Influence of Percutaneous Transhepatic Gallbladder Aspiration and Drainage for Severe Acute Cholecystitis on the Surgical Outcomes of Subsequent Laparoscopic Cholecystectomy: Post Hoc Analysis of the CSGO-HBP-017 (CSGO-HBP-017C).

OBJECTIVE: Percutaneous transhepatic gallbladder aspiration (PTGBA) and/or drainage (PTGBD) are useful approaches in the management of acute cholecystitis in patients who cannot tolerate surgery because of poor general condition or severe inflammation. However, reports regarding its effect on the surgical outcomes of subsequent laparoscopic cholecystectomy (LC) are sparse. The aim of this retrospective study was to investigate the influence of PTGBA on surgical outcomes of subsequent LC by comparing the only-PTGBA group, including patients who did not need the additional-PTGBD, versus the additional-PTGBD group, including those who needed the additional-PTGBD after PTGBA. PATIENTS AND METHODS: We conducted a post hoc analysis of our multi-institutional data. This study included 63 patients who underwent LC after PTGBA, and we compared the surgical outcomes between the only-PTGBA group (n = 56) and the additional-PTGBD group (n = 7). RESULTS: No postoperative complications occurred among the 63 patients, and the postoperative hospital stay was 11 ± 12 days. Fourteen patients (22.2%) had a recurrence of cholecystitis, of whom 7 patients (11.1%) needed the additional-PTGBD after PTGBA. Significantly longer operative time (245 ± 74 vs 159 ± 65 min, P = 0.0017) and postoperative hospital stay (22 ± 27 vs 10 ± 9 d, P = 0.0118) and greater intraoperative blood loss (279 ± 385 vs 70 ± 208 mL, P = 0.0283) were observed among patients in the additional-PTGBD group compared with the only-PTGBA group, whereas the rates of postoperative complications (Clavien-Dindo grade ≥3: 0% each) and conversion to open surgery (28.6% vs 8.9%, P = 0.1705) were comparable. CONCLUSION: PTGBA for acute cholecystitis could result in good surgical outcomes of subsequent LC, especially regarding postoperative complications. However, we should keep in mind that the additional-PTGBD after PTGBA failure, which sometimes happened, would be associated with increased operative difficulty and longer recovery.

Effect of Preserving the Percutaneous Gallbladder Drainage Tube Before Laparoscopic Cholecystectomy on Surgical Outcome: Post Hoc Analysis of the CSGO-HBP-017.

BACKGROUND: When percutaneous transhepatic gallbladder drainage (PTGBD) is followed by laparoscopic cholecystectomy (LC), there is no consensus regarding whether the drainage tube should be preserved or removed before LC. We hypothesized that the surgical results of LC might differ between cases with PTGBD tube preservation versus removal. Here, we investigated how drainage tube preservation or removal affected the surgical outcome of LC. METHODS: Using data from our previous multicenter study, we compared LC outcomes after PTGBD between patients with PTGBD tube preservation versus removal. This study included 208 patients who underwent LC over 12 days after PTGBD. In 83 cases, the PTGBD tube was preserved until LC, and in 125 cases, the tube was removed before LC. The results were verified by propensity score matching with 50 patients in each group. RESULTS: Cases with tube preservation versus removal exhibited significantly longer surgery duration (174 ± 105 min vs 145 ± 61 min, P = .0118) and postoperative hospital stay (14 ± 16 days vs 7 ± 7 days, P < .0001), a significantly higher postoperative complication rate (13.2% vs 3.2%, P = .0061), and a marginally higher incidence of open conversion (12.0% vs 4.8%, P = .0547). Propensity score matching verified the inferior surgical outcomes in cases with tube preservation. CONCLUSIONS: These results imply that when LC is performed > 12 days after PTGBD, the surgical outcome may be inferior when the drainage tube is preserved rather than removed before LC.

Preoperative risk score to predict subtotal cholecystectomy after gallbladder drainage for acute cholecystitis: Secondary analysis of data from a multi-institutional retrospective study (CSGO-HBP-017B).

