Development of an alternative saliva test for diagnosis of SARS-CoV-2 using TRIzol: Adapting to countries with lower incomes looking for a large-scale detection program.

The use of saliva for the diagnosis of SARS-CoV-2 has shown to be a good alternative to nasopharyngeal swabs (NPS), since it permits self-collection, avoids the exposure of healthy persons to infected patients, reduces waiting times, eliminates the need of personal protective equipment and is non-invasive. Yet current saliva testing is still expensive due to the need of specialized tubes containing buffers to stabilize the RNA of SARS-CoV-2 and inactivate the virus. These tubes are expensive and not always accessible in sufficient quantities. We now developed an alternative saliva testing method, using TRIzol for extraction, viral inactivation, and storage of SARS-CoV-2 RNA, combined with RT-qPCR, which was comparable in its performance to NPS. Paired saliva samples and NPS were taken from 15 asymptomatic healthcare workers and one patient with SARS-CoV-2. Further 13 patients with SARS-CoV-2 were only saliva-tested. All the tests were performed according to CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. Saliva (4 mL) was taken in sterile 50 mL tubes, 1.5 mL TRIzol were added and mixed. Our results show that 5 µL of saliva RNA extracted with TRIzol allow for an adequate detection of the virus in patients positive for SARS-CoV-2 and was equally sensitive to NPS in TRIzol. We conclude that saliva testing using TRIzol is a recommendable method for diagnosis of SARS-CoV-2 since it has several advantages over currently used saliva tests: it can be done with normal sterile tubes, does not need cold-chain handling, is stable at room temperature, is non-invasive and less costly, making it more accessible for low-income countries. Cheaper saliva testing using TRIzol is especially relevant for low-income countries to optimize diagnosis and help define quarantine durations for families, healthcare workers, schools, and other public workplaces, thus decreasing infections and mortality caused by SARS-CoV-2.

BRAF V600E Expression by Immunohistochemistry in Colon Cancer and Clinico-pathologic Features Associated with BRAF-Mutated Colonic Cancers in Mexican Patients.

BACKGROUND: BRAF evaluation is currently limited to molecular techniques, which are expensive and not widely available to practicing pathologists. Our objective was to determine the diagnostic performance of immunohistochemistry (IHC) against BRAF V600E for BRAF mutation and the secondary objective was determining histopathological characteristics of colon carcinomas with BRAF mutated. METHODS: Cases of adenocarcinoma of the colon with a known BRAF mutation status were identified from the pathological files of our institution. RESULTS: We analyzed 135 cases, 13 cases had the BRAF mutation (9.6%) and 122 were non-mutated. The mutated cases expressed intense and diffusely the anti-antibody against BRAF V600E, and 119 (97.5%) of the 122 cases without mutation were negative and the remaining 3 were focal and weakly positive. The IHC demonstrated a sensitivity of 100%, specificity of 97.5%, positive predictive value of 81.3% (95% CI = 56.9 to 93.4%), negative predictive value of 100% (95% CI = 89 to 100%), and an overall accuracy of 97.8%. The only significant clinicopathological differences between cancers with BRAF mutated compared with BRAF non-mutated were that mutated had less lymph node metastases (23% vs. 68.1%) and the tumor size was greater (median 90 mm vs. 60 mm). The survival between groups was not statistically significant. CONCLUSION: IHC against BRAF V600E showed an excellent performance, making it feasible as an alternative for molecular examination. Tumors with BRAF mutated did not show distinctive clinico-pathological characteristics, except for a larger tumor size.

Addition of analysis of KRAS mutation or immunohistochemistry with MUC1 and carcinoembryonic antigen improves the diagnostic performance of fine needle aspiration cytology for the diagnosis of pancreatic carcinoma.

