Xpert Breast Cancer STRAT4 Assay using fine-needle aspiration biopsy samples in a resource-constrained setting: a prospective diagnostic accuracy study.

BACKGROUND: Use of fine-needle aspiration biopsy (FNAB) specimens on Xpert Breast Cancer STRAT4 Assay (STRAT4; Cepheid, Sunnyvale, CA, USA), a CE-marked in-vitro diagnostic medical device, could potentially increase access to breast cancer biomarker testing in resource-constrained settings. We aimed to assess the performance of a research use-only version of STRAT4 using FNAB specimens in Tanzania. METHODS: In this prospective diagnostic accuracy study, patients aged 18 years or older with palpable breast masses presenting to the FNAB Clinic at Muhimbili National Hospital (Dar es Salaam, Tanzania) were recruited consecutively. Patients who were pregnant, lactating, or had a previous diagnosis of breast cancer were excluded. STRAT4 testing was performed on off-label FNAB samples using four protocols: the 1 × protocol (using the standard lysate method) on FNAB smears (1 × FNAB), quick lysis and Maui protocols (both on FNAB smears), and the 1 × protocol on formalin-fixed paraffin-embedded (FFPE) cell block material (1 × cell block). For 1 × FNAB and 1 × cell block, tissue was processed using FFPE lysis reagent, incubated at 80°C with proteinase K, and followed by addition of 95% or higher ethanol. Quick lysis was processed using FFPE lysis reagent and 95% or higher ethanol, whereas Maui was processed using a proprietary research-use only lysis reagent. The primary outcomes were overall concordance, sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) of STRAT4 as compared with immunohistochemistry or immunohistochemistry plus fluorescence in-situ hybridisation performed on cell blocks using clinically validated protocols in a Clinical Laboratory Improvement Amendments-accredited laboratory at the University of California, San Francisco (San Francisco, CA, USA). FINDINGS: Between Nov 29, 2017, and Dec 17, 2020, 208 patients were enrolled. Of 208 cases, 51 (25%) were excluded from analysis because of insufficient tissue in the cell block or absent cell blocks, leaving 157 participants (all female) for analysis. For oestrogen receptor, 1 × FNAB had the best performance, with an overall concordance of 95% (95% CI 90-100), sensitivity of 94% (85-100), specificity of 97% (90-100), and AUC of 0·96 (0·81-1·00). For progesterone receptor, 1 × cell block had the best overall performance (overall concordance 89% [95% CI 84-95], sensitivity 91% [82-99], and specificity 89% [81-97], with an AUC of 0·93 [0·89-0·99]) and 1 × FNAB performed the best among the smear protocols, with a concordance of 84% (95% CI 74-93), sensitivity of 63% (43-82), specificity of 97% (92-100), and AUC of 0·91 (0·72-0·97). For HER2, Maui had the highest agreement, with an overall concordance of 93% (95% CI 89-98), sensitivity of 96% (88-100), specificity of 92% (87-98), and AUC of 0·95 (0·98-1·00). For Ki67, Maui had the best performance of smear protocols, with a concordance of 73% (95% CI 64-82), sensitivity of 70% (58-81), specificity of 81% (66-96), and AUC of 0·80 (0·54-0·82). INTERPRETATION: Processing FNAB samples with STRAT4 is feasible in Tanzania, and performance for the oestrogen receptor is robust. Further optimisation of STRAT4 for FNAB has the potential to improve timely access to breast cancer diagnostics in resource-constrained settings. FUNDING: US National Institutes of Health; UCSF Global Cancer Program, Helen Diller Family Comprehensive Cancer Center; UCSF Department of Pathology; and Cepheid.

Prognostic impact of HER2 biomarker levels in trastuzumab-treated early HER2-positive breast cancer.

