Livestock hauler and dairy farmer perspectives about cull dairy cattle transport and cattle transport regulations in British Columbia, Canada.

Dairy cows are usually culled and transported from the farm when they no longer meet the farm's standards for production or are not needed for milk production. Some cows are transported while in poor condition and may deteriorate further during transport. In February 2020, Canadian federal animal transport regulations were revised with the aim to minimise risks to livestock during transport; changes that may impact cull dairy cows included defining compromised cattle and limiting their maximum transport time. This study conducted semi-structured interviews with dairy farmers (n = 6) and cattle haulers (n = 4) in British Columbia, Canada, to gain an in-depth understanding of the effect of the regulations on their practices when shipping and transporting dairy cows to slaughter. Interviews were transcribed in Otter.ai and thematically coded in NVivo 12. While farmer and hauler participants recognised the importance of animal welfare during transport and described practices such as shipping mobile animals to reduce the risk that cows would become non-ambulatory during transport, they also described little change in shipping and transport practices due to the new regulations. Among interviewed participants, barriers to compliance with the regulations appear to be low knowledge of, and mixed or negative attitudes towards the regulations. Participants also described how they felt a lack of communication along the transport chain and limited transport and slaughter infrastructure made compliance difficult. Possible suggestions to remedy these barriers include providing educational resources about the regulations and encouraging communication about cow fitness for transport between responsible parties in the transport chain.

Perioperative Outcomes after Regional Versus General Anesthesia for Above the Knee Amputations.

BACKGROUND: Nontraumatic lower extremity amputation (LEA) remains a common procedure among patients who frequently have significant comorbidities. Patients undergoing above knee amputation (AKA) have the highest rates of mortality in this cohort, yet there is little evidence to support selection between peripheral nerve block or neuraxial regional anesthesia (RA) versus general anesthesia (GA) techniques. The objective of this study was to determine whether RA (neuraxial or peripheral nerve block) techniques were associated with more favorable outcomes versus general anesthesia among patients undergoing AKA. METHODS: This is a retrospective cohort study using propensity-matched groups. Patients undergoing AKA were identified in the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) data set and grouped according to anesthetic type as either RA or GA. Patients undergoing AKA with RA were propensity matched to similar patients who had GA. Primary outcome was 30-day mortality. Secondary outcomes were numerous and included cardiac, pulmonary, infectious, and bleeding complications, as well as length of stay. Among a subset of patients for whom readmission data were available, rate of readmission was compared as a secondary outcome. RESULTS: Nine thousand nine hundred ninety-nine patients were identified in the ACS-NSQIP database. One thousand three hundred twelve received a regional anesthetic, and the remainder had a general anesthetic. Factors significantly associated with GA included younger age (70 vs. 75 years; P < 0.001), higher body mass index (26.5 vs. 25.4; P < 0.001), and ethnically white (62.4% vs. 57%; P < 0.001). Before matching, patients receiving RA were less likely to be smokers (22% vs. 29%; P < 0.001), have a bleeding disorder (15% vs 30%; P < 0.001), or have a diagnosis of sepsis (26% vs 34%; P < 0.001). Propensity score matching produced a cohort composed of 1,916 patients equally divided between RA and GA. We found no difference with respect to the primary end point of 30-day mortality (11.7% vs 11.7%; odds ratio [OR] 1.01; P = 0.943) nor was there any difference with respect to secondary outcomes. Among patients for whom readmission data were available, there was no statistically significant difference between rates of readmission between the groups (15.6% for RA vs. 12.7% for GA; OR 1.26, confidence interval 0.87-1.828, P = 0.221). CONCLUSIONS: The present investigation did not detect any difference between regional and general anesthetic with respect to morbidity or mortality among patients undergoing AKA. This data set did not allow us to address other relevant markers including pain control or phantom limb syndrome.

Outcomes After Transcatheter Aortic Valve Replacement: A Propensity Matched Retrospective Cohort Study.

