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.

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.

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.

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.

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.

On finding and using identifiable parameter combinations in nonlinear dynamic systems biology models and COMBOS: a novel web implementation.

Parameter identifiability problems can plague biomodelers when they reach the quantification stage of development, even for relatively simple models. Structural identifiability (SI) is the primary question, usually understood as knowing which of P unknown biomodel parameters p1,…, pi,…, pP are-and which are not-quantifiable in principle from particular input-output (I-O) biodata. It is not widely appreciated that the same database also can provide quantitative information about the structurally unidentifiable (not quantifiable) subset, in the form of explicit algebraic relationships among unidentifiable pi. Importantly, this is a first step toward finding what else is needed to quantify particular unidentifiable parameters of interest from new I-O experiments. We further develop, implement and exemplify novel algorithms that address and solve the SI problem for a practical class of ordinary differential equation (ODE) systems biology models, as a user-friendly and universally-accessible web application (app)-COMBOS. Users provide the structural ODE and output measurement models in one of two standard forms to a remote server via their web browser. COMBOS provides a list of uniquely and non-uniquely SI model parameters, and-importantly-the combinations of parameters not individually SI. If non-uniquely SI, it also provides the maximum number of different solutions, with important practical implications. The behind-the-scenes symbolic differential algebra algorithms are based on computing Gröbner bases of model attributes established after some algebraic transformations, using the computer-algebra system Maxima. COMBOS was developed for facile instructional and research use as well as modeling. We use it in the classroom to illustrate SI analysis; and have simplified complex models of tumor suppressor p53 and hormone regulation, based on explicit computation of parameter combinations. It's illustrated and validated here for models of moderate complexity, with and without initial conditions. Built-in examples include unidentifiable 2 to 4-compartment and HIV dynamics models.

B7-H3 associated with tumor progression and epigenetic regulatory activity in cutaneous melanoma.

B7-H3, a cell surface transmembrane glycoprotein, was assessed for its functional and prognostic role in cutaneous melanoma progression. B7-H3 expression in melanoma cells was shown to be related to specific downstream signal transduction events as well as associated with functional epigenetic activity. B7-H3 expression and prognostic utility were shown by reverse transcription and real-time PCR and immunohistochemistry analysis on individual melanoma specimens and then verified in clinically annotated melanoma stage III and stage IV metastasis tissue microarrays in a double-blind study. B7-H3 messenger RNA expression was shown to be significantly increased with stage of melanoma (P<0.0001) and significantly associated with melanoma-specific survival in both stage III (P<0.0001) and stage IV (P<0.012) melanoma patients. B7-H3 expression was related to migration and invasion; overexpression of B7-H3 increased migration and invasion, whereas knockdown of B7-H3 reduced cell migration and invasion. MiR-29c expression was shown to inversely regulate B7-H3 expression. Furthermore, we demonstrated that melanoma B7-H3 expression was correlated to phosphorylated signal transducer and activator of transcription-3 activity level in melanoma tissues and cell lines. These studies demonstrate that B7-H3 is a significant factor in melanoma progression and events of metastasis.

Association between circulating tumor cells and prognosis in patients with stage III melanoma with sentinel lymph node metastasis in a phase III international multicenter trial.