BACKGROUND: Overexpression of human epidermal growth factor receptor 2 (HER2) caused by HER2 gene amplification is a driver in breast cancer tumorigenesis. We aimed to investigate the prognostic significance of manual scoring and digital image analysis (DIA) algorithm assessment of HER2 copy numbers and HER2/CEP17 ratios, along with ERBB2 mRNA levels among early-stage HER2-positive breast cancer patients treated with trastuzumab. METHODS: This retrospective study comprised 371 early HER2-positive breast cancer patients treated with adjuvant trastuzumab, with HER2 re-testing performed on whole tumor sections. Digitized tumor tissue slides were manually scored and assessed with uPath HER2 Dual ISH image analysis, breast algorithm. Targeted ERBB2 mRNA levels were assessed by the Xpert® Breast Cancer STRAT4 Assay. HER2 copy number and HER2/CEP17 ratio from in situ hybridization assessment, along with ERBB2 mRNA levels, were explored in relation to recurrence-free survival (RFS). RESULTS: The analysis showed that patients with tumors with the highest and lowest manually counted HER2 copy number levels had worse RFS than those with intermediate levels (HR = 2.7, CI 1.4-5.3, p = 0.003 and HR = 2.1, CI 1.1-3.9, p = 0.03, respectively). A similar trend was observed for HER2/CEP17 ratio, and the DIA algorithm confirmed the results. Moreover, patients with tumors with the highest and the lowest values of ERBB2 mRNA had a significantly worse prognosis (HR = 2.7, CI 1.4-5.1, p = 0.003 and HR = 2.8, CI 1.4-5.5, p = 0.004, respectively) compared to those with intermediate levels. CONCLUSIONS: Our findings suggest that the association between any of the three HER2 biomarkers and RFS was nonlinear. Patients with tumors with the highest levels of HER2 gene amplification or ERBB2 mRNA were associated with a worse prognosis than those with intermediate levels, which is of importance to investigate in future clinical trials studying HER2-targeted therapy.

Improving detection of carcinoma in situ in bladder cancer: urinary cytology vs the Xpert® BC Monitor.

OBJECTIVE: To investigate and compare the performance of urinary cytology and the Xpert BC Monitor test in the detection of bladder cancer in various clinically significant patient cohorts, including patients with carcinoma in situ (CIS), in a prospective multicentre setting, aiming to identify potential applications in clinical practice. PATIENTS AND METHODS: A total of 756 patients scheduled for transurethral resection of bladder tumour (TURBT) were prospectively screened between July 2018 and December 2020 at six German University Centres. Central urinary cytology and Xpert BC Monitor tests were performed prior to TURBT. The diagnostic performance of urinary cytology and the Xpert BC Monitor was evaluated according to sensitivity (SN), specificity (SC), negative predictive value (NPV) and positive predictive value (PPV). Statistical comparison of urinary cytology and the Xpert BC Monitor was conducted using the McNemar test. RESULTS: Of 756 screened patients, 733 (568 male [78%]; median [interquartile range] age 72 [62-79] years) were included. Bladder cancer was present in 482 patients (65.8%) with 258 (53.5%) high-grade tumours. Overall SN, SC, NPV and PPV were 39%, 93%, 44% and 92% for urinary cytology, and 75%, 69%, 59% and 82% for the Xpert BC Monitor. In patients with CIS (concomitant or solitary), SN, SC, NPV and PPV were 59%, 93%, 87% and 50% for urinary cytology, and 90%, 69%, 95% and 50% for the Xpert BC Monitor. The Xpert BC Monitor missed four tumours (NPV = 98%) in patients with solitary CIS, while potentially avoiding 63.3% of TURBTs in inconclusive or negative cystoscopy and a negative Xpert result. CONCLUSION: Positive urinary cytology may indicate bladder cancer and should be taken seriously. The Xpert BC Monitor may represent a useful diagnostic tool for correctly identifying patients with solitary CIS and unsuspicious or inconclusive cystoscopy.

Temporal and spatial variation in hydrogen sulfide (H2S) emissions during holopelagic Sargassum spp. decomposition on beaches.

BACKGROUND: Since 2011, over 30 tropical Atlantic nations have experienced substantial landings of holopelagic Sargassum spp. Its decomposition results in the production of hydrogen sulfide (H2S), which, in elevated concentrations, can pose a threat to human health. This study aims to enhance our understanding of the temporal and spatial variability in H2S emissions during the decomposition of Sargassum on beaches. The primary objective is to assess potential exposure risks for local populations, tourists, and cleanup workers. METHODS: H2S levels were monitored using a SENKO sensor (SGTP-H2S; limit of detection 0.1-100 ppm; resolution 0.1 ppm) at four distances from Sargassum accumulation points of (0, 10, 30, and 40 m) in Puerto Morelos, Mexico, during 2022 and 2023. RESULTS: Elevated concentrations of H2S were detected beneath the Sargassum piles, with 23.5% of readings exceeding 5 ppm and occasional spikes above 100 ppm. Above the piles, 87.3% of the measurements remained below 2 ppm, and the remainder fell between 2.1 and 5.2 ppm. At 10 m from the shoreline, 90% of measurements registered below 0.1 ppm, and the remaining 10% were below 2 ppm. Readings at 30 and 40 m consistently recorded levels below 0.1 ppm. H2S concentrations positively correlated with Sargassum pile height, the temperature beneath the piles, and wind speed. CONCLUSIONS: Our findings suggest no immediate and significant exposure risk for residents or tourists. However, Sargassum cleanup workers face a higher exposure risk, potentially encountering concentrations above 5 ppm for nearly one-fourth of the working time.

