasma.

Myocardial revascularization with the lateral costal artery.

The lateral costal artery is a branch of the internal mammary artery that occurs in 27% of cadaver series. The similarities to the internal mammary artery and its easy accessibility would suggest its use as a bypass conduit for coronary revascularization. We describe the harvesting and utilization of this artery as an in situ coronary bypass graft, and we have initiated a study examining the exact frequency of this anomalous vessel. The availability of the lateral costal artery either unilaterally or bilaterally adds another dimension to the concept of complete arterial conduit myocardial revascularization.

Cryopreserved veins in myocardial revascularization: possible mechanism for their increased failure.

BACKGROUND: Cryopreserved veins are used as conduits for myocardial revascularization. However, a high failure rate associated with their use has been reported anecdotally. METHODS: To find an explanation for the poor performance of cryopreserved vein grafts, we conducted a retrospective 5-year study on all patients at a single institution in whom cryopreserved vein grafts were used. We further performed in vitro studies measuring cell adhesion, nitric oxide production, and contractile capacity of saphenous vein, internal thoracic artery, and cryopreserved veins. RESULTS; Forty-one patients were identified in whom one or more cryopreserved veins were used as a last resort. Sixteen had events (death or recatheterization). Seven deaths occurred (17%). Event-free survival was 50% at 12 months. Activated granulocyte/monocyte endothelial adherence could be lowered in internal thoracic arteries and saphenous veins with morphine incubation (50% and 57%, respectively), but not in cryopreserved veins. Simultaneous increases in nitric oxide release were also found in internal thoracic arteries and saphenous veins, but not cryopreserved veins. In addition, cryopreserved veins showed a diminished contractile capacity under experimental conditions. CONCLUSIONS: In this highly select group of patients, cryopreserved veins had a high early failure rate, which may be partially due to the inability of the endothelium to participate in immunovascular processes.

Improved sternal closure using steel bands: early experience with three-year follow-up.

BACKGROUND: Use of stainless steel wires in median sternotomy closure is at times associated with serious complications. In view of this, the efficacy and safety of a stainless steel band designed for fixation and approximation of the sternum in cardiothoracic procedures was evaluated in a prospective, randomized study. METHODS: Forty-eight patients undergoing open heart operations that involved a median sternotomy were studied. Group I (n = 21) was closed with four to six steel bands, and group II (n = 27) with six to eight standard stainless steel wires. The average age of the patients and the risk factors predisposing to dehiscence were similar in both groups. RESULTS: One postoperative death occurred in each group due to cardiac failure. In group I, the mean length of the postoperative hospital stay was 10.2 +/- 1.76 days (+/- 2 standard errors), whereas in group II the mean was 13.9 +/- 3.4 days (+/- 2 standard errors). Banded patients complained less of postoperative pain, although statistical significance was not achieved. No problems arose in either group during the 3-year follow-up. CONCLUSIONS: The steel bands, compared with wires, provided not only effective fixation, but a reduction in both postoperative pain and postoperative hospital stay. The band is now being studied in a larger group of patients to evaluate the incidence and type of complications associated with its use, as well as length of postoperative hospital stay.

Heparin-coated bypass circuits in cardiopulmonary bypass: improved biocompatibility or not.

Heparin bonding to bypass circuits has been found to reduce bleeding complications. Here, this process is reviewed with special attention to markers of inflammation and clinical outcome. Indicators of inflammation (i.e. cytokine levels, elastase and complement components) are decreased when using heparin bonded circuits compared to conventional bypass circuits. The decrease in the levels of these response modifiers appears minimal. Clinical outcomes, other than bleeding complications, have not been studied to any great extent with this technology. These lower levels of the various biological response modifiers are not correlated with lower levels of complications or shorter hospital stays. We conclude based on this data that it is not clear if this decrease translates into a clinical benefit in routine operative cases that require cardiopulmonary bypass.

Aprotinin diminishes inflammatory processes.