OBJECTIVES: To examine patient acuity and perioperative outcomes in a contemporary cohort of patients undergoing either transcatheter aortic valve replacement (TAVR) or surgical aortic valve replacement (SAVR). DESIGN: A retrospective propensity-matched cohort study with univariable logistic regression to assess postoperative outcomes. SETTING: Hospitals participating in the American College of Surgeons National Surgical Quality Improvement Program. PARTICIPANTS: The study comprised 2,043 patients who underwent either TAVR or SAVR that was reported in the American College of Surgeons-National Surgical Quality Improvement Program. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS: Age greater than 65 years, patients with dyspnea with moderate exertion or dependence in activities of daily living, high American Society of Anesthesiologists physical status classification, and history of chronic obstructive pulmonary disease were associated with TAVR, whereas body mass index greater than 25 was associated with SAVR. After propensity matching, no differences in 30-day mortality, length of stay, or most postoperative outcomes were observed between the 2 cohorts. Patients undergoing TAVR were less likely to require a perioperative blood transfusion and on an individual patient basis had a lower number of complications than patients in the SAVR group. CONCLUSIONS: Patients undergoing TAVR have similar mortality, length of stay, and risk for postoperative complications as do patients undergoing SAVR, but patients undergoing TAVR are less likely to have blood transfused.

Gene editing in animals: What does the public want to know and what information do stakeholder organizations provide?

Organizations involved with gene editing may engage with the public to share information and address concerns about the technology. It is unclear, however, if the information shared aligns with what people want to know. We aimed to understand what members of the public want to know about gene editing in animals by soliciting their questions through an open-ended survey question and comparing them with questions posed in Frequently Asked Question (FAQ) webpages developed by gene editing stakeholder organizations. Participants (338 USA residents) asked the most questions about gene editing in general and animal welfare. In contrast, FAQ webpages focused on regulations. The questions survey participants asked demonstrate a range of knowledge and interests. The discrepancy between survey participant questions and the information provided in the FAQ webpages suggests that gene editing stakeholders might engage in more meaningful public engagement by soliciting actual questions from the public and opening up opportunities for real dialogue.

Epigenetic regulation of cancer stem cell genes in triple-negative breast cancer.

Expression of specific breast cancer stem cells (BCSCs) is seen in aggressive tumors, but their regulation is unclear. Epigenetic changes influence gene expression and are implicated in breast cancer progression. We hypothesized that promoter methylation regulates specific BCSC-related genes [CD44, CD133, CD24, MSH1 (alias, Musashi-1), and ALDH1] and that this epigenetic profile can identify aggressive subtypes, such as triple-negative breast cancer (TNBC). Methylation analysis was performed using MassARRAY EpiTYPER sequencing; CpG-rich sites were identified in the promoter regions of BCSC genes, except ALDH1. These sites were screened by treatment with 5-aza-2'-deoxycytidine in four TN and five non-TNBC cell lines. The specific regulatory CpG site demonstrating the most significant inverse correlation between CpG site methylation and mRNA expression was identified for CD44, CD133, and Musashi-1, but not for CD24. Methylation of CD44, CD133, and Musashi-1 was evaluated in 91 American Joint Committee on Cancer stage I to III primary breast cancer tumors, and these sites were significantly hypomethylated in TNBC versus non-TNBC. The IHC staining of primary tumors with the highest and lowest methylation levels revealed the strongest staining in hypomethylated specimens, suggesting that hypomethylation leads to gene activation. We demonstrate that methylation is a significant mechanism regulating CD44, CD133, and Musashi-1, and that gene hypomethylation correlates with TNBC. Assessment of epigenetic changes in BCSC genes may provide a more accurate classification of TNBC and could be developed as potential therapeutic targets.

Assessment of prognostic circulating tumor cells in a phase III trial of adjuvant immunotherapy after complete resection of stage IV melanoma.