PURPOSE: The outcomes of patients with melanoma who have sentinel lymph node (SLN) metastases can be highly variable, which has precluded establishment of consensus regarding treatment of the group. The detection of high-risk patients from this clinical setting may be helpful for determination of both prognosis and management. We report the utility of multimarker reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) detection of circulating tumor cells (CTCs) in patients with melanoma diagnosed with SLN metastases in a phase III, international, multicenter clinical trial. PATIENTS AND METHODS: Blood specimens were collected from patients with melanoma (n = 331) who were clinically disease-free after complete lymphadenectomy (CLND) before entering onto a randomized adjuvant melanoma vaccine plus bacillus Calmette-Guérin (BCG) versus BCG placebo trial from 30 melanoma centers (United States and international). Blood was assessed using a verified multimarker RT-qPCR assay (MART-1, MAGE-A3, and GalNAc-T) of melanoma-associated proteins. Cox regression analyses were used to evaluate the prognostic significance of CTC status for disease recurrence and melanoma-specific survival (MSS). RESULTS: Individual CTC biomarker detection ranged from 13.4% to 17.5%. There was no association of CTC status (zero to one positive biomarkers v two or more positive biomarkers) with known clinical or pathologic prognostic variables. However, two or more positive biomarkers was significantly associated with worse distant metastasis disease-free survival (hazard ratio [HR] = 2.13, P = .009) and reduced recurrence-free survival (HR = 1.70, P = .046) and MSS (HR = 1.88, P = .043) in a multivariable analysis. CONCLUSION: CTC biomarker status is a prognostic factor for recurrence-free survival, distant metastasis disease-free survival, and MSS after CLND in patients with SLN metastasis. This multimarker RT-qPCR analysis may therefore be useful in discriminating patients who may benefit from aggressive adjuvant therapy or stratifying patients for adjuvant clinical trials.

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.

AIM1 and LINE-1 epigenetic aberrations in tumor and serum relate to melanoma progression and disease outcome.

Aberrations in the methylation status of noncoding genomic repeat DNA sequences and specific gene promoter region are important epigenetic events in melanoma progression. Promoter methylation status in long interspersed nucleotide element-1 (LINE-1) and absent in melanoma-1 (AIM1; 6q21) associated with melanoma progression and disease outcome was assessed. LINE-1 and AIM1 methylation status was assessed in paraffin-embedded archival tissue (PEAT; n = 133) and in melanoma patients' serum (n = 56). LINE-1 U-Index (hypomethylation) and AIM1 were analyzed in microdissected melanoma PEAT sections. The LINE-1 U-Index of melanoma (n = 100) was significantly higher than that of normal skin (n = 14) and nevi (n = 12; P = 0.0004). LINE-1 U-Index level was elevated with increasing American Joint Committee on Cancer (AJCC) stage (P<0.0001). AIM1 promoter hypermethylation was found in higher frequency (P = 0.005) in metastatic melanoma (65%) than in primary melanomas (38%). When analyzed, high LINE-1 U-Index and/or AIM1 methylation in melanomas were associated with disease-free survival (DFS) and overall survival (OS) in stage I/II patients (P = 0.017 and 0.027, respectively). In multivariate analysis, melanoma AIM1 methylation status was a significant prognostic factor of OS (P = 0.032). Furthermore, serum unmethylated LINE-1 was at higher levels in both stage III (n = 20) and stage IV (n = 36) patients compared with healthy donors (n = 14; P = 0.022). Circulating methylated AIM1 was detected in patients' serum and was predictive of OS in stage IV patients (P = 0.009). LINE-1 hypomethylation and AIM1 hypermethylation have prognostic utility in both melanoma patients' tumors and serum.

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.

Lymphovascular invasion of colorectal cancer is correlated to SPARC expression in the tumor stromal microenvironment.

As an integral component of the microenvironment in colorectal cancer (CRC), stromal cells can influence tumor progression. Found in the extracellular matrix of CRC, secreted protein acidic and rich in cysteine (SPARC) is expressed in stromal and CRC cells. While SPARC's influence on CRC is not clear, we hypothesized that epigenetically regulated SPARC expression in the microenvironment stromal cells of CRC can affect primary CRC progression and is influenced by lymphovascular invasion (LVI). Quantitative immunohistochemistry (IHC) analysis of paraffin-embedded (n=72) from 37 LVI-positive and 35 LVI-negative primary CRCs was performed. MassARRAY sequencing was performed to assess the methylation status of the promoter region in 22 LVI-positive and 20 LVI-negative CRC and to identify specific CpG island(s) regulating SPARC expression. SPARC in CRC cells was not correlated with LVI, whereas SPARC in the microenvironment stromal cells was inversely related to LVI (P < 0.0001). There was a direct relationship between LVI and 6 specific CpG site methylation in the SPARC promoter region of stromal cells (P = 0.017) but not in CRC cells. Stromal SPARC expression inversely correlated with VEGF-A expression in CRC (P = 0.003) and positively correlated with HSP27 expression (P = 0.009). The results suggested that the epigenetic regulation of SPARC expression in tumor cells versus stromal cells of CRC is significantly different. Stromal cell SPARC expression is epigenetically influenced by LVI of CRC tumors, and may play a significant role in primary CRC progression.

