Detection of activated KRAS from cancer patient peripheral blood using a weighted enzymatic chip array.

BACKGROUND: The KRAS oncogene was one of the earliest discoveries of genetic alterations in colorectal and lung cancers. Moreover, KRAS somatic mutations might be used for predicting the efficiency of anti-EGFR therapeutic drugs. The purpose of this research was to improve Activating KRAS Detection Chip by using a weighted enzymatic chip array (WEnCA) platform to detect activated KRAS mutations status in the peripheral blood of non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC) patients in Taiwan. METHODS: Our laboratory developed an Activating KRAS Detection Chip and a WEnCA technique that can detect activated KRAS mutation status by screening circulating cancer cells in the surrounding bloodstream. We collected 390 peripheral blood samples of NSCLC patients (n = 210) and CRC patients (n = 180) to evaluate clinical KRAS activation using this gene array diagnosis apparatus, an Activating KRAS Detection Chip and a WEnCA technique. Subsequently, we prospectively enrolled 88 stage III CRC patients who received adjuvant FOLFOX-4 chemotherapy with or without cetuximab. We compared the chip results of preoperative blood specimens and their relationship with disease control status in these patients. RESULTS: After statistical analysis, the sensitivity of WEnCA was found to be 93%, and the specificity was found to be 94%. Relapse status and chip results among the stage III CRC patients receiving FOLFOX-4 plus cetuximab (n = 59) and those receiving FOLFOX-4 alone (n = 29) were compared. Among the 51 stage III CRC patients with chip negative results who were treated with FOLFOX-4 plus cetuximab chemotherapy, the relapse rate was 33.3%; otherwise, the relapse rate was 48.5% among the 23 out of 88 patients with chip negative results who received FOLFOX-4 alone. Negative chip results were significantly associated to better treatment outcomes in the FOLFOX-4 plus cetuximab group (P = 0.047). CONCLUSIONS: The results demonstrated that the WEnCA technique is a sensitive and convenient technique that produces easy-to-interpret results for detecting activated KRAS from the peripheral blood of cancer patients. We suggest that the WEnCA technique is also a potential tool for predicting responses in CRC patients following FOLFOX-4 plus cetuximab chemotherapy.

Decreasing relapse in colorectal cancer patients treated with cetuximab by using the activating KRAS detection chip.

The KRAS oncogene was among the first genetic alterations in colorectal cancer (CRC) to be discovered. Moreover, KRAS somatic mutations might be used for predicting the efficiency of anti-epidermal growth factor receptor therapeutic drugs. Because the KRAS mutations are similar in the primary CRC and/or the CRC metastasis, KRAS mutation testing can be performed on both specimen types. The purpose of this study was to investigate the clinical advantage of using a KRAS pathway-associated molecule analysis chip to analyze CRC patients treated with cetuximab. Our laboratory developed a KRAS pathway-associated molecule analysis chip and a weighted enzymatic chip array (WEnCA) technique, activating KRAS detection chip, which can detect KRAS mutation status by screening circulating cancer cells in the bloodstream. We prospectively enrolled 210 stage II-III CRC patients who received adjuvant oxaliplatin plus infusional 5-fluorouracil/leucovorin (FOLFOX)-4 chemotherapy with or without cetuximab. We compared the chip results of preoperative blood specimens with disease control status in these patients. Among the 168 CRC patients with negative chip results, 119 were treated with FOLFOX-4 plus cetuximab chemotherapy, and their relapse rate was 35.3 % (42/119). In contrast, the relapse rate was 71.4 % among the patients with negative chip results who received FOLFOX-4 treatment alone (35/49). Negative chip results were significantly correlated with better treatment outcomes in the FOLFOX-4 plus cetuximab group (P < 0.001). We suggest that the activating KRAS detection chip is a potential tool for predicting clinical outcomes in CRC patients following FOLFOX-4 treatment with or without cetuximab therapy.

Significant overexpression of DVL1 in Taiwanese colorectal cancer patients with liver metastasis.