OBJECTIVE: To verify circulating tumor cell (CTC) prognostic utility in stage IV resected melanoma patients in a prospective international phase III clinical trial. BACKGROUND: Our studies of melanoma patients in phase II clinical trials demonstrated prognostic significance for CTCs in patients with AJCC stage IV melanoma. CTCs were assessed to determine prognostic utility in follow-up of disease-free stage IV patients pre- and during treatment. METHODS: After complete metastasectomy, patients were prospectively enrolled in a randomized trial of adjuvant therapy with a whole-cell melanoma vaccine, Canvaxin, plus Bacille Calmette-Guerin (BCG) versus placebo plus BCG. Blood specimens obtained pretreatment (n = 244) and during treatment (n = 214) were evaluated by quantitative real-time reverse-transcriptase polymerase chain reaction (qPCR) for expression of MART-1, MAGE-A3, and PAX3 mRNA biomarkers. Univariate and multivariate Cox analyses examined CTC biomarker expression with respect to clinicopathological variables. RESULTS: CTC biomarker(s) (≥ 1) was detected in 54% of patients pretreatment and in 86% of patients over the first 3 months. With a median follow-up of 21.9 months, 71% of patients recurred and 48% expired. CTC levels were not associated with known prognostic factors or treatment arm. In multivariate analysis, pretreatment CTC (> 0 vs. 0 biomarker) status was significantly associated with disease-free survival (DFS; HR 1.64, P = 0.002) and overall survival (OS; HR 1.53, P = 0.028). Serial CTC (>0 vs. 0 biomarker) status was also significantly associated with DFS (HR 1.91, P = 0.02) and OS (HR 2.57, P = 0.012). CONCLUSION: CTC assessment can provide prognostic discrimination before and during adjuvant treatment for resected stage IV melanoma patients.

Direct serum assay for microRNA-21 concentrations in early and advanced breast cancer.

BACKGROUND: MicroRNAs (miRs) are a class of small noncoding RNAs whose expression changes have been associated with cancer development and progression. Current techniques to isolate miRs for expression analysis from blood are inefficient. We developed a reverse-transcription quantitative real-time PCR (RT-qPCR) assay for direct detection of circulating miRs in serum. We hypothesized that serum concentrations of miR-21, a biomarker increased in breast tumors, would correlate with the presence and extent of breast cancer. METHODS: The RT-qPCR applied directly in serum (RT-qPCR-DS) assay for circulating miR-21 was tested in sera from 102 patients with different stages of breast cancer and 20 healthy female donors. RESULTS: The assay was sensitive for detection of miR-21 in 0.625 µL of serum from breast cancer patients. For differentiation of samples from patients with locoregional breast cancer from those from healthy donors, the odds ratio was 1.796 and the area under the curve was 0.721. In a multivariate analysis that included standard clinicopathologic prognostic factors, high circulating miR-21 concentrations correlated significantly (P < 0.001) with visceral metastasis. CONCLUSIONS: A novel RT-qPCR-DS can improve the efficiency of miR assessment. Use of this assay to detect circulating miR-21 has diagnostic and prognostic potential in breast cancer.

Development of sporadic microsatellite instability in colorectal tumors involves hypermethylation at methylated-in-tumor loci in adenoma.

Microsatellite instability (MSI) and genomic hypermethylation of methylated-in-tumor (MINT) loci are both strong prognostic indicators in a subgroup of patients with sporadic colorectal cancer (CRC). The present study was designed to determine whether the methylation of MINT loci during the progression of adenoma to CRC is related to MSI in CRC cases. Methylation index (MI) was measured by absolute quantitative assessment of methylated alleles at seven MINT loci in primary CRC with contiguous adenomatous and normal tissues of 79 patients. Results were then validated in primary CRC tissues from an independent group of 54 patients. Increased MI of both MINT loci 1 and 31 was significantly associated with MSI in CRC and was specific for adenoma. Total MI and the number of methylated loci were threefold (P=0.02) and fivefold (P=0.004) higher, respectively, in adenomas associated with microsatellite-stable CRC versus microsatellite-unstable CRC. MINT MI was found to be correlated with mismatch repair protein expression, MSI, BRAF (V600E) mutation status, mut-L homologue 1 methylation status, and disease-specific survival in the second independent validation group of patients. MI of specific MINT loci may be prognostic indicators of colorectal adenomas that will develop into sporadic microsatellite-unstable CRCs. Increased MINT locus methylation appears to precede MSI and may have utility in defining clinical pathology in the absence of features of malignant invasive tumors.