INTRODUCTION: Subtotal cholecystectomy (STC) has become recognized as a "bailout procedure" to prevent bile duct injury in patients undergoing laparoscopic cholecystectomy (LC). Predictors of conversion to STC have not been identified because LC difficulty varies based on pericholecystic inflammation. We analyzed data from patients enrolled in a previously performed multi-institutional retrospective study of the optimal timing of LC after gallbladder drainage for acute cholecystitis (AC). These patients presumably had a considerable degree of pericholecystic inflammation. METHODS: In total, 347 patients who underwent LC after gallbladder drainage for AC were analyzed to examine preoperative and perioperative factors predicting conversion to STC. RESULTS: Three hundred patients underwent total cholecystectomy (TC) and 47 underwent conversion to STC. Eastern Cooperative Oncology Group Performance Status (ECOG PS) (P < .01), severity of cholecystitis (P = .04), previous history of treatment for common bile duct stones (CBDS) (P < .01), and surgeon experience (P = .03) were significantly associated with conversion to STC. Logistic regression analyses showed that ECOG PS (odds ratio 0.2; P < .0001) and previous history of treatment for CBDS (odds ratio 0.37; P = .0073) were independent predictors of conversion to STC. Our predictive risk score using these two variables suggested that a score ≥2 could discriminate between TC and STC (P < .0001). CONCLUSION: Poor ECOG PS and previous history of treatment for CBDS were significantly associated with conversion to STC after gallbladder drainage for AC.

Optimal timing of laparoscopic cholecystectomy after gallbladder drainage for acute cholecystitis: A multi-institutional retrospective study.

BACKGROUND: There is no consensus on the optimal timing of laparoscopic cholecystectomy (LC) after gallbladder drainage for acute cholecystitis (AC). To obtain evidence for a consensus, we investigated surgical outcomes of LC after gallbladder drainage with respect to the time elapsed from gallbladder drainage to surgery in a multi-institutional retrospective study. METHODS: This study enrolled 347 patients who underwent LC after gallbladder drainage for AC at 15 institutions. Surgical outcome of LC was investigated in the cases based on the interval from gallbladder drainage to surgery. RESULTS: The median interval from gallbladder drainage to surgery of the patients was 34 days, with a mean ± standard deviation of 58 ± 99 days. Patients were divided into four groups based on quartiles of the interval: Group A, cases with an interval of 1-12 days; Group B, cases with an interval of 13-34 days; Group C, cases with an interval of 35-73 days; and Group D, cases with an interval of ≥74 days. Surgical outcomes, which were evaluated with respect to intraoperative blood loss, operation time, postoperative hospital stay, rate of intraoperative accident, conversion from laparoscopic to open surgery, and postoperative complication, were worse in Group B than in the other groups. The finding was verified by propensity score-matched analysis. CONCLUSIONS: Surgical outcome of LC after gallbladder drainage for AC was inferior in Group B compared with the other groups. This finding could be useful for determining the optimal timing of LC after gallbladder drainage for AC.

[A case of a solitary splenic metastasis from gastric cancer].

A 69-year-old man was brought to our hospital's emergency room with a chief complaint of hematemesis, which had been caused by advanced gastric cancer on the lesser curvature of the stomach's upper body. Subsequently, total gastrectomy with lymph node dissection (D2) was performed. A pathological diagnosis of gastric adenocarcinoma, U, Less, type 2, 100×70mm, tub2, pT3, int, INFb, ly0, v0, pN0 (0/24), pPM0 (30mm), pDM0 (30mm), fStage IIA, was then established. After discharge, the patient was treated with S-1 as adjuvant chemotherapy at a dose of 120mg per day. However, a decrease in the platelet count prompted termination of chemotherapy, which lasted for three courses. Ten months after surgery, serum CEA levels increased to 116.6ng/ml, with enhanced CT showing a solitary splenic tumor with a diameter of 48×52mm suggestive of gastric cancer recurrence. PET/CT revealed no other tumors suggestive of gastric cancer recurrence. Given that only a solitary splenic metastatic tumor was detected, splenectomy was performed eleven months after surgery. Histological findings were the same as the previous gastric cancer, with peritoneal washing cytology being suspicious. Chemotherapy with the SOX regimen (S-1 at a dose of 120mg per day and oxaliplatin at a dose of 100mg/m2) was then started. The patient remained recurrence-free for a half year. Except during the terminal phase, only a few cases of a splenic metastasis from gastric cancer have been reported. We consider splenectomy to be potentially useful for patients with a solitary splenic metastasis from gastric cancer, through which prolonged prognosis could be expected.