Multi-Institutional Study of Pathologist Reading of the Programmed Cell Death Ligand-1 Combined Positive Score Immunohistochemistry Assay for Gastric or Gastroesophageal Junction Cancer.

The assessment of the expression of programmed cell death ligand-1 (PD-L1) using immunohistochemistry (IHC) has been controversial since its introduction. The methods of assessment and the range of assays and platforms contribute to confusion. Perhaps the most challenging aspect of PD-L1 IHC is the combined positive score (CPS) method of interpretation of IHC results. Although the CPS method is prescribed for more indications than any other PD-L1 scoring system, its reproducibility has never been rigorously assessed. In this study, we collected a series of 108 gastric or gastroesophageal junction cancer cases, stained them using the Food and Drug Administration-approved 22C3 assay, scanned them, and then circulated them to 14 pathologists at 13 institutions for the assessment of interpretative concordance for the CPS system. We found that higher cut points (10 or 20) performed better than a CPS of <1 or >1. We used the Observers Needed to Evaluate Subjective Tests algorithm to assess how the CPS system might perform in the real-world setting and found that the cut points of <1 or >1 showed an overall percent agreement of only 30% among the pathologist raters, with a plateau occurring at 8 raters. The raters performed better at higher cut points. However, the best cut point of <20 versus that of >20 was still disappointing, with a plateau at an overall percent agreement of 70% (at 7 raters). Although there is no ground truth for CPS, we compared the score with quantitative messenger RNA measurement and showed no relationship between the score (at any cut point) and messenger RNA amount. In summary, we showed that CPS shows high subjective variability among pathologist readers and is likely to perform poorly in the real-world setting. This system may be the root cause of the poor specificity and relatively low predictive value of IHC companion diagnostic tests for PD-1 axis therapies that use the CPS system.

Mid-term Follow Up of a Highly Porous Acetabular Component for Primary Total Hip Arthroplasty.

INTRODUCTION: As implant technology has continued to improve over the past decade, there has been an increase in the utilization of highly porous metal substrate acetabular components for primary total hip arthroplasty (THA). These implants have several theoretical benefits including a lower modulus of elasticity, which may result in a reduction in stress shielding, a higher coefficient of friction, which may enable better initial implant fixation, as well as higher porosity that may facilitate improved biological fixation. Although these components are implanted frequently, there are some studies that have posed concerns regarding radiographic evidence of loosening. Therefore, the purpose of this study was to assess: 1) The quality of fixation of porous metal acetabular components based on radiographs; 2) clinical outcomes; and 3) revision rates. MATERIALS AND METHODS: A total of 159 patients (169 hips) who had undergone a primary THA utilizing a porous metal primary acetabular cup with minimum two-year follow up were assessed. The study cohort consisted of 51% women, had a mean age of 65 years (range, 30 to 92 years), a mean body mass index (BMI) of 29kg/m2 (range, 15 to 54), and a mean follow up of approximately four years (range, three to six years). Acetabular revision for component failure was documented. Radiographic assessments were independently performed by two fellowship-trained arthroplasty surgeons to determine implant stability and radiolucencies. Clinical evaluations were made by assessing the hip disability and osteoarthritis outcome score (HOOS-Jr) survey scores. Failure was defined as the need to revise the acetabular component, for either septic or aseptic pathology. RESULTS: At final follow up, one patient had definitive loosening, one had probable loosening, and three patients had possible loosening. Only 3.0% had radiolucencies or radiosclerotic lesions in at least one zone. Of these patients, three developed progressive radiolucencies. All patients achieved excellent postoperative HOOS-Jr scores, and no significant differences were noted between patients who did not have loosening compared to patients who had possible or probable loosening. Only two patients underwent revision for aseptic loosening of the cup (success rate for this implant was 98.8% [2/169]). DISCUSSION: There is a paucity of studies focused on the results of this porous metal substrate acetabular component, with some of the current literature reporting conflicting outcomes. Our study reported a low acetabular revision rate of only 1.2% at an approximate mean follow up of four years. The incidence of radiolucencies and progressive radiolucencies were lower (3.0%) than has been found in some studies. Overall, the results of this study support the utilization of this acetabular component in appropriately indicated patients. CONCLUSION: These data show a low rate of acetabular revision at mean four-year follow up.