Prognostic molecular biomarkers for cutaneous malignant melanoma.

Molecular signatures of melanoma have propelled new approaches to early diagnosis, monitoring of treatment response, and targeted therapy. This review discusses messenger RNA (mRNA), genomic, and epigenomic melanoma biomarkers in blood and tissue specimens. The major focus is on tissue-based molecular assays to upstage sentinel lymph nodes (SLNs), and blood-based assays to detect melanoma progression by monitoring levels of circulating tumor cells (CTC) and circulating DNA.

Utility of circulating B-RAF DNA mutation in serum for monitoring melanoma patients receiving biochemotherapy.

PURPOSE: Somatic B-RAF gene mutation has been identified in many malignancies and detected at a high frequency in cutaneous malignant melanoma. However, the significance of the B-RAF mutation (B-RAFmt) in terms of its prognostic and predictive capabilities for treatment response or disease outcome is not known. We hypothesized that circulating serum B-RAFmt (B-RAFsmt) at V600E, detected in serum, predicts response in melanoma patients receiving concurrent biochemotherapy. EXPERIMENTAL DESIGN: A real-time clamp quantitative reverse transcription-PCR assay was designed to assess B-RAFsmt by peptide nucleic acid clamping and a locked nucleic acid hybrid probe. Normal (n = 18) and American Joint Committee on Cancer stage I to IV melanoma patients (n = 103) were evaluated. These included stage IV patients (n = 48) with blood drawn before and after biochemotherapy. Patients were classified as biochemotherapy responders or nonresponders. Responders (n = 24) had a complete or partial response following biochemotherapy; nonresponders (n = 24) developed progressive disease. RESULTS: Of the 103 melanoma patients, 38 (37%) had B-RAFsmt DNA, of which 11 of 34 (32%) were stage I or II, and 27 of 69 (39%) were stage III or IV. Of the 48 biochemotherapy patients, 10 of 24 (42%) patients were positive for the B-RAFsmt in the respective responder and nonresponder groups before treatment. After biochemotherapy, B-RAFsmt was detected in only 1 of 10 patients (10%) in the responder group and 7 of 10 patients (70%) in the nonresponder group. B-RAFsmt is associated with significantly worse (P = 0.039) overall survival in patients receiving biochemotherapy. CONCLUSION: These studies show the presence and utility of circulating B-RAFsmt DNA in melanoma patients.

Postoperative outcomes in patients with a do-not-resuscitate (DNR) order undergoing elective procedures.

STUDY OBJECTIVE: Do-not-resuscitate (DNR) status has been shown to be an independent risk factor for mortality in the post-operative period. Patients with DNR orders often undergo elective surgeries to alleviate symptoms and improve quality of life, but there are limited data on outcomes for informed decision making. DESIGN: Retrospective cohort study. SETTING: A multi-institutional setting including operating room, postoperative recovery area, inpatient wards, and the intensive care unit. PATIENTS: A total of 566 patients with a DNR status and 316,431 patients without a DNR status undergoing elective procedures using the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) from 2012. INTERVENTIONS: Patients undergoing elective surgical procedures. MEASUREMENTS: We analyzed the risk-adjusted 30-day morbidity and mortality outcomes for the matched DNR and non-DNR cohorts undergoing elective surgeries. MAIN RESULTS: DNR patients had significantly increased odds of 30-day mortality (OR 2.51 [1.55-4.05], p < 0.001) compared with non-DNR patients. In the DNR versus non-DNR cohort there was no significant difference in the occurrence of a number of 30-day complications, the rate of resuscitative measures undertaken, including cardiac arrest requiring CPR, reintubation, or return to the OR. The most common complications in both DNR and non-DNR patients undergoing elective procedures were transfusion, urinary tract infection, reoperation, and sepsis. Finally, the DNR patients had a significantly increased total length of hospital stay (7.65 ±â€¯9.55 vs. 6.87 ±â€¯9.21 days, p = 0.002). CONCLUSIONS: DNR patients, as compared with non-DNR patients, have increased post-operative mortality but not morbidity, which may arise from unmeasured severity of illness or transition to comfort care in accordance with a patient's wishes. The informed consent process for elective surgeries in this patient population should include a discussion of acceptable operative risk.