Implantation of esophageal cancer onto post-dissection ulcer after gastric endoscopic submucosal dissection.

A case in which implantation of esophageal squamous cell carcinoma onto a post-dissection gastric ulcer was strongly suspected is presented. A 72-year-old man with alcoholic liver cirrhosis underwent esophagogastroduodenoscopy (EGD). Esophageal cancer (EC) (Mt, 20 mm, 0-Is) and gastric cancer (GC) (antrum, 15 mm, 0-IIc) were identified. Biopsy specimens revealed moderately differentiated squamous cell carcinoma (SCC) and differentiated adenocarcinoma, respectively. The GC was resected by endoscopic submucosal dissection (ESD) [14 mm × 9 mm, type 0-IIc, tub1, pT1a(M), ly0, v0, HM(-), VM(-)]. Two months after ESD, radiation therapy was started for the EC, and an almost complete response was obtained. Nine months after the ESD, a follow-up EGD showed a submucosal tumor-like lesion with ulceration, located immediately under the post-ESD scar, and biopsy specimens showed moderately differentiated SCC. There were no similar lesions suggesting hematogenous or lymphatic metastasis in the stomach.

Water immersion colonoscopy facilitates straight passage of the colonoscope through the sigmoid colon without loop formation: randomized controlled trial.

BACKGROUND AND AIM: One of the major causes of pain during colonoscopy is looping of the instrument during insertion through the sigmoid colon, which causes discomfort by stretching the mesentery. There are many studies in colonoscope techniques, but they have not been assessed objectively with respect to colonoscope passage through the sigmoid colon without loop formation. The aim of the present study was to determine whether cap-fitted colonoscopy and water immersion increase the success rate of insertion through the sigmoid without loop formation. METHODS: A total of 1005 patients were randomized to standard colonoscopy, cap-fitted colonoscopy or water immersion technique. All examinations were carried out under a magnetic endoscope imaging device. Main outcome was the success rate of insertion without loop formation. RESULTS: Success rate of insertion without loop formation was 37.5%, 40.0%, and 53.8% in the standard, cap, and water groups, respectively (standard vs water P = 0.00014, cap vs water P = 0.00186). There were no significant differences among the groups regarding cecal intubation rate, cecal intubation time and number of polyps ≥5 mm per patient. CONCLUSIONS: Water immersion increases the success rate of insertion through the sigmoid colon without loop formation. This practical technique, requiring only preparation of a cap and water, is useful without compromising cecal intubation rate, cecal intubation time, or polyp detection rate.

The role of estrogen receptor in melanoma.

The estrogen receptor (ER) belongs to the group of sex hormone receptors and binds the biologically active form of estrogen, 17beta-estradiol. Expression of ER in tumor tissue is a well-established prognostic marker in breast cancer. The role of ER has been extensively studied in several other types of human cancers. This report investigates the potential role of ER as a surrogate marker for predicting melanoma progression, response to therapy, and patient survival. In addition, the authors review what is known, so far, about ER signaling pathways and their potential role in carcinogenesis and progression of cutaneous melanoma. Possibilities and limitations of using ER as a therapeutic target in the treatment of melanoma is also discussed.

Estrogen receptor-alpha methylation predicts melanoma progression.

