Early intubation and decreased in-hospital mortality in patients with coronavirus disease 2019.

BACKGROUND: Some academic organizations recommended that physicians intubate patients with COVID-19 with a relatively lower threshold of oxygen usage particularly in the early phase of pandemic. We aimed to elucidate whether early intubation is associated with decreased in-hospital mortality among patients with novel coronavirus disease 2019 (COVID-19) who required intubation. METHODS: A multicenter, retrospective, observational study was conducted at 66 hospitals in Japan where patients with moderate-to-severe COVID-19 were treated between January and September 2020. Patients who were diagnosed as COVID-19 with a positive reverse-transcription polymerase chain reaction test and intubated during admission were included. Early intubation was defined as intubation conducted in the setting of ≤ 6 L/min of oxygen usage. In-hospital mortality was compared between patients with early and non-early intubation. Inverse probability weighting analyses with propensity scores were performed to adjust patient demographics, comorbidities, hemodynamic status on admission and time at intubation, medications before intubation, severity of COVID-19, and institution characteristics. Subgroup analyses were conducted on the basis of age, severity of hypoxemia at intubation, and days from admission to intubation. RESULTS: Among 412 patients eligible for the study, 110 underwent early intubation. In-hospital mortality was lower in patients with early intubation than those with non-early intubation (18 [16.4%] vs. 88 [29.1%]; odds ratio, 0.48 [95% confidence interval 0.27-0.84]; p = 0.009, and adjusted odds ratio, 0.28 [95% confidence interval 0.19-0.42]; p < 0.001). The beneficial effects of early intubation were observed regardless of age and severity of hypoxemia at time of intubation; however, early intubation was associated with lower in-hospital mortality only among patients who were intubated later than 2 days after admission. CONCLUSIONS: Early intubation in the setting of ≤ 6 L/min of oxygen usage was associated with decreased in-hospital mortality among patients with COVID-19 who required intubation. Trial Registration None.

Emergency gastrointestinal surgery in patients undergoing antithrombotic therapy in a single general hospital: a propensity score-matched analysis.

BACKGROUND: This study aimed to review and evaluate the surgical outcomes, particularly intraoperative severe blood loss and postoperative blood complications, of emergency gastrointestinal surgery in patients undergoing antithrombotic therapy (AT). Emergency surgeries for patients with antithrombotic medication have been increasing in the aging population. However, the effect of AT on intraoperative blood loss and perioperative complications remains unclear. METHODS: We retrospectively reviewed 732 patients who underwent emergency gastrointestinal surgery between April 2014 and March 2019. Patients were classified into AT group and Non-AT group, and propensity score-matched analysis was performed to compare the short surgical outcomes between the groups. Additionally, risk factors in severe estimated blood loss (EBL) and postoperative bleeding complications were assessed. RESULTS: Altogether, 64 patients received AT; 50 patients and 12, and 2 were given antiplatelet and anticoagulant, and both drugs, respectively. After propensity score matching, EBL (101 vs. 99 mL; p = 0.466) and postoperative complications (14 vs. 16 patients; p = 0.676) were similar between the groups (63 patients matched paired). Intraoperative severe bleeding (EBL ≥ 492 mL) occurred in 44 patients. Multivariate analysis using the full cohort revealed that antithrombotic drug use was not an independent risk factor for severe bleeding and postoperative bleeding complications. CONCLUSIONS: This study demonstrated antithrombotic drugs do not adversely affect the perioperative outcomes of emergency gastrointestinal surgery.

Emergency laparoscopic ileocecal resection for a low-grade appendiceal mucinous neoplasm with impending rupture: A case report.