Microphthalmia transcription factor as a molecular marker for circulating tumor cell detection in blood of melanoma patients.

PURPOSE: Microphthalmia transcription factor (Mitf), which is important in melanocyte development and melanoma growth, was assessed using real-time quantitative reverse transcription-PCR assay to investigate its expression as a marker for circulating melanoma cells in blood and determine the correlation with disease stage and survival in melanoma patients. EXPERIMENTAL DESIGN: In optimization studies for Mitf, we tested 15 melanoma cell lines, 41 peripheral blood lymphocytes from healthy volunteers, and 21 metastatic melanoma tissues. Blood specimens were procured from 90 patients with stage I (n = 20), stage II (n = 20), stage III (n = 28), and stage IV (n = 22) melanoma. Blood specimens were also obtained at four bleed intervals from 58 patients enrolled in a prospective multicenter trial of biochemotherapy before and after surgical treatment of American Joint Committee on Cancer stage III melanoma. RESULTS: Under the optimized conditions, Mitf was negative in healthy peripheral blood lymphocytes and positive in all melanoma cell lines and 18 (86%) melanoma tissues. In the 90 patients, the rate of Mitf detection was higher with increasing American Joint Committee on Cancer stage (P < 0.0001). In the 58 patients treated with biochemotherapy and surgery, Mitf detection decreased with treatment (P = 0.019). Mitf detection after treatment was associated with a significantly lower relapse-free (P < 0.0001) and overall (P = 0.001) survival and was a significant independent prognostic factor for relapse-free (risk ratio, 5.63; P = 0.0004) and overall (risk ratio, 5.36; P = 0.005) survival. CONCLUSIONS: Mitf detection in blood can indicate subclinical metastatic disease and predict treatment outcome in melanoma patients.

Chemokine receptor CXCR4 expression in colorectal cancer patients increases the risk for recurrence and for poor survival.

PURPOSE: Liver metastasis is the predominant cause of colorectal cancer (CRC) related mortality. Chemokines, soluble factors that orchestrate hematopoetic cell movement, have been implicated in directing cancer metastasis, although their clinical relevance in CRC has not been defined. Our hypothesis was that the chemokine receptor CXCR4 expressed by CRC is a prognostic factor for poor disease outcome. METHODS: CRC cell lines (n = 6) and tumor specimens (n = 139) from patients with different American Joint Committee on Cancer (AJCC) stages of CRC were assessed. Microarray screening of select specimens and cell lines identified CXCR4 as a prominent chemokine receptor. CXCR4 expression in tumor and benign specimens was assessed by quantitative real-time reverse transcription polymerase chain reaction and correlated with disease recurrence and overall survival. RESULTS: High CXCR4 expression in tumor specimens (n = 57) from AJCC stage I/II patients was associated with increased risk for local recurrence and/or distant metastasis (risk ratio, 1.35; 95% CI, 1.09 to 1.68; P = .0065). High CXCR4 expression in primary tumor specimens (n = 35) from AJCC stage IV patients correlated with worse overall median survival (9 months v 23 months; RR, 2.53; 95% CI, 1.19 to 5.40; P = .016). CXCR4 expression was significantly higher in liver metastases (n = 39) compared with primary CRC tumors (n = 100; P < .0001). CONCLUSION: CXCR4, a well-characterized chemokine receptor for T-cells, is differentially expressed in CRC. CXCR4 gene expression in primary CRC demonstrated significant associations with recurrence, survival, and liver metastasis. The CXCR4-CXCL12 signaling mechanism may be clinically relevant for patients with CRC and represents a potential novel target for disease-directed therapy.

Predictive utility of circulating methylated DNA in serum of melanoma patients receiving biochemotherapy.