The role of estrogen receptor alpha (ER-alpha) in melanoma is unknown. ER-alpha expression may be regulated in melanoma via hypermethylation of promoter CpG islands. We assessed ER-alpha hypermethylation in primary and metastatic melanomas and sera as a potential tumor progression marker. ER-alpha methylation status in tumor (n = 107) and sera (n = 109) from American Joint Committee on Cancer (AJCC) stage I to IV melanoma patients was examined by methylation-specific PCR. The clinical significance of serum methylated ER-alpha was assessed among AJCC stage IV melanoma patients receiving biochemotherapy with tamoxifen. Rates of ER-alpha methylation in AJCC stage I, II, and III primary melanomas were 36% (4 of 11), 26% (5 of 19), and 35% (8 of 23), respectively. Methylated ER-alpha was detected in 42% (8 of 19) of stage III and 86% (30 of 35) of stage IV metastatic melanomas. ER-alpha was methylated more frequently in metastatic than primary melanomas (P = 0.0003). Of 109 melanoma patients' sera in AJCC stage I, II, III, and IV, methylated ER-alpha was detected in 10% (2 of 20), 15% (3 of 20), 26% (5 of 19), and 32% (16 of 50), respectively. Serum methylated ER-alpha was detected more frequently in advanced than localized melanomas (P = 0.03) and was the only factor predicting progression-free [risk ratio (RR), 2.64; 95% confidence interval (95% CI), 1.36-5.13; P = 0.004] and overall survival (RR, 2.31; 95% CI, 1.41-5.58; P = 0.003) in biochemotherapy patients. Hypermethylated ER-alpha is a significant factor in melanoma progression. Serum methylated ER-alpha is an unfavorable prognostic factor.

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.

Association of circulating tumor cells with serum tumor-related methylated DNA in peripheral blood of melanoma patients.

Although previous studies have separately shown the utility of circulating tumor cells (CTC) or cell-free tumor-related DNA in blood of cancer patients, there has been no investigation of their association and/or the prognostic value of combining these assessments. To date, the true source of tumor-related DNA in serum remains unknown. We hypothesized that CTC is a possible origin of serum tumor-related methylated DNA and their combination can predict disease outcome. To test this hypothesis, we obtained matched pairs of peripheral blood lymphocytes and serum specimens simultaneously from 50 American Joint Committee on Cancer stage IV melanoma patients before administration of biochemotherapy. Peripheral blood leukocytes were analyzed for three mRNA markers of CTC: MART-1, GalNAc-T, and MAGE-A3. Sera were analyzed for two methylated DNA markers: RASSF1A and RAR-beta2. CTC were detected in 13 of 15 (86%) patients with serum tumor-related methylated DNA and only in 13 of 35 (37%) patients without methylated DNA (P = 0.001). The number of CTC markers detected significantly correlated with methylated DNA (P = 0.008). CTC and methylated DNA were significantly correlated with biochemotherapy-treated patients' outcome. Patients with both CTC and methylated DNA showed significantly poorer response to biochemotherapy (P = 0.02) and worse time to progression and overall survival (P = 0.009 and 0.02, respectively). The correlation between CTC and serum tumor-related methylated DNA and the significant effect of this correlation on disease outcome indicate that a composite molecular assessment in blood may be a useful determinant of disease status and efficacy of systemic therapy for melanoma.

Molecular mechanisms of metastasis.

A major topic covered at the First International Symposium on Cancer Metastasis and the Lymphovascular System was the molecular mechanisms of metastasis. This has become of major interest in recent years as we have discovered new metastasis-related genes and gained understanding of the molecular events of lymphatic metastasis. The symposium covered new aspects and important questions related to the events of metastasis in both humans and animals. The basic and clinical related research covered in this topic represented many disciplines. The presentations showed novel findings and at the same time, raised many new unanswered questions, indicating the limited knowledge we still have regarding the molecular events of metastasis. The hope is that further unraveling of the direct and indirect molecular events of lymphatic metastasis will lead to new approaches in developing effective therapeutics.

Molecular upstaging of sentinel lymph nodes in melanoma: where are we now?