Longitudinal follow-up and performance validation of an mRNA-based urine test (Xpert® Bladder Cancer Monitor ) for surveillance in patients with non-muscle-invasive bladder cancer.

OBJECTIVE: To evaluate the performance of the Xpert Bladder Cancer Monitor (Xpert; Cepheid, Sunnyvale, CA, USA) test as a predictor of tumour recurrence in patients with non-muscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS: Patients (n = 429) undergoing surveillance for NMIBC underwent Xpert, cytology, and UroVysion testing. Patients with a positive Xpert and a negative cystoscopy result (positive-negative [PN] group, n = 66) and a control group of double negative patients (negative Xpert and cystoscopy results [NN] group) were followed for 12 months (±90 days). RESULTS: Histology-confirmed recurrences were detected in 58 patients (13.5%). Xpert had an overall sensitivity of 60.3% and a specificity of 76.5%. The sensitivity for high-grade (HG) cancer was 87% with a negative predictive value (NPV) of 99%. Urine cytology showed an overall sensitivity of 23.2% (47.6% sensitivity for HG tumours) and a specificity of 88.3%. In the PN group, 32% (n = 21) developed a recurrence within 12 months, 11 of which were HG tumours. In the NN control group, 14% (n = 9) developed a recurrence and only two were HG tumours. The hazard ratio for developing recurrence in the PN group was 2.68 for all tumours and 6.84 for HG cancer. CONCLUSIONS: The Xpert test has a high sensitivity for detecting the recurrence of cancer and a high NPV for excluding HG cancer. In addition, the data suggest that patients with a positive Xpert assay in the setting of negative cystoscopy are at high risk for recurrence and need close surveillance.

Analysis of racial disparities in acute type A aortic dissection repair at a rural tertiary academic medical center.

BACKGROUND: To determine if racial disparities exist between African Americans (AA) and Non-Hispanic Whites (NHW) for patients undergoing repair of acute type A aortic dissection (ATAAD) at a rural tertiary academic medical center. METHODS: There were 215 consecutive AA and NHW patients who underwent ATAAD repair at our institution from 1999 to 2019 included in a retrospective analysis of our Society of Thoracic Surgeons Adult Cardiac Surgery Database. Statistical analysis was performed with a p value of less than .05 considered statistically significant. RESULTS: Patients undergoing ATAAD repair were 47% AA despite comprising only 27% of the total population in our region. AAs were significantly younger (54.0 vs. 61.2 years), were more likely to be hypertensive (94.1% vs. 79.7%), had higher creatinine levels (1.7 vs. 1.1 mg/dL), and higher body mass index (30.8 vs. 28.1 kg/m2 ) (all p values < .006). There were no significant differences in type of repair or intraoperative variables. A logistic regression analysis showed AAs had an increased rate of postoperative acute renal failure not requiring hemodialysis when compared to NHWs (20.8% vs. 10.6%, p value = .042). Thirty-day mortality was not significantly different (15.7% vs. 13.4%) nor was 1-year survival (78% vs. 79%) in AAs and NHWs, respectively. CONCLUSIONS: Despite AAs having more medical comorbidities at presentation, there were no differences in short- and intermediate-term survival. In our catchment of 1.8 million people, AAs appear to undergo ATAAD repair at a disproportionate rate versus NHWs. These findings may alter strategies for surveillance and prevention of aortic disease in this high-risk population.

Bovine leukemia virus discovered in human blood.

