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.

[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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Allelic imbalance of APAF-1 locus at 12q23 is related to progression of colorectal carcinoma.

APAF-1 gene, located at chromosome locus 12q23, is a key factor in the mitochondrial apoptotic pathway downstream of p53, and is a potential tumor suppressor gene. We hypothesized that APAF-1 gene dysfunction due to allelic imbalance (AI) contributes to the development and progression of colorectal carcinoma (CRC). AI at APAF-1 locus and microsatellite instability (MIN) in CRCs and adenomas were assessed by multiple microsatellite markers. The frequency of AI significantly increased with tumor progression; 0 of 33 (0%) adenomas, 14 of 49 (29%) primary CRCs, and 18 of 34 (53%) liver metastases had AI. A total of 12 metastases were matched with corresponding primary CRCs; in 11 of 12 (92%) pairs, the metastasis had same AI status as the corresponding primary tumor. APAF-1 mRNA transcription level was significantly decreased with AI in liver metastases (P=0.009). Promoter hypermethylation was found in three of 35 (9%) primary CRCs and one of 15 (7%) liver metastases by methylation-specific PCR but was not correlated with AI. MIN was observed in 11 of 49 (23%) primary CRCs and was a favorable prognostic factor. Our results suggest that APAF-1 gene haploinsufficiency caused by AI increases with tumor progression, and relates to hepatic metastasis.

Epigenetic inactivation of ID4 in colorectal carcinomas correlates with poor differentiation and unfavorable prognosis.

PURPOSE: ID4 gene is a member of the inhibitor of DNA binding (ID) family proteins that inhibit DNA binding of basic helix-loop-helix transcription factors. The epigenetic inactivation of ID4 gene on colorectal cancer (CRC) development and its clinical significance was assessed. EXPERIMENTAL DESIGN: In CRC cell lines, ID4 methylation status of the promoter region was assessed by methylation-specific PCR and bisulfite sequencing. The mRNA expression level was assessed by quantitative real-time reverse transcription-PCR. The methylation status of 9 normal epithelia, 13 adenomas, 92 primary CRCs, and 26 liver metastases was assessed by methylation-specific PCR. ID4 protein expression was assessed by immunohistochemistry analysis of tissue specimen. RESULTS: CRC cell lines were shown to be hypermethylated, and mRNA expression was suppressed and could be restored by 5-aza-cytidine treatment. In clinical specimens from normal epithelia, adenomas, primary CRCs, and liver metastases, the frequency of ID4 hypermethylation was 0 of 9 (0%), 0 of 13 (0%), 49 of 92 (53%), and 19 of 26 (73%), respectively, with a significant elevation according to CRC pathological progression. Methylation status of primary CRCs significantly correlated with histopathological tumor grade (P = 0.028). Immunohistochemistry analysis showed ID4 expression of normal colon epithelia, adenomas, and unmethylated primary CRCs but not hypermethylated CRC specimens. Among 76 American Joint Committee on Cancer stage I to IV patients who had undergone curative surgical resection, overall survival was significantly poorer in patients with hypermethylated ID4 bearing tumors (P = 0.0066). CONCLUSIONS: ID4 gene is a potential tumor suppressor gene for which methylation status may play an important role in the CRC progression.