PURPOSE: Currently, no validated blood-based assays accurately predict treatment response or outcome in melanoma patients. We hypothesized that methylation of tumor-related genes detected in serum DNA could predict disease outcome and therapeutic response in patients receiving concurrent biochemotherapy (BC) for metastatic melanoma. PATIENTS AND METHODS: American Joint Committee on Cancer stage IV melanoma patients (N = 50) had blood drawn before administration of BC. Patients (n = 47) were classified as BC responders or nonresponders. Responders (n = 23) demonstrated a complete or partial response following BC; nonresponders (n = 24) demonstrated progressive disease. Hypermethylation of Ras association domain family 1 (RASSF1A), retinoic acid receptor-beta2 (RAR-beta2), and O6-methylguanine DNA methyltransferase (MGMT) genes were assessed by methylation-specific polymerase chain reaction. RESULTS: Circulating methylated RASSF1A was significantly less frequent for responders (three of 23 patients; 13%) than nonresponders (10 of 24 patients; 42%; P = .028). Patients with RASSF1A, RAR-beta2, or at least one serum methylated gene had significantly worse overall survival than patients with no methylated genes (log-rank, P = .013, .021, and .01, respectively). Methylated RASSF1A was the only factor that significantly correlated with overall survival and BC response (risk ratio, 2.38; 95% CI, 1.16 to 4.86; P = .018; odds ratio = 0.21; 95% CI, 0.05 to 0.90; P = .036). CONCLUSION: Detection of circulating methylated DNA in serum can predict response to BC and disease outcome.

X-Linked inhibitor of apoptosis protein expression level in colorectal cancer is regulated by hepatocyte growth factor/C-met pathway via Akt signaling.

PURPOSE: The inhibitor of the apoptosis protein (IAP) family members, such as the X-linked IAP (XIAP), survivin, and livin, are essential for cell survival and antiapoptosis in colorectal cancer cells. We hypothesized that the hepatocyte growth factor (HGF) activation in colorectal cancer via c-Met receptor regulates IAP proteins through Akt signaling. EXPERIMENTAL DESIGN: The level of IAPs and C-Met mRNA expression was assessed using a quantitative real-time reverse transcriptase-PCR (RT-PCR) assay on colorectal normal mucosa (n = 13), adenomas (n = 6), and colorectal cancer tumors (n = 50). The role of HGF/C-Met pathway through Akt and XIAP was investigated by small interfering RNA (siRNA) and quantitative RT-PCR analysis of colorectal cancer lines. RESULTS: Of the IAPs, only XIAP showed significant correlation to tumor development and progression. XIAP mRNA level in primary colorectal cancer was significantly higher than that in colorectal normal mucosa (P = 0.01); liver metastases was significantly higher than primary colorectal cancer tumors (P = 0.04); and primary colorectal cancer N1/N2 cases were significantly higher than N0 cases (P = 0.008). HGF stimulation of colorectal cancer lines enhanced XIAP mRNA expression but not other IAPs. Activation of XIAP expression by HGF was inhibited by siRNA targeting Akt1 and Akt2. CONCLUSIONS: Activation of C-MET enhances XIAP through the Akt pathway. XIAP up-regulation was shown to be correlated to colorectal cancer tumor progression. The Akt-XIAP pathway may be a potential molecular target for regulating colorectal cancer progression.

Expression of differentiation melanoma-associated antigen genes is associated with favorable disease outcome in advanced-stage melanomas.

Cutaneous melanomas have been found to express several immunogenic differentiation melanoma-associated antigens (MAAs) that have been suggested to play an important role in disease outcome. Adaptive host immunity to MAAs has shown some level of control on melanoma progression. To date, there has been no definitive report correlating the level of differentiated MAAs gene expression in melanomas with overall disease outcome. Metastasis of melanoma to distant visceral organ sites usually indicates a survival of less than 1 year; however, a subset of patients who undergo cytoreductive surgery of distant metastases survive for a longer period. We hypothesized that the gene expression level of differentiation MAAs in metastatic melanoma (AJCC stage IV) lesions would be predictive of survival. We focused on three known differentiation MAAs: tyrosinase (TYR), TYR-related protein 2 (TRP-2), and melanoma antigen recognized by T cells 1 (MART-1); all three of them are known to induce immune responses in melanoma patients and are frequently expressed in melanomas. A quantitative reverse-transcriptase RealTime PCR (qRT) assay was developed for these MAAs to assess mRNA expression in metastatic melanoma tumors obtained from cytoreductive surgery of AJCC stage IV melanoma patients (n = 35). Patients were followed up for over 60 months. There was a variation in mRNA copy levels for individual MAAs in melanoma tumors. Elevated MAA mRNA copy levels of TYR and TRP-2 significantly (P < 0.03 and < 0.009, respectively) correlated with improved overall survival. Patients having at least one MAA expressed in their tumors had a significantly (P = 0.01) better overall survival (median 16 months). These studies demonstrate that levels of differentiated MAA mRNA expression of advanced-stage metastatic melanomas can be used as molecular predictive factors of disease outcome. The studies also imply that an assessment of melanoma tumor MAAs may provide a stratification factor targeted for active-specific immunotherapy.