INTRODUCTION AND IMPORTANCE: We report the case of a patient with a low-grade appendiceal mucinous neoplasm (LAMN) who underwent emergency laparoscopic ileocecal resection to avoid the metastatic spread of tumor cells due to an impending rupture. CASE PRESENTATION: A 55-year-old woman presented to our hospital with pain in the right lower quadrant of the abdomen. Computed tomography revealed a markedly tense appendiceal mucinous tumor with surrounding inflammation, and laboratory test results showed elevated serum C-reactive protein (7.47 mg/dL), indicating impending rupture of the appendix. Magnetic resonance imaging revealed nodules inside the appendix, suggesting the possibility of appendiceal cancer. We performed emergency laparoscopic ileocecal resection with regional lymph node dissection. The tumor was pathologically diagnosed as a LAMN without rupture. CLINICAL DISCUSSION: LAMN is classified as a clinically malignant tumor because it can cause pseudomyxoma peritonei due to perforation or the presence of residual tissue. Although an appendectomy would be appropriate for LAMN if the tumor margin is secured, ileocecal resection with lymph node dissection is necessary when preoperative discrimination of appendiceal cancer is impossible. CONCLUSION: Further studies of preoperative imaging for appropriate differential diagnosis were necessary.

[Two Cases of Pneumocystis Jirovecii Pneumonia during Chemotherapy for Gastrointestinal Cancer].

BACKGROUND: In recent years, there has been an increasing incidence of Pneumocystis jirovecci pneumonia(PCP)in immunosuppressed non-HIV patients. However, only a few studies on PCP developed during chemotherapy for gastrointestinal cancer have been reported. Case 1: A 72-year-old man was complaining of dyspnea during chemotherapy for unresectable gastric cancer. The patient showed high ß-D-glucan levels, and his sputum tested positive for sputum Pneumocystis PCR. Even after TMP-SMX administration, the patient's respiratory condition worsened; hence, intubation was needed. Finally, he died without showing any improvement. Case 2: A 75-year-old man underwent chemotherapy for a recurrence of cecal cancer and received steroid pulse for adverse events of optic neuritis. However, his respiratory condition worsened. Furthermore, his sputum tested positive for Pneumocystis PCR. Intensive care including TMP-SMX administration followed to improve his condition. DISCUSSION: PCP with non-HIV has a more acute onset and a poorer prognosis than that with HIV. It is necessary to identify PCP when there is a rapid progression of respiratory symptoms and pneumonia in cancer patients undergoing chemotherapy or steroid treatment.

[A Case of Duodenal Cancer with Advanced Lymph Node Metastasis Treated with mFOLFOX6 Preoperative Chemotherapy].

A 60-year-old man underwent surgical distal gastrectomy 10 years prior to receiving treatment for stomach cancer. He visited our hospital with complaints of abdominal fullness and weight loss. Abdominal computed tomography(CT)revealed intestinal blockage starting at the duodenum near the Treitz ligament. During upper alimentary canal endoscopy, a type 2 tumor coveringthe entire circumference of the horizontal duodenum was found, and biopsy results indicated that the tumor was a well-differentiated adenocarcinoma. Although no distant metastasis was observed in the abdominal CT scan, multiple attached bulky lymph nodes were observed leadingto a suspicion of metastasis. Finally, the patient was diagnosed as having progressive duodenal cancer accompanied with advanced lymph node metastasis. A gastrojejunal bypass surgery with laparoscopy was performed. When the patient's overall condition improved, mFOLFOX6 chemotherapy was started, and 6 courses were completed. As the sizes of the primary tumor and bulky lymph nodes decreased, surgery to completely remove the cancer was performed. UFT/UZEL supplemental postoperative chemotherapy was administered for 6 months. The patient remained alive, with no remissions 3 years post-surgery.

[Two Cases of Peritoneal Dissemination of Gastric Cancer Treated with CART Followed by Chemotherapy].