BACKGROUND: Bovine leukemia virus (BLV) infection is widespread in cattle globally and is present in marketed beef and dairy products. Human infection with BLV has been reported in breast and lung cancer tissues and was significantly associated with breast cancer in 3 case-control studies. The purpose of this current research was to determine if BLV is present in human blood cells and if antibodies to BLV are related to blood cell infection. METHODS: Standard liquid PCR and Sanger DNA sequencing were used to test for BLV in buffy coat cells (leukocytes and platelets) of blood specimens from 95 self-selected female subjects. Enzyme-linked immunosorbent assay (ELISA) for IgG, IgM, and IgA was used to detect antibodies to BLV in the plasma of the corresponding blood samples. RESULTS: BLV DNA was detected in the buffy coat cells of blood in 33/95 (38%) of the subjects by PCR and DNA sequencing. IgG antibodies were detected in 30/95(32%), IgM in 55/95(58%), and IgA in 30/95(32%) of the subjects. There was no significant correlation between presence of the antibodies and presence of BLV DNA. CONCLUSIONS: This first report of BLV in human blood raises the question of whether infection of leukocytes could conceivably lead to leukemia as it does in infected cattle. Also, system wide circulation of infected blood cells could facilitate BLV transit to various internal tissues/organs with potential for their infection and subsequent development of cancer. The most likely route of BLV transmission to humans would be zoonotic, as a foodborne infection. Although eradicated from cattle in some countries, BLV still has a high rate of infection in the Americas, the Middle East, and parts of Europe and Asia. This report of BLV in the blood layer containing human leukocytes/platelets adds important information which could be useful to elucidate possible routes of transmission of BLV to humans and to prevent further human infection.

Household fuel use and pulmonary tuberculosis in western Nepal: A case-control study.

BACKGROUND: Whether cooking with solid fuels, as occurs widely in developing countries, including Nepal, is a risk factor for pulmonary tuberculosis (PTB) is uncertain. Epidemiologic studies have produced variable results. This case-control study sought to resolve this issue with a large sample size and a population-based control group. METHODS: PTB cases (N = 581), aged 18-70 were recruited from diagnostic centers in Kaski and neighboring districts of Nepal. Population-based controls (N = 1226) were recruited. Persons who had previously been diagnosed with TB were excluded. Questionnaires were administered at participants' homes. RESULTS: Using liquefied petroleum gas (LPG) as the cookstove reference fuel, for women the odds ratio (OR) for having a primary cookstove that used wood was 0.21 (95% CI: 0.08,0.52); for men the corresponding OR was 0.80 (0.37, 1.74). For biogas, the OR for women was 0.24 (0.06,0.87) and for men, 1.41 (0.61, 3.23). CONCLUSIONS: The unexpected finding of a higher risk for women using LPG cookstoves, relative to wood or biogas-burning cookstoves, may be attributable to excluding persons with prior TB. A possible explanation is that emissions, such as ultrafine particles, formed during LPG combustion promote PTB manifestation in infected people who have not previously had PTB. The damage from the initial PTB leaves them susceptible to the PTB-promoting effects of smoke from wood fires. Further studies, excluding participants who have previously had TB are needed to confirm these findings. Use of exhaust hoods to the outdoors for all stoves, well-ventilated kitchens, and gas stoves raised above ground would reduce exposures.

Household fuel use and latent tuberculosis infection in a Nepali population.

BACKGROUND: The risk of developing latent tuberculosis infection (LTBI) associated with cooking with solid fuels is unknown. This study examined the relationship between household fuel uses and LTBI in adults living in Nepal, a country with a high incidence of tuberculosis. METHODS: Participants were 1088 adults aged 18-70 years, members of the control group of a population-based case-control study of pulmonary TB (PTB) in people without previous TB, living in Kaski and neighboring districts of Nepal. Participants were interviewed in their homes with a standardized questionnaire. Blood samples were tested for LTBI using an interferon-gamma release assay. Multivariate unconditional logistic regression was used to examine associations between household fuel sources and LTBI. RESULTS: The overall prevalence of LTBI in the study population was 36%. Using liquefied petroleum gas (LPG) as the baseline cooking fuel type, the adjusted odds ratio (aOR) for using a primary wood cookstove was 1.13 (95%CI: 0.73,1.77) for all participants and, in women only, 1.14 (0.62, 2.09). Corresponding figures for biogas stoves were 0.64 (0.34,1.20) and 0.59 (0.24,1.45), respectively. Household sources of air pollution positively associated with LTBI included traditional oil lamps (diyos) used during power outages, for which the aOR in all participants was 2.53 (1.20, 5.31), although the number of users was small. Use of candles for lighting was also associated with increased risk of LTBI among men (aOR = 1.61, 95% CI:1.01, 2.56). CONCLUSIONS: No association was found between use of wood for cooking and LTBI. However, there was some evidence that biogas cookstoves were associated with reduced odds of LTBI. Some exposures at the time of actual infection will have been different than the current exposures used in the analysis, biasing results towards the null. Results are sufficient for the use of diyos to be discouraged for lighting purposes. Overall, results suggest that household cooking fuel use is likely to have more effect on moving from the infected state to PTB than on becoming infected with the M. tuberculosis complex. Further research, including longitudinal studies with serial LTBI testing would be useful to more accurately assess the relationships between exposures and infection.

Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer.

An on-demand, closed RT-qPCR, the GeneXpert (GX) system, has the potential to provide biomarker information in low-resourced settings and elsewhere. We used this system with a research use only version of the Breast Cancer STRAT4 cartridge that measures the mRNA expression levels of ERBB2, ESR1, PGR, and MKi67. Here we evaluated the impact of non-macrodissected (non m-d) versus macrodissected (m-d) samples using STRAT4 on formalin-fixed, paraffin-embedded (FFPE) core needle biopsies. Two cohorts were assessed: (1) 60 FFPE infiltrating ductal carcinoma (IDCA) cases and (2) 20 FFPE IDCA cases with ductal carcinoma in situ (DCIS) with a range of HER2 expression as determined by clinical immunohistochemistry and fluorescence in situ hybridization (IHC/FISH). We observed about half of the core needle biopsy area as invasive tumor in both IDCA (mean = 51.5%) and IDCA with DCIS (mean = 53.5%) cohorts, but also found the mRNA levels were independent of tumor area. We found excellent agreement of the mRNA transcript level between the paired samples, m-d versus non m-d, for ERBB2, ESR1, PGR, and MKi67 for both the IDCA and IDCA with DCIS cohorts. No significant difference (P > 0.99) was observed when we compared the mRNA transcript level between the paired samples m-d versus non m-d. In addition, we noted a significant concordance (P < 0.001) between RT-qPCR and IHC/FISH for HER2-positivity, ER-positivity, and PR-positivity, independent of specimen dissection. These data suggest that mRNA expression for ERBB2, ESR, and PGR is sufficiently low in surrounding tissue cells such that macrodissection is not required for assessment of key breast cancer mRNA markers and is independent of the amount of input tumor. This approach may be valuable in settings lacking pathology expertise or using specimen types, such as fine-needle aspirates, where it may be challenging to separate non-tumor from tumor tissue.

Maternal residential pesticide use and risk of childhood leukemia in Costa Rica.

Evidence suggests that early-life exposure to pesticides inside the home may be associated with childhood leukemia, however data from Latin American countries are limited. We examined whether self-reported maternal residential pesticide use and nearby pesticide applications-before and after child's birth-were associated with acute lymphoblastic leukemia (ALL) in the Costa Rican Childhood Leukemia Study (CRCLS), a population-based case-control study (2001-2003). Cases (n = 251 ALL) were diagnosed between 1995 and 2000 (age <15 years at diagnosis) and were identified through the Costa Rican Cancer Registry and National Children's Hospital. Population controls (n = 577) were drawn from the National Birth Registry. We fitted unconditional logistic regression models adjusted for child sex, birth year, and socioeconomic status to estimate the exposure-outcome associations and also stratified by child sex. We observed that self-reported maternal insecticide use inside the home in the year before pregnancy, during pregnancy, and while breastfeeding was associated with increased odds of ALL among boys [adjusted Odds Ratio (aOR) = 1.63 (95% confidence interval [95% CI]: 1.05-2.53), 1.75 (1.13-2.73), and 1.75 (1.12-2.73), respectively. We also found evidence of exposure-response relationships between more frequent maternal insecticide use inside the home and increased odds of ALL among boys and girls combined. Maternal report of pesticide applications on farms or companies near the home during pregnancy and at any time period were also associated with ALL. Our study in Costa Rica highlights the need for education to minimize pesticide exposures inside and around the home, particularly during pregnancy and breastfeeding.

Comparison of central laboratory assessments of ER, PR, HER2, and Ki67 by IHC/FISH and the corresponding mRNAs (ESR1, PGR, ERBB2, and MKi67) by RT-qPCR on an automated, broadly deployed diagnostic platform.

