Mortality reduction and cumulative excess incidence (CEI) in the prostate-specific antigen (PSA) screening era.

The extent to which PSA screening is related to prostate cancer mortality reduction in the United States (US) is controversial. US Surveillance, Epidemiology, and End Results Program (SEER) data from 1980 to 2016 were examined to assess the relationship between prostate cancer mortality and cumulative excess incidence (CEI) in the PSA screening era and to clarify the impact of race on this relationship. CEI was considered as a surrogate for the intensity of prostate cancer screening with PSA testing and subsequent biopsy as appropriate. Data from 163,982,733 person-years diagnosed with 544,058 prostate cancers (9 registries, 9% of US population) were examined. Strong inverse linear relationships were noted between CEI and prostate cancer mortality, and 317,356 prostate cancer deaths were avoided. Eight regions of the US demonstrated prostate cancer mortality reduction of 46.0-63.7%. On a per population basis, the lives of more black men than white men were saved in three of four registries with sufficient black populations for comparison. Factor(s) independent of CEI (potential effects of treatment advances) explained 14.6% of the mortality benefit (p-value = 0.3357) while there was a significant main effect of CEI (effect = -0.0064; CI: [-0.0088, -0.0040]; p-value < 0.0001). Therefore, there is a strong relationship between CEI and prostate cancer mortality reduction that was not related to factors independent of screening utilization. Minority populations have experienced large mortality reductions in the context of PSA mass utilization.

Efficacy of a Second Course of Radiation for Patients With Metachronous Hepatocellular Carcinoma.

PURPOSE: Liver-directed radiation therapy is an effective treatment for hepatocellular carcinoma (HCC), but metachronous lesions develop outside the irradiated field in >50% of patients. We hypothesized that irradiation of these new lesions would produce an outcome like that of patients receiving a first course (C1) of treatment. METHODS AND MATERIALS: We included patients with HCC who received a second course (C2) of radiation therapy >1 month after C1. Toxicity was defined as Child-Pugh score increase ≥2 within 6 months posttreatment (binary model) and as the change in albumin-bilirubin during the year after treatment (longitudinal model). Overall survival (OS) and local failure (LF) were captured at the patient and lesion level, respectively; both were summarized with Kaplan-Meier estimates. Predictors of toxicity and OS were assessed using generalized linear mixed and Cox regression models, respectively. RESULTS: Of 340 patients with HCC, 47 underwent irradiation for metachronous HCC, receiving similar prescription dose in C1/C2. Median follow-up was 17 months after C1 and 15 months after C2. Twenty-two percent of patients experienced toxicity after C1, and 25% experienced toxicity after C2. Worse baseline albumin-bilirubin predicted toxicity in both binary (odds ratio, 2.40; 95% CI, 1.46-3.94; P = .0005) and longitudinal models (P < .005). Two-year LF rate was 11.2% after C1 and 8.3% after C2; tumor dose (hazard ratio [HR], 0.982; 95% CI, 0.969-0.995; P = .007) and tumor size (HR, 1.135; 95% CI, 1.068-1.206; P < .005) predicted LF. Two-year OS was 46.0% after C1 and 42.6% after C2; tumor dose (HR, 0.986; 95% CI, 0.979-0.992; P < .005) and tumor size (HR, 1.049; 95% CI, 1.010-1.088; P = .0124) predicted OS. Reirradiation was not associated with toxicity (P > .7), LF (P = .79), or OS (P = .39). CONCLUSIONS: In this largest series in the Western hemisphere, we demonstrate that irradiation for metachronous HCC offers low rates of LF with acceptable toxicity and OS like that of patients receiving a C1. These findings support judicious selection of patients for reirradiation in metachronous HCC.

Cannabis Use in Patients Seen in an Academic Radiation Oncology Department.