BACKGROUND: Cell-free and concentrated ascites reinfusion therapy(CART)is useful for relief of the symptoms caused by malignant ascites. We experienced 2 cases of untreated gastric cancer with massive ascites due to peritoneal dissemination, to whom chemotherapy was successfully introduced as a result of improvement of general conditions achieved by CART. Case 1: A 56-year-old woman with massive ascites was introduced for the treatment of gastric cancer. After a CART, oral ingestion became possible and S-1 plus oxaliplatin(SOX)therapy was introduced. Three courses of SOX therapy were possible until just before her death with 6 times of maintenance CART in total. Case 2: An 80-year-old man was introduced for the same reason. After a CART, he was treated with 4 courses of trastuzumab plus capecitabine plus oxaliplatin(Tra plus CapeOX)therapy with 5 times of maintenance CART in total. DISCUSSION: CART is useful for alleviating symptoms caused by malignant ascites and makes systemic chemotherapy possible because it improves and maintains the general conditions.

Laparoscopic surgery for repeated cecal volvulus with intestinal malrotation: A case report.

This is the first report of laparoscopic surgery for an advanced-age patient with cecal volvulus accompanied by intestinal malrotation. A 96-year-old woman who had previously undergone laparotomy for cecal volvulus underwent emergency laparoscopic surgery for recurrent volvulus. Because the cecum was about to rupture but not ischemic, we untwist the intestinal volvulus and fixed the cecum to the abdominal wall with a single suture. Five days after the surgery, the volvulus between the suture and the hepatic flexure of the colon recurred. We performed a second laparoscopic surgery in which we fixed the right side of the colon to the abdominal wall after diagnosing intestinal malrotation. Given its positioning, the sign of malrotation would not have been visible on preoperative CT images. As shown by this case, intestinal malrotation might lie behind the repeated cecal volvulus, and laparoscopic surgery may be a good option for volvulus with intestinal malrotation, even in cases with obstruction.

Quantitative analysis of methylation of genomic loci in early-stage rectal cancer predicts distant recurrence.

PURPOSE: There are no accurate prognostic biomarkers specific for rectal cancer. Epigenetic aberrations, in the form of DNA methylation, accumulate early during rectal tumor formation. In a preliminary study, we investigated absolute quantitative methylation changes associated with tumor progression of rectal tissue at multiple genomic methylated-in-tumor (MINT) loci sequences. We then explored in a different clinical patient group whether these epigenetic changes could be correlated with clinical outcome. PATIENTS AND METHODS: Absolute quantitative assessment of methylated alleles was used to assay methylation changes at MINT 1, 2, 3, 12, 17, 25, and 31 in sets of normal, adenomatous, and malignant tissues from 46 patients with rectal cancer. Methylation levels of these biomarkers were then assessed in operative specimens of 251 patients who underwent total mesorectal excision (TME) without neoadjuvant radiotherapy in a multicenter clinical trial. RESULTS: Methylation at MINT 2, 3, and 31 increased 11-fold (P = .005), 15-fold (P < .001), and two-fold (P = .02), respectively, during adenomatous transformation in normal rectal epithelium. Unsupervised grouping analyses of quantitative MINT methylation data of TME trial patients demonstrated two prognostic subclasses. In multivariate analysis of node-negative patients, this subclassification was the only predictor for distant recurrence (hazard ratio [HR], 4.17; 95% CI, 1.72 to 10.10; P = .002), cancer-specific survival (HR, 3.74; 95% CI, 1.4 to 9.43; P = .003), and overall survival (HR, 2.68; 95% CI, 1.41 to 5.11; P = .005). CONCLUSION: Methylation levels of specific MINT loci can be used as prognostic variables in patients with American Joint Committee on Cancer stage I and II rectal cancer. Quantitative epigenetic classification of rectal cancer merits evaluation as a stratification factor for adjuvant treatment in early disease.

Epigenetic silencing of cyclooxygenase-2 affects clinical outcome in gastric cancer.