The presence of lymph node metastasis is the best predictor of disease progression and overall survival in patients who have melanoma. Lymphatic mapping and selective lymphadenectomy allows directed pathologic analysis of the node or nodes most likely to have metastatic disease. To diagnose metastatic disease in SLNs reliably requires a coordinated effort by nuclear medicine physicians, surgeons, and pathologists. Errors may occur if quality assurance is not emphasized at any point during the process. This, along with the presence of occult metastatic disease, may lead to disease recurrence and progression, even when SLN histologically are free of disease. Molecular up-staging of occult malignant disease has the potential to provide important information to facilitate the diagnosis, surveillance, and treatment of cancer. The detection of occult tumor cells in SLNs and blood provides a powerful tool for assessing early regional and systemic disease spread in patients who have AJCC stage II and III-not only melanoma but also other solid tumors. The use of varying panels of markers from different laboratories has hampered the interpretation of data and made it difficult to unravel the merits of molecular up staging. Molecular approaches have made a major impact on the field of infectious disease and should one day be of equal usefulness in the diagnosis of cancer.

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.

Aberrant hypermethylation of ID4 gene promoter region increases risk of lymph node metastasis in T1 breast cancer.

ID4 gene is a member of the inhibitor of DNA-binding (ID) family, which inhibits DNA binding of basic helix-loop-helix transcription factors. Certain human primary breast cancers reportedly have low or no expression of ID4 protein, but its role in carcinogenesis and cancer progression is unknown. To determine its possible role, we examined epigenetic inactivation of ID4 gene by promoter hypermethylation in human breast cell lines and T1 breast cancer tissues. Methylation status of ID4 promoter CpG island was assessed by methylation-specific PCR (MSP); ID4 mRNA level was assessed by quantitative real-time RT-PCR. Of eight cell lines, two were fully methylated, four were partially methylated, and two were not methylated. ID4 mRNA level was suppressed in fully methylated cell lines. ID4 hypermethylation was observed in 16 of 24 (67%) node-positive and seven of 36 (19%) node-negative T1 primary breast cancers matched by patient age and tumor diameter. It was a significant risk factor for nodal metastasis (OR 13.1, P=0.0004). ID4 mRNA level was suppressed in hypermethylated cancer specimens (P=0.014). ID4 may play an important suppressive role in tumor progression, and its silencing by hypermethylation may increase the risk of regional lymph node metastasis.

A mouse model of early-stage peritoneal metastasis: optimal RT-PCR-based method for detection of peritoneal micrometastases.

The presence of peritoneal micrometastasis in the abdomen is a poor prognostic factor in advanced gastric cancer. However, there is no standardized method for detection of peritoneal micrometastases. The aim of this study was to establish an animal model mimicking early phase peritoneal dissemination of advanced gastric cancer and then to use this model to compare the sensitivity and specificity of reverse transcription-polymerase chain reaction (RT-PCR) with peritoneal lavage fluid, randomly collected omentum with or without milky spots stained with activated carbon particles for detection of disseminated cancer cells. MKN-45-EGFP gastric cancer cells (1 x 10(3), 5 x 10(3) and 1 x 10(4)) were injected intraperitoneally into 4-week-old female BALBc nu/nu mice on day 0. Mice were sacrificed at 7 or 14 days postinjection. Peritoneal seeding was assessed by fluorescence stereomicroscopy. The sensitivities of RT-PCR with peritoneal lavage fluid, omentum with or without milky spots, were compared. Peritoneal seeding was confirmed in 8 of 10 mice injected with 1 x 10(4) MKN-45-EGFP cells by fluorescence stereomicroscopy. On days 7 and 14, the rate of the detection of CEA mRNA was 0, 50.0, and 87.5% in the omentum, omentum with milky spots and peritoneal lavage fluid, respectively. On days 7 and 14, the average level of CEA mRNA was 0, 51113+/-28225, and 3556+/-2842 in the omentum, omentum with milky spots and peritoneal lavage fluid, respectively. Molecular diagnosis using peritoneal lavage fluid is more sensitive than that using the omentum.

Multimarker quantitative real-time PCR detection of circulating melanoma cells in peripheral blood: relation to disease stage in melanoma patients.