PURPOSE: The methods (IHC/FISH) typically used to assess ER, PR, HER2, and Ki67 in FFPE specimens from breast cancer patients are difficult to set up, perform, and standardize for use in low and middle-income countries. Use of an automated diagnostic platform (GeneXpert®) and assay (Xpert® Breast Cancer STRAT4) that employs RT-qPCR to quantitate ESR1, PGR, ERBB2, and MKi67 mRNAs from formalin-fixed, paraffin-embedded (FFPE) tissues facilitates analyses in less than 3 h. This study compares breast cancer biomarker analyses using an RT-qPCR-based platform with analyses using standard IHC and FISH for assessment of the same biomarkers. METHODS: FFPE tissue sections from 523 patients were sent to a College of American Pathologists-certified central reference laboratory to evaluate concordance between IHC/FISH and STRAT4 using the laboratory's standard of care methods. A subset of 155 FFPE specimens was tested for concordance with STRAT4 using different IHC antibodies and scoring methods. RESULTS: Concordance between STRAT4 and IHC was 97.8% for ESR1, 90.4% for PGR, 93.3% for ERBB2 (IHC/FISH for HER2), and 78.6% for MKi67. Receiver operating characteristic curve (ROC) area under the curve (AUC) values of 0.99, 0.95, 0.99, and 0.85 were generated for ESR1, PGR, ERBB2, and MKi67, respectively. Minor variabilities were observed depending on the IHC antibody comparator used. CONCLUSION: Evaluation of breast cancer biomarker status by STRAT4 was highly concordant with central IHC/FISH in this blinded, retrospectively analyzed collection of samples. STRAT4 may provide a means to cost-effectively generate standardized diagnostic results for breast cancer patients in low- and middle-income countries.

Genome-Scale Single Nucleotide Resolution Analysis of DNA Methylation in Human Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts in the kidney and end stage renal disease by the fourth or fifth decade of life. Mutations in the PKD1 gene account for 85% of all cases of ADPKD. No curative therapy exists for patients affected by this disease and an underexplored avenue for the treatment of ADPKD is the targeting of epigenetic changes that occur during cystogenesis. Limited data exists on alterations in DNA methylation that are associated with ADPKD. Given the similarities between cyst growth and neoplasia, and the fact that 2 DNA methylation inhibitors are already Food and Drug Administration-approved for the treatment of myelodysplastic syndrome, we hypothesized that global DNA methylation patterns in ADPKD would parallel that observed in neoplasia, and which may provide an opportunity to treat ADPKD with epigenetic inhibitors. To address this hypothesis, we undertook a global DNA methylation analysis of human ADPKD kidney. METHODS: We generated single nucleotide resolution methylomes of cortical kidney tissue from individuals with ADPKD, and from non-ADPKD kidney tissue, using reduced representation bisulfite sequencing. Using quantitative reverse transcription polymerase chain reaction, we investigated expression of the PKD1 gene in both ADPKD and non-ADPKD kidney. RESULTS: We have shown that ADPKD-derived genomic DNA exhibits global hypomethylation when compared with non-ADPKD kidney, a pattern commonly observed in DNA methylation studies of tumor-derived tissue. We have also identified 13 discrete regions that are significantly differentially methylated in ADPKD compared to non-ADPKD, and 8 of these are gene specific. The PKD1 gene shows an increase in methylation at the 3' end of the gene body, but in contrast to previously published data, this is not associated with a decrease in PKD1 mRNA expression. CONCLUSION: This genome-scale single nucleotide resolution analysis of DNA methylation in human polycystic kidneys suggests that DNA methylation differences at specific loci are associated with ADPKD. Further exploration into the significance of these preliminary results may shed light on the disease process, and potentially reveal new therapeutic possibilities.

Kitchen PM2.5 concentrations and child acute lower respiratory infection in Bhaktapur, Nepal: The importance of fuel type.

BACKGROUND: Globally, solid fuels are used by about 3 billion people for cooking and a smaller number use kerosene. These fuels have been associated with acute lower respiratory infection (ALRI) in children. Previous work in Bhaktapur, Nepal, showed comparable relationships of biomass and kerosene cooking fuels with ALRI in young children, compared to those using electricity for cooking. We examine the relationship of kitchen PM2.5 concentrations to ALRI in those households. METHODS: ALRI cases and age-matched controls were enrolled from a cohort of children 2-35 months old. 24-h PM2.5 was measured once in each participant's kitchen. The main analysis was carried out with conditional logistic regression, with PM2.5 measures specified both continuously and as quartiles. RESULTS: In the kitchens of 393 cases and 431 controls, quartiles of increasing PM2.5 concentration were associated with a monotonic increase in odds ratios (OR): 1.51 (95% CI: 1.00, 2.27), 2.22 (1.47, 3.34), 2.48 (1.63, 3.77), for the 3 highest exposure quartiles. The general kitchen concentration-response shape across all stoves was supralinear. There was evidence for increased risk with biomass stoves, but the slope for kerosene stoves was steeper, the highest quartile OR being 5.36 (1.35, 21.3). Evidence for increased risk was also found for gas stoves. CONCLUSION: Results support previous reports that biomass and kerosene cooking fuels are both ALRI risk factors, but suggests that PM2.5 from kerosene is more potent on a unit mass basis. Further studies with larger sample sizes and preferably using electricity as the baseline fuel are needed.