PURPOSE: Overexpression of cyclooxygenase-2 (COX-2) in gastric cancer has been shown to enhance tumor progression. We investigated whether silencing by promoter region hypermethylation of the COX-2 gene contributes to disease outcome in gastric cancer. MATERIALS AND METHODS: COX-2 methylation status was initially assessed by capillary array electrophoresis methylation-specific polymerase chain reaction (CAE-MSP) and COX-2 protein expression by immunohistochemistry (IHC) in 40 primary gastric cancer tissues in a pilot study. Prognostic end points of correlative studies of COX-2 methylation status were time to recurrence, overall survival, and standard clinicopathologic features. CAE-MSP analysis was then validated in a second independent gastric cancer population (n = 137). RESULTS: COX-2 methylation was detected in 23% and 28% of the pilot and validation patient groups, respectively. COX-2 expression (IHC) in gastric tumors inversely correlated with COX-2 gene methylation status in the pilot study (P = .02). COX-2 methylation in tumors was significantly associated with lower T, N, and TNM stage in the validation patient group (P = .02, P = .006, and P = .008, respectively). Patients with COX-2 methylated tumors had significantly longer time to recurrence and improved overall survival in a multivariate analysis in the validation patient group (hazard ratio[HR], 0.49; 95% CI, 0.24% to 0.99%; HR, 0.62; 95% CI, 0.38% to 0.99%, respectively). CONCLUSION: Hypermethylation of COX-2 gene promoter was identified as an independent prognostic factor in gastric cancer patients. The results suggest promoter hypermethylation to be an important regulatory mechanism of COX-2 expression in gastric cancer and an important prognostic biomarker.

Assessment of methylation events during colorectal tumor progression by absolute quantitative analysis of methylated alleles.

To date, the epigenetic events involved in the progression of colorectal cancer are not well described. To study, in detail, methylation during colorectal cancer development in high-risk adenomas, we developed an assay combining in situ (on-slide) sodium bisulfite modification (SBM) of paraffin-embedded archival tissue sections with absolute quantitative assessment of methylated alleles (AQAMA). We tested the performance of the assay to detect methylation level differences between paired pre-malignant and malignant colorectal cancer stages. AQAMA assays were used to measure methylation levels at MINT (methylated in tumor) loci MINT1, MINT2, MINT12, and MINT31. Assay performance was verified on cell line DNA and standard cDNA. On-slide SBM, allowing DNA methylation assessment of 1 to 2 mm(2) of paraffin-embedded archival tissue, was employed. Methylation levels of adenomatous and cancerous components within a single tissue section in 72 colorectal cancer patients were analyzed. AQAMA was verified as accurately assessing CpG island methylation status in cell lines. The correlation between expected and measured cDNA methylation levels was high for all four MINT AQAMA assays (R >or= 0.966, P<0.001). Methylation levels at the four loci increased in 11% and decreased in 36% of specimens comparing paired adenoma and cancer tissues (P<0.0001 by Kolmogorov-Smirnov test). Single-PCR AQAMA provided accurate methylation level measurement. Variable MINT locus methylation level changes occur during malignant progression of colorectal adenoma. Combining AQAMA with on-slide SBM provides a sensitive assay that allows detailed histology-oriented analysis of DNA methylation levels and may give new, accurate insights into understanding development of epigenetic aberrancies in colorectal cancer progression.

Hypermethylation of adenosine triphosphate-binding cassette transporter genes in primary hyperparathyroidism and its effect on sestamibi imaging.