Many of the recent reports concerning cytokine levels in cardiopulmonary bypass have documented changes in the levels of these trauma indicators. In the present report, we also document their levels but in the presence of Aprotinin. Aprotinin is a protease inhibitor used not only to diminish bleeding, but also to diminish elements of the diffuse inflammatory response associated with this type of surgery. We report in plasma obtained from 20 patients that initially interleukin-8 (IL-8) levels (53.4 +/- 7 pg/ml) plasma to 185.5 +/- 30 pg/ml) increased 20 min from the start of surgery. This is followed by IL-6 (5.3 +/- 1.1 to 200 +/- 50 pg/ml) peaking 15 h post surgery. These levels return to normal by day 3 postop. IL-1 beta and tumour necrosis factor (TNF) levels remained at baseline for the observation period. Associated with these changes in cytokine levels is the activity state of immunocytes (granulocytes and monocytes) noted by conformational changes obtained from computer-assisted microscopy. The cells exhibited an ameboid conformation and became mobile (67%), peaking at 120 min after surgery began and returned to a more rounded conformation with only 6% exhibiting the ameboid conformation by day three. In in-vitro experiments, where immunocytes not exposed to cardiopulmonary bypass were exposed to plasma obtained from patients having undergone this surgery, their activity level rose to 65%. In the same experiment, when Aprotinin was added to the cell-plasma mixture, the level of activation dramatically dropped to 25%. Thus, aprotinin was found at high doses to lower cytokine and cellular activation associated with the acute inflammatory responses of cardiopulmonary bypass, suggesting that this may be initiated by hyperstimulated immunocytes.

Ventricular reconstruction with vascular prosthesis after aneurysmectomy. Ability to defibrillate and utilize the automatic internal cardioverter defibrillator.

Adjunct surgical procedures for patients with malignant ventricular tachyarrhythmias who require the automatic internal cardioverter defibrillator include aortocoronary artery bypass graft surgery, valvular repair or replacement, subendocardial resection, and aneurysmectomy. Ventricular reconstruction and its compatibility with the AICD are described in a 64-year-old man who required treatment of refractory ventricular tachycardia as well as resection of a large anteroapical aneurysm. Reconstruction using a dacron patch preserved left ventricular geometry but did not adversely affect the ability to defibrillate and allowed successful utilization of the automatic defibrillator.

Emergency coronary revascularization using polytetrafluoroethylene conduits in a patient in cardiogenic shock.

There is growing awareness that a particular subset of patients with coronary artery disease who need surgical revascularization do not have autologous vein or internal mammary artery available or surgically applicable. The polytetrafluoroethylene graft has had limited use in aortocoronary bypass procedures. We describe a case of cardiogenic shock secondary to severe coronary artery disease and aortic stenosis, where the use of this synthetic graft contributed to a successful outcome. Angiographic patency was documented at 3 months follow-up. Our experience, plus a review of the literature, supports the use of polytetrafluoroethylene grafts during aortocoronary artery bypass to salvage infarcting or ischemic myocardium, when traditional autologous bypass conduits are not available or applicable.

Intrapleural instillation of urokinase in the treatment of loculated pleural effusions in children.

The authors report on the use of intrapleural instillation of urokinase in the treatment of loculated pleural effusions in two pediatric patients. Urokinase helps to lyse fibrin by converting plasminogen to plasmin. The intrapleural instillation of urokinase is safe and effective for promoting drainage of loculated intrapleural effusions, and it proved a useful option in the treatment of persistent loculations.

Transmural myocardial infarction with coexisting critical aortic stenosis as an etiology for early myocardial rupture.

Myocardial rupture is the most important cause of post-infarct sudden death after myocardial infarction other than shock and dysrhythmias. Usually unrecognized, pseudoaneurysm formation is a delayed consequence of myocardial rupture in a small portion of patients who will remain at high risk for late rupture and death. Clinical studies have defined a profile of the patient who is at increased risk for post-infarct myocardial rupture. We believe that an additional factor, ventricular outflow tract obstruction, may add to the risk of having a post infarct rupture. A high degree of suspicion by the clinician accompanied by the timely performance of diagnostic tests may help to decrease the mortality from this catastrophic event.