Profiling epigenetic inactivation of tumor suppressor genes in tumors and plasma from cutaneous melanoma patients.

Aberrant methylation of CpG islands in promoter regions of tumor suppressor genes (TSG) has been demonstrated in epithelial origin tumors. However, the methylation profiling of tumor-related gene promoter regions in cutaneous melanoma tumors has not been reported. Seven known or candidate TSGs that are frequently hypermethylated in carcinomas were assessed by methylation-specific polymerase chain reaction (MSP) in 15 melanoma cell lines and 130 cutaneous melanoma tumors. Four TSGs were frequently hypermethylated in 86 metastatic tumor specimens: retinoic acid receptor-beta2 (RAR-beta2) (70%), RAS association domain family protein 1A (RASSF1A) (57%), and O6-methylguanine DNA methylatransferase (MGMT) (34%), and death-associated protein kinase (DAPK) (19%). Hypermethylation of MGMT, RASSF1A, and DAPK was significantly lower in primary melanomas (n=20) compared to metastatic melanomas. However, hypermethylation of RAR-beta2 was 70% in both primary and metastatic melanomas. Cell lines had hypermethylation profiles similar to those of metastatic melanomas. The analysis of these four markers of metastatic tumors demonstrated that 97% had > or =1 gene(s) and 59% had > or =2 genes hypermethylated. The methylation of genes was verified by bisulfite sequencing. The mRNA transcripts could be re-expressed in melanoma cell lines having hypermethylated genes following treatment with 5'-aza 2'-deoxycytidine (5Aza-dC). Analysis of melanoma patients' plasma (preoperative blood; n=31) demonstrated circulating hypermethylated MGMT, RAR-beta2, and RASSF1A DNA for at least one of the markers in 29% of the patients. Our findings indicate that the incidence of TSG hypermethylation increases during tumor progression. Methylation of TSG may play a significant role in cutaneous melanoma progression.

Prognostic significance of molecular upstaging of paraffin-embedded sentinel lymph nodes in melanoma patients.

PURPOSE: Detection of micrometastases in sentinel lymph nodes (SLNs) is important for accurate staging and prognosis in melanoma patients. However, a significant number of patients with histopathology-negative SLNs subsequently develop recurrent disease. We hypothesized that a quantitative realtime reverse transcriptase polymerase chain reaction (qRT) assay using multiple specific mRNA markers could detect occult metastasis in paraffin-embedded (PE) SLNs to upstage and predict disease outcome. PATIENTS AND METHODS: qRT was performed on retrospectively collected PE SLNs from 215 clinically node-negative patients who underwent lymphatic mapping and sentinel lymphadenectomy for melanoma and were followed up for at least 8 years. PE SLNs (n = 308) from these patients were sectioned and assessed by qRT for mRNA of four melanoma-associated genes: MART-1 (antigen recognized by T cells-1), MAGE-A3 (melanoma antigen gene-A3 family), GalNAc-T (beta1-->4-N-acetylgalactosaminyl-transferase), and Pax3 (paired-box homeotic gene transcription factor 3). RESULTS: Fifty-three (25%) patients had histopathology-positive SLNs by hemotoxylin and eosin and/or immunohistochemistry. Of the 162 patients with histopathology-negative SLNs, 48 (30%) had nodes that expressed at least one of the four qRT markers, and these 48 patients also had a significantly increased risk of disease recurrence by a Cox proportional hazards model analysis (P <.0001; risk ratio, 7.48; 95% CI, 3.70 to 15.15). The presence of > or = one marker in histopathology-negative SLNs was also a significant independent prognostic factor by multivariate analysis for overall survival (P =.0002; risk ratio, 11.42; 95% CI, 3.17 to 41.1). CONCLUSION: Molecular upstaging of PE histopathology-negative SLNs by multiple-marker qRT assay is a significant independent prognostic factor for long-term disease recurrence and overall survival of patients with early-stage melanoma.