High concordance of a closed-system, RT-qPCR breast cancer assay for HER2 mRNA, compared to clinically determined immunohistochemistry, fluorescence in situ hybridization, and quantitative immunofluorescence.

Historically, mRNA measurements have been tested on several commercially available platforms, but none have gained broad acceptance for assessment of HER2. An mRNA measurement, as a continuous value, has the potential for use in adjudication of the equivocal category. Here we use a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay in a closed, single-use cartridge, automated system. Multiple cores (1 mm in diameter) were retrospectively collected from 80 formalin-fixed paraffin-embedded (FFPE) tissue blocks with invasive breast cancer seen by Yale Pathology Labs between 1998 and 2011. Tissue cores were processed with a FFPE lysis kit to create lysates that were tested with the automated RT-qPCR assay. Results for IHC and FISH were extracted from the pathology reports and quantitative immunofluorescence (QIF) for each case was measured as previously described. Quality control testing showed that the GX platform RT-qPCR shows no case to case cross contamination on material from routine histology practices. Concordance between RT-qPCR and IHC/FISH was 91.25% (sensitivity=0.87; specificity=0.94; PPV=0.89; NPV=0.92) using a pre-defined delta Ct cut-off (dCt≥-1) for HER2. Concordance (OPA) between RT-qPCR and QIF was 94% (sensitivity=0.90; specificity=0.96; PPV=0.93; NPV=0.94) using dCt≥-1 and a previously defined cut-point for positivity by QIF. In conclusion, the closed system RT-qPCR assay shows >90% concordance with the ASCO/CAP HER2 IHC/FISH scoring. Additionally, the RT-qPCR assay is highly concordant (94%) with the continuous variable HER2 QIF assay, and may better reflect the true continuum of HER2 receptor status in invasive breast cancer. These initial results suggest that fast, closed system molecular assays may have future value for the adjudication of the ASCO/CAP HER2 equivocal category or possibly routine usage in time constrained or low resource settings.

Lens Opacity and Hydrogen Sulfide in a New Zealand Geothermal Area.

PURPOSE: Hydrogen sulfide (H2S) is a highly toxic gas with well-established, acute irritation effects on the eye. The population of Rotorua, New Zealand, sited on an active geothermal field, has some of the highest ambient H2S exposures in the world. Evidence from ecological studies in Rotorua has suggested that H2S is associated with cataract. The purpose of the present study was, using more detailed exposure characterization, clinical examinations, and anterior eye photography, to more directly investigate this previously reported association. METHODS: Enrolled were 1637 adults, ages 18 to 65, from a comprehensive Rotorua primary care medical register. Patients underwent a comprehensive ophthalmic examination, including pupillary dilation and lens photography to capture evidence of any nuclear opacity, nuclear color, and cortical and posterior subcapsular opacity. Photographs were scored for all four outcomes on the LOCS III scale with decimalized interpolation between the exemplars. H2S exposure for up to the last 30 years was estimated based on networks of passive samplers set out across Rotorua and knowledge of residential, workplace, and school locations over the 30 years. Data analysis using linear and logistic regression examined associations between the degree of opacification and nuclear color or cataract (defined as a LOCS III score ≥2.0) in relation to H2S exposure. RESULTS: No associations were found between estimated H2S exposures and any of the four ophthalmic outcome measures. CONCLUSIONS: Overall, results were generally reassuring. They provided no evidence that H2S exposure at the levels found in Rotorua is associated with cataract. The previously found association between cataract and H2S exposure in the Rotorua population seems likely to be attributable to the limitations of the ecological study design. These results cannot rule out the possibility of an association with cataract at higher levels of H2S exposure.