BACKGROUND: Detection of melanoma cells in circulation may be important in assessing tumor progression. The objective of this study was to develop a specific, reliable multimarker quantitative real-time reverse transcription-PCR (qRT) assay for detecting melanoma cells in patients' blood. METHODS: We developed qRT assays for the mRNA of four melanoma-associated markers: MART-1, GalNAc-T, PAX-3, and MAGE-A3. In optimization studies, we tested 17 melanoma cell lines and 49 peripheral blood leukocyte (PBL) samples from volunteers. We performed RNA and melanoma cell dilution studies to assess the detection limits and imprecision of the assays. We measured the mRNAs in blood specimens from 94 melanoma patients [American Joint Committee on Cancer (AJCC) stage I, n = 20; II, n = 20; III, n = 32; IV, n = 22]. RESULTS: All markers were frequently detected in melanoma cell lines, whereas none of the markers was detected in PBLs from volunteers. The qRT assay could detect 1 melanoma cell in 10(7) PBLs in the melanoma cell-dilution studies. Markers were detected in 15%, 30%, 75%, and 86% of melanoma patients with AJCC stage I, II, III, and IV disease, respectively. The number of positive markers and AJCC stage were significantly correlated (Spearman correlation coefficient = 0.58; P < 0.0001). CONCLUSIONS: Multimarker qRT can detect circulating melanoma cells in blood. Measurement of the studied molecular markers in blood may be useful in detection of metastasis and monitoring treatment response of melanoma patients.

Epigenetic up-regulation of C-C chemokine receptor 7 and C-X-C chemokine receptor 4 expression in melanoma cells.

Histone deacetylation and DNA methylation establish epigenetic modifications, which through chromatin remodeling may result in gene silencing. We hypothesized that chemokine receptors C-C chemokine receptor 7 (CCR7) and C-X-C chemokine receptor 4 (CXCR4) on melanoma cells undergo epigenetic regulation. We investigated whether a histone deacetylase inhibitor and a demethylating agent influence CCR7 and CXCR4 expression on melanoma cells. Initially, microarray analysis was done to screen changes in chemokine receptor expression on melanoma cells after treatment with trichostatin A (TSA) and 5-Aza-2-deoxycytidine (5-Aza). CCR7 and CXCR4 mRNA expression were uniformly altered and selected for further investigation. Quantitative real-time reverse transcription-PCR assay, immunohistochemistry, and Western blot analysis were used to assess changes in mRNA and protein expression induced by TSA and 5-Aza in melanoma lines. Cell migration assays were conducted to assess the effects of altered CCR7 and CXCR4 expression on cell function. Treatment with TSA or 5-Aza increased gene expression of both CCR7 and CXCR4 in melanoma lines. TSA was the strongest enhancer. With combined treatment, CCR7 and CXCR4 mRNA expression was also up-regulated. Immunohistochemistry after combined treatment showed enhanced staining of both CCR7 and CXCR4 compared with control cells. Melanoma cell migration in TSA- and 5-Aza-treated cells was 7- and 2-fold higher than control cells for CCR7 and CXCR4, respectively. In summary, a histone deacetylase inhibitor and a demethylating agent up-regulated CCR7 and CXCR4 expression on melanoma cells. This increase in chemokine receptor expression correlated with functional activity. Most importantly, we have identified an epigenetic mechanism that may endogenously regulate chemokine receptor expression on melanoma cells.

Synthesis of universal unmethylated control DNA by nested whole genome amplification with phi29 DNA polymerase.

Optimization of highly sensitive methods to detect methylation of CpG islands in gene promoter regions requires adequate methylated and unmethylated control DNA. Whereas universal methylated control DNA is available, universal unmethylated control (UUC) DNA has not been made because demethylase is not available to remove methyl groups from all methylated cytosines. On the basis that DNA synthesized by DNA polymerase does not contain methylated cytosines, we developed a method to create UUC DNA by nested whole genome amplification (WGA) with phi29 DNA polymerase. Contamination of the template genomic DNA in UUC was only 3.1 x 10(-7), below the detection limit of sensitive methods used for methylation studies such as methylation-specific PCR. Assessment of microsatellite markers demonstrated that even nested phi29 WGA achieves highly accurate and homogeneous amplification with very low amounts of genomic DNA as an initial template. The UUC DNA created by nested phi29 WGA is practically very useful for methylation analysis.