PURPOSE: Cannabis use rates are increasing in the United States. Patients with cancer use cannabis for many reasons, even without high-quality supporting data. This study sought to characterize cannabis use among patients seen in radiation oncology in a state that has legalized adult nonmedical use cannabis and to identify key cannabis-related educational topics. METHODS AND MATERIALS: Cannabis history was documented by providers using a structured template at patient visits in an academic radiation oncology practice October 2020 to November 2021. Cannabis use data, including recency/frequency of use, reason, and mode of administration, were summarized, and logistic regression was used to explore associations between patient and disease characteristics and recent cannabis use. A multivariable model employed stepwise variable selection using the Akaike Information Criterion. RESULTS: Of 3143 patients total, 91 (2.9%) declined to answer cannabis use questions, and 343 (10.9%) endorsed recent use (≤1 month ago), 235 (7.5%) noted nonrecent use (>1 month ago), and 2474 (78.7%) denied history of cannabis use. In multivariable analyses, those ≥50 years old (odds ratio [OR], 0.409; 95% confidence interval [CI], 0.294-0.568; P < .001) or with history of prior courses of radiation (OR, 0.748; 95% CI, 0.572-0.979; P = .034) were less likely, and those with a mental health diagnosis not related to substance use (OR, 1.533; 95% CI, 1.171-2.005; P = .002) or who smoked tobacco (OR, 3.003; 95% CI, 2.098-4.299; P < .001) were more likely to endorse recent cannabis use. Patients reported pain, insomnia, and anxiety as the most common reasons for use. Smoking was the most common mode of administration. CONCLUSIONS: Patients are willing to discuss cannabis use with providers and reported recent cannabis use for a variety of reasons. Younger patients new to oncologic care and those with a history of mental illness or tobacco smoking may benefit most from discussions about cannabis given higher rates of cannabis use in these groups.

Age, race, insurance type, and digital divide index are associated with video visit completion for patients seen for oncologic care in a large hospital system during the COVID-19 pandemic.

INTRODUCTION: The COVID-19 pandemic drove rapid adoption of telehealth across oncologic specialties. This revealed barriers to telehealth access and telehealth-related disparities. We explored disparities in telehealth access in patients with cancer accessing oncologic care. MATERIALS/METHODS: Data for all unique patient visits at a large academic medical center were acquired pre- and intra-pandemic (7/1/2019-12/31/2020), including visit type (in-person, video, audio only), age, race, ethnicity, rural/urban (per zip code by Federal Office of Rural Health Policy), distance from medical facility, insurance, and Digital Divide Index (DDI; incorporates technology/internet access, age, disability, and educational attainment metrics by geographic area). Pandemic phases were identified based on visit dynamics. Multivariable logistic regression models were used to examine associations of these variables with successful video visit completion. RESULTS: Data were available for 2,398,633 visits for 516,428 patients across all specialties. Among these, there were 253,880 visits from 62,172 patients seen in any oncology clinic. Dramatic increases in telehealth usage were seen during the pandemic (after 3/16/2020). In multivariable analyses, patient age [OR: 0.964, (95% CI 0.961, 0.966) P<0.0001], rural zip code [OR: 0.814 (95% CI 0.733, 0.904) P = 0.0001], Medicaid enrollment [OR: 0.464 (95% CI 0.410, 0.525) P<0.0001], Medicare enrollment [OR: 0.822 (95% CI 0.761, 0.888) P = 0.0053], higher DDI [OR: 0.903 (95% CI 0.877, 0.930) P<0.0001], distance from the facility [OR: 1.028 (95% CI 1.021, 1.035) P<0.0001], black race [OR: 0.663 (95% CI 0.584, 0.753) P<0.0001], and Asian race [OR: 1.229 (95% CI 1.022, 1.479) P<0.0001] were associated with video visit completion early in the pandemic. Factors related to video visit completion later in the pandemic and within sub-specialties of oncology were also explored. CONCLUSIONS: Patients from older age groups, those with minority backgrounds, and individuals from areas with less access to technology (high DDI) as well as those with Medicare or Medicaid insurance were less likely to use video visits. With greater experience through the pandemic, disparities were not mitigated. Further efforts are required to optimize telehealth to benefit all patients and avoid increasing disparities in care delivery.

Rectal sparing in prostate radiotherapy with combination-brachytherapy and hydrogel spacer.