BACKGROUND: Pancreatic adenocarcinoma (PAC) is a health problem because of high lethality, increasing incidence and the absence of an early diagnosis. Biopsy by fine needle aspiration guided by endoscopic ultrasound has allowed obtaining tissue for cytopathological analysis, but there are several problems with their interpretation. We aimed to compare the diagnostic performance of the cytopathological analysis with the addition of either an immunohistochemical (IHC) panel or the KRAS mutation for the diagnosis of PAC. METHODS: We evaluated 62 pancreatic lesions by fine needle aspiration guided by endoscopic ultrasound, applying an IHC panel with mucin (MUC)-1, MUC4, carcinoembryonic antigen (CEA) and p53. All cases also had a KRAS mutation determination. Three cytopathologists blinded to clinical data and the KRAS status reviewed the cytology independently. We calculated diagnostic performances for the cytology alone, cytology+IHC and cytology+KRAS to show the best method to diagnose PAC. RESULTS: From 62 samples, 50 (80.6%) were PAC and 12 benign lesions. The cytopathological analysis correctly interpreted 26 malignant and 12 non-neoplastic cases (sensitivity 52%, specificity 100% and diagnostic accuracy 61.3%). The KRAS mutation was present in 88% of PAC. The cytology+ KRAS mutation increased the sensitivity by 10% and the diagnostic accuracy by 8%. The sensitivity increased by 2% adding either MUC1 or CEA to the cytology, and the diagnostic accuracy by 10 or 18%, respectively. CONCLUSION: The addition of IHC either with CEA or MUC1 improved the diagnostic performance of the cytology alone to diagnose PAC. The cytology + IHC evaluation was superior to the cytology + KRAS mutation to diagnose PAC.

Plasma neutrophil gelatinase-associated lipocalin and factors related to acute kidney injury and mortality in critically ill cancer patients.

RATIONALE: Acute kidney injury (AKI) is a frequent complication in critically ill cancer patients. OBJECTIVES: To assess plasma neutrophil gelatinase-associated lipocalin (NGAL) levels and risks factors associated with AKI and mortality. METHODS: We recruited 96 critically ill cancer patients and followed them prospectively. Plasma NGAL levels were determined at intensive care unit (ICU) admission and at 48 hours. We generated receiver operating characteristic curves to assess the ability of NGAL to predict AKI. Logistic regression analysis was performed to determine risks factors associated with AKI. Cox-regression analysis was performed to evaluate 6-month mortality. MEASUREMENTS AND MAIN RESULTS: From 96 patients, 60 (63%) developed AKI and 33 (55%) were classified as stages 2 and 3. In patients without AKI at admission, plasma NGAL levels revealed an area under the curve (AUC) = 0.522 for all AKI stages and an AUC = 0.573 for stages 2 and 3 AKI (85% sensitivity and 67% specificity for a 50.66 ng/mL cutoff). We identified sequential organ failure assessment (SOFA) score (without renal parameters) at admission as an independent factor for developing stages 2 and 3 AKI, and haemoglobin as a protective factor. We observed that metastatic disease, dobutamine use and stage 3 AKI were independent factors associated with 6-month mortality. CONCLUSIONS: In our cohort of critically ill cancer patients, NGAL did not predict AKI. SOFA score was a risk factor for developing AKI, and haemoglobin level was a protective factor for developing AKI. The independent factors associated with 6-month mortality included metastatic disease, dobutamine use, lactate and stage 3 AKI.

Medullary colonic carcinoma with microsatellite instability has lower survival compared with conventional colonic adenocarcinoma with microsatellite instability.

INTRODUCTION: Colorectal medullary carcinoma (MC) is a rare subtype of poorly differentiated adenocarcinoma (PDA) with unclear prognostic significance. Microsatellite instable (MSI) colorectal carcinomas have demonstrated better prognosis in clinical stage II. AIM: To analyze the survival and clinicopathological characteristics of MCs versus PDAs with MSI in clinical stage III. MATERIAL AND METHODS: We studied 22 cases of PDAs with MSI versus 10 MCs. RESULTS: Of the 10 MCs, 7 patients were men; the mean age was 57.8 ±5.6 years. The mean tumor size was 9.6 ±4.1 cm, and the primary site was the right colon in 9; 7 patients showed lymph node metastases (LNM) and lymphovascular invasion (LVI). Of the 22 PDA cases, 12 (54.5%) were women with a mean age of 75 ±16.1 years. The mean tumor size was 6.4 ±3.2 cm. Twelve (54.5%) presented in the right colon, 21 (95.5%) showed LNM and 7 (31.8%) LVI. Follow-up was 32 ±8 months, with a 5-year overall survival of 42.9% for MCs and 76.6% for PDAs (p = 0.048). Univariate analysis found local recurrence (p = 0.001) and medullary subtype (p = 0.043) associated with lower survival. CONCLUSIONS: Medullary carcinomas were of greater tumor size and associated with more LVI and worse survival versus PDAs with MSI in stage III.

Extramural perineural invasion in pT3 and pT4 rectal adenocarcinoma as prognostic factor after preoperative chemoradiotherapy.