Undetected micrometastasis plays a key role in the metastasis of cancer in colorectal cancer (CRC) patients. The aim of this study is to identify a biomarker of CRC patients with liver metastasis through the detection of circulating tumor cells (CTCs). Microarray and bioinformatics analysis of 10 CRC cancer tissue specimens compared with normal adjacent tissues revealed that 31 genes were up-regulated (gene expression ratio of cancer tissue to paired normal tissue > 2) in the cancer patients. We used a weighted enzymatic chip array (WEnCA) including 31 prognosis-related genes to investigate CTCs in 214 postoperative stage I-III CRC patients and to analyze the correlation between gene expression and clinico-pathological parameters. We employed the immunohistochemistry (IHC) method with polyclonal mouse antibody against DVL1 to detect DVL1 expression in 60 CRC patients. CRC liver metastasis occurred in 19.16% (41/214) of the patients. Using univariate analysis and multivariate proportional hazards regression analysis, we found that DVL1 mRNA overexpression had a significant, independent predictive value for liver metastasis in CRC patients (OR: 5.764; 95% CI: 2.588-12.837; p < 0.0001 on univariate analysis; OR: 3.768; 95% CI: 1.469-9.665; p = 0.006 on multivariate analysis). IHC staining of the immunoreactivity of DVL1 showed that DVL1 was localized in the cytoplasm of CRC cells. High expression of DVL1 was observed in 55% (33/60) of CRC tumor specimens and was associated significantly with tumor depth, perineural invasion and liver metastasis status (all p < 0.05). Our experimental results demonstrated that DVL1 is significantly overexpressed in CRC patients with liver metastasis, leading us to conclude that DVL1 could be a potential prognostic and predictive marker for CRC patients.

Clinical Detection of Chronic Rhinosinusitis through Next-Generation Sequencing of the Oral Microbiota.

Chronic rhinosinusitis (CRS) is the chronic inflammation of the sinus cavities of the upper respiratory tract, which can be caused by a disrupted microbiome. However, the role of the oral microbiome in CRS is not well understood. Polymicrobial and anaerobic infections of CRS frequently increased the difficulty of cultured and antibiotic therapy. This study aimed to elucidate the patterns and clinical feasibility of the oral microbiome in CRS diagnosis. Matched saliva and nasal swabs were collected from 18 CRS patients and 37 saliva specimens from normal volunteers were collected for 16S rRNA sequencing. The α-diversity of the saliva displayed no significant difference between control and CRS patients, whereas the ß-diversity was significantly different (p = 0.004). Taxonomic indices demonstrated that Veillonella dispar, Rothia mucilaginosa, and Porphyromonas endodontalis were enriched, while Campylobacter and Cardiobacterium were reduced in the saliva of CRS patients. These microbial markers could significantly distinguish CRS patients from control (AUC = 0.939). It is noted that the 16S rRNA results of the nasal swab were consistent with the nasopharynx aerobic culture, and additionally detected multiple pathogens in CRS patients. In summary, these results indicated these oral microbiomes may provide a novel signal for CRS detection and that NGS may be an alternative approach for CRS diagnosis.

Faster waning of the rubella-specific immune response in young pregnant women immunized with MMR at 15 months.

PROBLEM: Vaccination is the best protection against rubella and congenital rubella infection. Although a high rate of immunization coverage is achieved in Taiwan, it is unknown if the vaccine-induced immunity persists from the age of vaccination to childbearing age. METHODS OF STUDY: A total of 5,988 prenatal rubella IgG test results of young pregnant women aged 19-23 years old from six hospitals during January 2001 to December 2008 and January 2013 to December 2017 were analyzed. We compared the rubella seropositivity rates and titers in these women who were vaccinated with MMR vaccine in four different vaccination age cohorts. RESULTS: The overall rubella seropositivity rate was 87.4% (95% CI: 86.6%-88.3%), and the mean rubella IgG level was 39 IU/mL among young pregnant women aged 19-23 years. Women in the elementary cohort had the highest rubella positivity of 90.8% (95% CI: 89.6%-91.9%), and levels gradually decrease to 84.6% (95% CI: 82.4%-86.7%) in 15-month plus cohort. The average rubella IgG was only 25 IU/mL for the 15-month plus cohort. Women in cohorts immunized at younger age exhibited significantly lower chances of being seropositive relative to women in older cohort after adjusting other factors (all P < .01). CONCLUSION: The rubella seropositivity rate and rubella IgG levels were low among young women aged 19-23 years, especially in cohorts immunized at younger age. As rubella immunity wanes over time, a third dose of MMR may be a protective strategy for women who conceive later in life.