CONTEXT: Retention of technetium-(99m)-sestamibi ((99m)Tc-sestamibi) by parathyroid adenomas appears to be due to the loss of at least one membrane transporter, multidrug resistance 1 (MDR1), and possibly another, multidrug resistance-associated protein 1 (MRP1). OBJECTIVE: The objective was to determine whether hypermethylation of either gene plays a role in their expression and (99m)Tc-sestamibi retention. DESIGN: This was a retrospective study on a convenience sample of paraffin-embedded parathyroid glands. SETTING: The study was performed at the John Wayne Cancer Institute at Saint John's Health Center (Santa Monica, CA). PATIENTS: Forty-eight patients with primary hyperparathyroidism and five patients without parathyroid disease undergoing thyroid surgery provided 27 adenomatous, 10 hyperplastic, and 16 normal parathyroid glands. INTERVENTION: We performed immunohistochemistry, real-time quantitative RT-PCR, and methylation-specific PCR for MDR1 and MRP1 on archival parathyroid tissue and correlated these results with the patient's (99m)Tc-sestamibi scan. MAIN OUTCOME MEASURE: The main outcome measure was to determine whether hypermethylation of the genes for either transporter is associated with loss of their expression and with a positive (99m)Tc-sestamibi scan. RESULTS: The MDR1 gene was methylated in none of 12 normal glands, 19 of 27 adenomas, and three of 10 hyperplastic glands. Methylation of the MRP1 gene was uncommon (five of 48 tested glands). Methylation of the gene affected the transcript level only for MDR1. Among all glands, hypermethylation for MDR1 was more likely in (99m)Tc-sestamibi-positive scans (P < 0.001). CONCLUSION: In parathyroid tissue, hypermethylation of the MDR1 gene decreases its expression and is associated with increased detection of parathyroid adenomas by (99m)Tc-sestamibi parathyroid scans.

Higher amount of free circulating DNA in serum than in plasma is not mainly caused by contaminated extraneous DNA during separation.

Circulating DNA isolated from serum and plasma has been shown to be a useful biomarker in various diseases including cancer. Serum reportedly contains a higher amount of free circulating DNA than it does in plasma. The underlying reason for this is unclear, but important because it may have clinical implications in interpreting results and using the appropriate resource. Twenty-four pairs of serum and plasma samples were collected from patients with tumors, and free circulating DNA was quantified by real-time quantitative PCR (qPCR) for the ALU repeats, which had a sensitivity of 0.1 pg/microL of DNA in serum/plasma. The possibility of DNA loss was eliminated because ALU-qPCR does not require DNA purification from serum/plasma. The DNA concentrations of serum and plasma samples were 970 +/- 730 pg/microL and 180 +/- 150 pg/microL (mean +/- SD), respectively. The amount of DNA in paired serum and plasma specimens was positively correlated (R = 0.72 and P = 0.0002). An estimated 8.2% of total DNA in serum was extraneous; the concentration of DNA was 6.1 +/- 3.5 (mean +/- SD)-fold higher in serum than in paired plasma after subtraction of it. Contribution of extraneous DNA from cells in blood ruptured during the separation step was minor for explaining the difference between serum and plasma. A possible explanation was unequal distribution of DNA during separation from whole blood. We advocate that serum is a better specimen source for circulating cancer-related DNA as a biomarker.

Lymphatic mapping establishes the role of BRAF gene mutation in papillary thyroid carcinoma.

OBJECTIVE: To define the role of BRAF gene mutation in the progression of papillary thyroid carcinoma. SUMMARY BACKGROUND DATA: BRAF gene mutation is frequently detected in papillary thyroid carcinoma. Its role in pathogenesis or progression is under investigation. METHODS: Patients who underwent thyroidectomy and sentinel lymph node biopsy for papillary thyroid cancer were accrued. BRAF mutation was assessed in primary tumors and matched sentinel lymph nodes by a quantitative real-time PCR assay. RESULTS: Tissue specimens from 103 consecutive patients were evaluated. BRAF mutation of the primary tumor was detected in 34 (33%) patients. In 26 of 34 (76%) patients with BRAF mutation, concomitant lymph node metastasis was detected. On the contrary, in 69 patients with BRAF mutation-negative primary tumors, only 12 (17%) patients had lymph node metastasis (chi, P < 0.0001). BRAF mutation was detected in 20 of 26 (77%) lymph node metastases matched to BRAF mutation-positive primary tumors; it was not detected in lymph node metastases matched to BRAF mutation-negative primary tumors. Univariate analysis identified age, stage, tumor size, and BRAF mutation as prognostic factors for lymph node metastasis. In multivariate analysis, only BRAF mutation remained a significant prognostic factor for lymph node metastasis (odds ratio = 10.8, 95% confidence interval, 3.5-34.0, P < 0.0001). CONCLUSIONS: BRAF mutation may be a key genetic factor for the metastatic progression of papillary thyroid carcinoma. The study demonstrates that this gene mutation is a significant risk factor for locoregional lymph node metastasis and has potential utility as a surrogate marker.