Perineural invasion (PNI) is widely studied in malignant tumors, and its prognostic significance is well demonstrated in the head and neck and prostate carcinomas, but its significance in rectal cancer is controversial. Most studies have focused on evaluating mural PNI (mPNI); however, extramural PNI (ePNI) may influence the prognosis after rectal cancer resection. We evaluated the prognostic value of ePNI compared with mPNI and with non-PNI, in rectal resections after preoperative chemoradiotherapy in 148 patients with pT3 and pT4 rectal carcinomas. PNI was identified in 35 patients (23.6%), 60% of which were in the mPNI group. Factors associated with PNI were tumor invasion depth, lymph node metastasis, lymphovascular invasion, and venous invasion; patients with PNI were more likely to have positive resection margins (65.7% versus 11.6%). ePNI, compared with mPNI, was associated with female sex (64.3% versus 28.6%), positive surgical margins (42.8% versus 28.6%), recurrence (50% versus 28.6%), and death (92.9% versus 28.6%). The 5-year disease-specific survival rate was 78.1% for patients without PNI, compared with 63.7% for the mPNI group and 26.4% for the ePNI group (P<.001). On multivariate analysis, the independent adverse prognostic factors were ePNI (odds ratio [OR], 22.17; 95% confidence interval [CI], 17.03-24.58; P<.001), overall recurrence (OR, 9.19; CI, 6.11-10.63; P=.002), clinical stage IV (OR, 8.56; CI, 6.34-9.47; P=.003), and positive surgical margin (OR, 3.95; CI, 2.00-4.28; P=.047). In conclusion, we demonstrated the prognostic effect of ePNI for disease-specific survival in surgically resected pT3-pT4 rectal cancer patients with preoperative chemoradiotherapy.

Outcome of young patients with rectal adenocarcinoma.

BACKGROUND: There is an increase in the incidence of rectal carcinoma (RC) in young patients. METHODS: We analyzed 175 patients with sporadic RC which were divided in two groups according their age: 24 patients ≤40 years and 151 patients >40 years and the two groups were compared in order to determine if the outcomes (especially overall 5-year survival) were different. RESULTS: Overall 5-year survival was similar between groups (67.1% for patients over 40 years and 70.4% for those under 40 years, P=0.803). The only differences found were in some clinicopathologic features: patients <40 years showed more dissected lymph nodes (LNs) (21 vs. 15, P=0.035) and more LN metastasis (54.2% vs. 39.1%, P=0.048). In multivariate analysis factors associated with worse survival were incomplete resection and no use of neoadjuvant therapy. Age did not demonstrate prognostic value (P=0.077). CONCLUSIONS: RC in people ≤40 years demonstrated greater number of LN harvested and LN metastases but oncologic outcomes, especially 5-year overall survival, were similar between groups.

Mesorectal Invasion Depth in Rectal Carcinoma Is Associated With Low Survival.

BACKGROUND: Most cases of rectal cancer (RC) in our institution are in pathologic stage T3. They are a heterogeneous group but have been classified in a single-stage category. We performed the present study to validate the prognostic significance of the mesorectal extension depth (MED) in T3 RC measured in millimeters beyond the muscularis propria plane. MATERIALS AND METHODS: We performed a retrospective analysis of 104 patients with T3 RC who had undergone curative surgery after a course of preoperative chemoradiotherapy at a tertiary referral cancer hospital. The patients were grouped by MED (T3a, < 1 mm; T3b, 1-5 mm; T3c > 5-10 mm; and T3d > 10 mm). The clinicopathologic data and disease-free survival were analyzed. RESULTS: The 5-year disease-free survival rate according to the T3 subclassification was 87.5% for those with T3a, 57.9% for T3b, 38.7% for T3c, and 40.3% for those with T3d tumors (P = .050). On univariate and multivariate analysis, the prognostic factors affecting survival were overall recurrence (hazard ratio [HR], 3.670; 95% confidence interval [CI], 1.710-7.837; P = .001), histologic grade (HR, 2.204; 95% CI, 1.156-4.199; P = .016), mesorectal invasion depth (HR, 1.885; 95% CI, 1.164-3.052; P = .010), and lymph node metastasis (HR, 1.211; 95% CI, 1.015-1.444; P = .033). CONCLUSION: MED is a significant prognostic factor in patients with T3 RC who have undergone neoadjuvant chemoradiotherapy, especially when the MED is > 5 mm. The MED could be as important as other clinicopathologic factors in predicting disease-specific survival.