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.

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.

Downregulation of microRNA-29c is associated with hypermethylation of tumor-related genes and disease outcome in cutaneous melanoma.

Hypermethylation of the promoter region of tumor-related genes (TRGs) has been shown to silence gene expression during melanoma progression, whereas microRNA-29(miR-29) has been found to downregulate DNA methyltransferases DNMT3A and DNMT3B which were shown as essential to the methylation of TRGs. We hypothesized that the expression level of miR-29 is associated to TRG methylation status and may have prognostic utility in melanoma. AJCC stage I-IV cutaneous melanoma paraffin-embedded archival tissue (PEAT) specimens (n=149) were assessed. Expression of miR-29 isoforms a, b, and c were analyzed by reverse-transcription quantitative real-time polymerase chain reaction(RT-qPCR). Expression of DNMT3A and DNMT3B was assessed by immunohistochemistry(IHC) on defined clinically annotated tissue microarrays (TMA) of AJCC stage III melanoma lymph node metastases. Promoter region CpG island methylation status of RASSF1A, TFPI-2, RAR-ß, SOCS, GATA4 and genomic repeat sequence MINT17 and MINT31 were previously evaluated in melanoma tissues. Only miR-29c isoform expression was correlated to advancing AJCC stages in melanoma. miR-29c expression was significantly downregulated in AJCC stage IV melanoma tumors compared to primary melanomas. Hypermethylation status of TRGs and non-coding MINT loci in different stages of melanoma showed an inverse association with miR-29c expression. Overall, an increase in miR-29c expression inversely correlated to both DNMT3A and DNMT3B protein expression in melanomas. Expression of DNMT3B and miR-29c were significantly (p=0.004 and p=0.002, respectively) associated with overall survival(OS) in AJCC stage III melanoma patients by multivariate analysis. The studies demonstrated that both miR-29c and DNMT3B have significant roles in melanoma progression, and may be useful epigenetic biomarkers for disease outcome.

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.

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.

Chemokine receptor CXCR4 expression in patients with melanoma and colorectal cancer liver metastases and the association with disease outcome.

OBJECTIVE: To determine the role of chemokine receptor (CR) expression in patients with melanoma and colorectal cancer (CRC) liver metastases. SUMMARY BACKGROUND DATA: Murine and in vitro models have identified CR as potential factors in organ-specific metastasis of multiple cancers. Chemokines via their respective receptors have been shown to promote cell migration to distant organs. METHODS: Patients who underwent hepatic surgery for melanoma or CRC liver metastases were assessed. Screening cDNA microarrays of melanoma/CRC cell lines and tumor specimens were analyzed to identify CR. Microarray data were validated by quantitative real-time RT-PCR (qRT) in paraffin-embedded liver metastases. Migration assays and immunohistochemistry were performed to verify CR function and confirm CR expression, respectively. RESULTS: Microarray analysis identified CXCR4 as the most common CR expressed by both cancers. qRT demonstrated CXCR4 expression in 24 of 27 (89%) melanoma and 28 of 29 (97%) CRC liver metastases. In vitro treatment of melanoma or CRC cells with CXCL12, the ligand for CXCR4, significantly increased cell migration (P < 0.001). Low versus high CXCR4 expression in CRC liver metastases correlated with a significant difference in overall survival (median 27 months vs. 10 months, respectively; P = 0.036). In melanoma, low versus high CXCR4 expression in liver metastases demonstrated no difference in overall survival (median 11 months vs. 8 months, respectively; P = not significant). CONCLUSIONS: CXCR4 is expressed and functional on melanoma and CRC cells. The ligand for CXCR4 is highly expressed in liver and may specifically attract melanoma and CRC CXCR4 (+) cells. Quantitative analysis of CXCR4 gene expression in patients with liver metastases has prognostic significance for disease outcome.

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.

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.