Prediction of breast tumor progression by integrity of free circulating DNA in serum.

PURPOSE: Cell-free DNA circulating in serum is a candidate molecular biomarker for malignant tumors. Unlike uniformly truncated DNA released from apoptotic cells, DNA released from dead cancer cells varies in size. Serum DNA integrity, the ratio of longer fragments to total DNA, may be clinically useful for detecting breast cancer progression. PATIENTS AND METHODS: Serum samples from 51 healthy females and 83 females with primary breast cancers (eight American Joint Committee on Cancer stage 0, 24 stage I, 27 stage II, 21 stage III, and three stage IV) were assessed preoperatively. Serum DNA integrity was assessed by fragment length-dependent quantitative real-time polymerase chain reaction of ALU DNA repeats. RESULTS: Mean serum DNA integrity was significantly higher in patients with stage II, III, and IV breast cancers than in healthy females (P = .005, P < .0001, and P = .002, respectively). The receiver operating characteristic (ROC) curve for discriminating patients with stage II or more advanced breast cancers from healthy females had an area under the curve (AUC) of 0.79 (95% CI, 0.70 to 0.86). Mean serum DNA integrity was positively correlated to size of invasive cancers (r = 0.48; P < .0001) and significantly higher in the presence of lymphovascular invasion (LVI; 0.25 +/- 0.02 v 0.17 +/- 0.02; P < .0001) or lymph node (LN) metastasis (0.27 +/- 0.02 v 0.14 +/- 0.02; P < .0001). The ROC curve for discriminating LN metastasis had an AUC of 0.81 (95% CI, 0.72 to 0.89). Serum DNA integrity and LVI were significant for predicting LN metastasis in a multivariate analysis (P = .0002 and P < .0001, respectively). CONCLUSION: Integrity of serum circulating DNA is a promising molecular biomarker for detecting breast cancer tumor progression and regional LN metastases.

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.

Methylation of p16 and Ras association domain family protein 1a during colorectal malignant transformation.

Accurate assessment of gene methylation in formalin-fixed, paraffin-embedded archived tissue (FF-PEAT) by microdissection remains challenging because the tissue volume is small and DNA is damaged. In addition, methods for methylation assessment, such as methylation-specific PCR (MSP), require sodium bisulfite modification (SBM) on purified DNA, which causes major loss of DNA. On-slide SBM, in which DNA is modified in situ before isolation of tumor cells, eliminates DNA purification steps and allows histology-oriented assessment of gene methylation. This study describes a protocol and use of on-slide SBM using 20 FF-PEAT of colorectal cancers with intratumoral adenoma components to detect accumulation of gene methylation during colorectal malignant transformation. Deparaffinized tissue sections were incubated in sodium bisulfite solution for 8 hours at 60 degrees C, stained with hematoxylin, and then microdissected. Proteinase K lysate was directly used as a template in subsequent PCR. Using on-slide SBM, 282-bp-long bisulfite direct sequencing was possible. Yield of modified DNA was 2.6-fold greater than standard SBM on average. The mean conversion rate was 97%, and false-positive or false-negative results were not observed in subsequent MSP. Intratumoral heterogeneity by accumulation of p16 and Ras association domain family protein 1a methylation during malignant transformation were shown by MSP comparing cancer with adenoma parts within a single section. On-slide SBM is applicable in most methylation studies using FF-PEAT. It allows detailed, intratumoral analysis of methylation heterogeneity within solid tumors. On-slide SBM will significantly improve our approach and understanding of epigenetic events in minimal disease and the carcinogenic process.

Prospective surveillance effectively reduced rates of surgical site infection associated with elective colorectal surgery at a university hospital in Japan.