Pre-operative Neutrophils/Lymphocyte Ratio in Rectal Cancer Patients with Preoperative Chemoradiotherapy.

BACKGROUND: Several studies have reported that an elevation in neutrophils/lymphocyte ratio (NLR) is correlated with poor survival in patients with colorectal cancer, but in rectal cancer (RC), it has been reported only in a few studies. It is necessary to separate colon cancer and rectal cancer to clarify the prognostic significance of NLR, especially in patients who received chemoradiotherapy. METHODS: It is a comparative, observational retrospective study of a cohort of 175 patients. We grouped the patients into two based on their NLR (0-3 vs. > 3) to correlate with disease-specific survival (DSS) and pathologic complete response (pCR). RESULTS: The average NLR was 2.65 + 1.32 (range 0.58-6.89), and 144 (82.3%) patients had an NLR of 0-3. The median follow-up was 33.53 months. There were no differences in pCR between the two groups. The 5-year DSS was 78.8%. NLR did not correlate with survival. Mesorectal quality, pT3-4 tumors, lymph node metastasis, lymphovascular invasion, perineural invasion, positive margins and recurrence were statistically significant predictors of increased mortality in univariate analysis. In multivariate analysis, only overall recurrence correlated with poor survival. The analysis of the association of NLR with outcomes with different cut points (2.0, 2.5, 4 and 5) did not show differences in DSS and pCR. CONCLUSION: In our cohort, the NLR did not serve as a prognostic marker in patients with locally advanced rectal cancer and who received chemoradiotherapy and did not correlate with pCR as well.

Thyroid Transcription Factor-1 Expression in Adenocarcinomas of the Bile Duct.

BACKGROUND: The immunoreactivity of thyroid transcription factor-1 (TTF-1) is a very specific marker for lung and thyroid neoplasms; the expression of TTF-1 has also been demonstrated in extrapulmonary carcinomas. We examined the expression of TTF-1 in 15 intestinal-type adenocarcinomas of the extrahepatic bile duct. We then compared the expression to TTF-1 staining with other immunohistochemical markers including cytokeratin (CK) 7, CK20, caudal-type homeobox transcription factor 2 (CDX2), Napsin A, and MUC2. We additionally compared the clinicopathological prognostic factors with the TTF-1 expression status. RESULTS: Nuclear TTF-1 staining was detected in 2 cases (13.3%), and Napsin A was positive in the same 2 cases (13.3%). All cases were positive for CK20, CDX2, and MUC2; 5 cases were positive for CK7. There was no correlation between TTF-1 expression and the clinicopathological characteristics. CONCLUSIONS: To avoid potential pitfalls, TTF-1 should be interpreted in conjunction with the clinical setting, histology, and the results of markers such as CK7, CK20, Napsin A, and CDX2. This report is the first of TTF-1 positivity in adenocarcinomas from the extrahepatic biliary tract.

Mesorectal pathologic assessment in two grades predicts accurately recurrence, positive circumferential margin, and correlates with survival.

BACKGROUND AND OBJECTIVES: We aimed to determine the prognostic value of the mesorectum quality assessed in a two-grade system compared with a classic system. METHODS: Consecutive patients undergoing surgery for rectal cancer were included (n = 103). Mesorectum was assessed into three grades (classic system: complete, nearly complete, incomplete) and compared with a two-grade system (adequate, inadequate). RESULTS: Mesorectum was complete in 62 (60.25%) patients, nearly complete in 21, and incomplete in 20. Reassessment showed adequate mesorectum in 83 (80.5%) patients and inadequate in 20. A R0 resection was achieved in 90.4% of adequate mesorectum and in 65% of inadequate mesorectum (P = 0.006). Recurrence was present in 18% of adequate mesorectum patients as compared with 50% of inadequate mesorectum (P = 0.003). The classic system failed to accurately predict the 5-year survival rate between complete (78.9%) and nearly complete (86.2%) categories (P = 0.235); whereas a two grading system showed a 5-year survival rate of 80.8% for adequate versus 39.3% for inadequate (P = 0.034). CONCLUSION: High recurrence occurred in inadecuate mesorectum patients and was correlated with R1/R2 resections, positive margins, and decreased survival. We propose a simplified classification of mesorectum that correlates with survival and overall recurrence.