Molecular tumor markers in the blood: early prediction of disease outcome in melanoma patients treated with a melanoma vaccine.

PURPOSE: Patients with American Joint Committee on Cancer (AJCC) stage III melanoma are at high risk of recurrence and death. We hypothesized that a multiple-marker reverse transcriptase polymerase chain reaction (MM-RT-PCR) blood assay could predict, early in the course of therapy, those patients destined to experience treatment failure with a melanoma vaccine (MV) previously shown to improve survival in a phase II clinical trial. PATIENTS AND METHODS: After complete surgical resection, prospectively collected cryopreserved peripheral-blood lymphocyte specimens (n = 90) from the serial bleeds of 30 patients with AJCC stage III melanoma were studied by MM-RT-PCR, using the markers tyrosinase, melanoma antigen recognized by T cells-1 (MART-1), and universal melanoma antigen gene-A (uMAG-A). All patients were enrolled in a phase II MV trial during the period of blood draws, and were selected for this study in a blinded fashion. Median duration of clinical follow-up was 74 months for the 13 survivors and 11 months for the 17 nonsurvivors. RESULTS: The presence of at least one melanoma-specific RT-PCR marker was associated with an increased risk of disease recurrence (risk rate, 3.12; P =.02) and decreased risk of survival (relative rate, 2.62; P =.0496) by multivariate analysis. CONCLUSION: MM-RT-PCR of the blood provided early prediction of subsequent disease recurrence and death in clinically disease-free AJCC stage III melanoma patients enrolled in a MV phase II trial. On the basis of the results of this pilot study, the MM-RT-PCR blood assay should be considered as a clinically important monitoring tool for assessing patient response to adjuvant therapy, and in the surveillance of clinically disease-free patients for the earliest signs of recurrence.

Prediction of disease outcome in melanoma patients by molecular analysis of paraffin-embedded sentinel lymph nodes.

PURPOSE: A significant number of patients who develop recurrence after a histopathologically negative sentinel lymph node (SLN) biopsy will demonstrate occult metastases on re-evaluation of the SLNs with serial sectioning and immunohistochemistry. Reverse transcriptase polymerase chain reaction (RT-PCR) has been evaluated to improve disease staging and avoid false-negative findings in fresh or frozen-section SLNs. The purpose of this study was to develop a multimarker RT-PCR assay for assessing melanoma patients' archived paraffin-embedded (PE) SLNs. PATIENTS AND METHODS: Archived PE histopathologically positive (n = 37) and negative (n = 40) SLNs from patients with primary melanoma were analyzed using a semiquantitative multimarker RT-PCR assay. RESULTS: Marker expression in histopathologically positive and negative SLNs were as follows: 89%, 92%, 35%, and 43% (positive) and 40%, 33%, 5%, and 13% (negative) for tyrosinase, melanoma antigen recognized by T cells-1, tyrosinase-related protein-1, and tyrosinase-related protein-2, respectively. Twenty-five percent of histopathologically negative SLN patients were upstaged using at least two markers. Of these, 80% developed a recurrence. Furthermore, at a median follow-up of 55 months, patients with histopathologically negative SLNs who expressed zero or one marker had a significantly improved disease-free (P <.002) and overall (P <.03) survival versus those expressing two or more markers. CONCLUSION: These findings demonstrate the feasibility of a multimarker RT-PCR assay for evaluating archived PE SLNs. More significantly, identification of molecular risk factors can be detected in histopathologically negative SLNs for distinguishing early-stage melanoma patients with a worse prognosis.