At a university hospital in Japan, the introduction of prospective surveillance and subsequent interventions was effective in reducing the rate of surgical site infection associated with elective colorectal surgery from 27.5% to 17.8% of surgeries. Japan should both recognize the importance of broader surveillance for surgical site infection and establish its own nationwide surveillance database.

Increased integrity of free circulating DNA in sera of patients with colorectal or periampullary cancer: direct quantitative PCR for ALU repeats.

BACKGROUND: Cell-free DNA circulating in blood is a candidate biomarker for malignant tumors. Unlike uniformly truncated DNA released from apoptotic nondiseased cells, DNA released from dead cancer cells varies in size. We developed a novel method to measure the ratio of longer to shorter DNA fragments (DNA integrity) in serum as a potential biomarker for patients with colorectal cancer (CRC) or periampullary cancers (PACs). METHODS: Sera from 32 patients with CRC (3 stage I, 14 stage II, 6 stage III, and 9 stage IV patients), 19 patients with PACs (2 stage I, 9 stage II, 1 stage III, and 7 stage IV patients), and 51 healthy volunteers were assessed by quantitative real-time PCR of ALU repeats (ALU-qPCR) with 2 sets of primers (115 and 247 bp) amplifying different lengths of DNA. We used serum directly as a template for ALU-qPCR without DNA purification. DNA integrity was determined as ratio of qPCR results of 247-bp ALU over 115-bp ALU. RESULTS: ALU-qPCR had a detection limit of 0.01 pg of DNA. Eliminating DNA purification reduced technical artifacts and reagent/labor costs. Serum DNA integrity was significantly increased for stage I/II and III/IV CRC and stage I/II and III/IV PACs (P = 0.002, P = 0.006, P = 0.022, and P <0.0001, respectively). ROC curves for detecting CRC and PACs had areas under the curves of 0.78 and 0.80, respectively. CONCLUSIONS: Direct ALU-qPCR is a robust, highly sensitive, and high-throughput method to measure serum DNA integrity. DNA integrity is a potential serum biomarker for detection and evaluation of CRC and PACs.

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.

Serial monitoring of circulating melanoma cells during neoadjuvant biochemotherapy for stage III melanoma: outcome prediction in a multicenter trial.

PURPOSE: Circulating tumor cells (CTCs) in blood may be important in assessing tumor progression and treatment response. We hypothesized that quantitative real-time reverse transcriptase polymerase chain reaction using multimarker mRNA assays could detect CTCs and be used as a surrogate predictor of outcome in patients receiving neoadjuvant biochemotherapy (BC) for melanoma. PATIENTS AND METHODS: Blood specimens were collected at four sampling points from 63 patients enrolled on a prospective multicenter phase II trial of BC before and after surgical treatment of American Joint Committee on Cancer stage III melanoma. Each specimen was assessed by quantitative real-time reverse transcriptase polymerase chain reaction for expression of four melanoma-associated markers: melanoma antigen recognized by T cells 1; beta1 --> 4-N-acetylgalactosaminyltransferase; paired box homeotic gene transcription factor 3; and melanoma antigen gene-A3 family, and the changes of CTCs during treatment and prognostic effect of CTCs after overall treatment on recurrence and survival were investigated. RESULTS: At a median postoperative follow-up time of 30.4 months, 44 (70%) patients were clinically disease free. In relapse-free patients, the number of detected markers significantly decreased during preoperative BC (P = .036), during postoperative BC (P = .002), and during overall treatment (P < .0001). Marker detection after overall treatment was associated with significant decreases in relapse-free and overall survival (P < .0001). By multivariate analysis using a Cox proportional-hazards model, the number of markers detected after overall treatment was a significant independent prognostic factor for overall survival (risk ratio, 12.6; 95% CI, 3.16 to 50.5; P = .0003). CONCLUSION: Serial monitoring of CTCs in blood may be useful for indicating systemic subclinical disease and predicting outcome of patients receiving neoadjuvant BC for metastatic melanoma.