BACKGROUND: Though some techniques that facilitate rectal sparing such as brachytherapy and intensity modulated radiotherapy (IMRT) have been examined in detail, technical aspects of hydrogel spacer (HS) have been studied less exhaustively. We examined HS quality metrics and approaches to placement for superior dosimetric outcomes. MATERIALS AND METHODS: A single site retrospective review of radiation plans was conducted for patients who received combination-brachytherapy (CBT) with 90 Gy low-dose-rate implant followed by external beam radiotherapy (45 Gy/25 fractions) with operating room (OR) placed HS (2017-2021). A randomly selected set of patients that received CBT without HS over the same time period was used for comparison. Dosimetric outcomes included D1cc and D5% rectum. Dose gradients were quantified. Student's t-test was used for statistical comparisons. RESULTS: Sixty patients (30 with and 30 without HS) who received CBT for prostate cancer were examined. Those with HS had lower mean D1cc [65.31 Gy (SD = 13.53)] and D5% [53.20 Gy (SD = 10.18)] compared to those treated without HS [91.67 Gy (SD = 8.31) and 75.00 Gy (SD = 8.45), respectively, p < 0.001]. Patients with superior HS (average thickness ≥1 cm; n = 12) had lower mean D1cc [58.49 Gy (SD = 13.25, p = 0.026)] and D5% [48.69 Gy (SD = 9.85, p = 0.049)] than those with thinner HS. When dose gradients were considered, HS spanning the interface between the prostate and perirectal tissues to a thickness ≥1 cm can reduce rectal maximum dose to 50-60 Gy. CONCLUSIONS: Through effective use of CBT and HS, extreme rectal dose restriction is possible. The goal for HS placement should be thickness ≥1 cm from base to apex.

Pretreatment Levels of Soluble Tumor Necrosis Factor Receptor 1 and Hepatocyte Growth Factor Predict Toxicity and Overall Survival After 90Y Radioembolization: Potential Novel Application of Biomarkers for Personalized Management of Hepatotoxicity.

Liver function may be negatively affected by radiation for treatment of hepatic malignancy. Pretreatment blood cytokine levels are biomarkers for prediction of toxicity and survival after external-beam radiation therapy. We hypothesized that cytokines may also predict outcomes after radioembolization, enabling a biomarker-driven personalized approach to treatment. Methods: Pretherapy blood samples from patients enrolled on a prospective protocol evaluating 90Y radioembolization for management of intrahepatic malignancy were analyzed for 2 cytokines selected on the basis of prior studies in stereotactic body radiotherapy, soluble tumor necrosis factor receptor 1 (sTNFR1) and hepatocyte growth factor (HGF), via enzyme-linked immunosorbent assay, and key dosimetric parameters were derived from posttreatment 90Y PET/CT imaging. Toxicity was defined as a change in albumin-bilirubin score from baseline to follow-up (3-6 mo after treatment). Associations of cytokine levels, dose metrics, and baseline liver function with toxicity and overall survival were assessed. Results: Data from 43 patients treated with 90Y radioembolization for primary (48.8% [21/43]) or secondary (51.2% [22/43]) malignancy were assessed. Examined dose metrics and baseline liver function were not associated with liver toxicity; however, levels of sTNFR1 (P = 0.045) and HGF (P = 0.005) were associated with liver toxicity in univariate models. Cytokines were the only predictors of toxicity in multivariable models including dose metrics and prior liver-directed therapy. sTNFR1 (hazard ratio, 12.3; 95% CI, 3.5-42.5, P < 0.001) and HGF (hazard ratio, 7.5; 95% CI, 2.4-23.1, P < 0.001) predicted overall survival, and findings were similar when models were controlled for absorbed dose and presence of metastatic disease. Conclusion: Pretreatment cytokine levels predict liver toxicity and overall survival. These pathways can be targeted with available drugs, an advantage over previously studied dose metrics and liver function tests. Interventions directed at the TNFα-axis should be considered in future studies for prevention of liver toxicity, and HGF should be explored further to determine whether its elevation drives toxicity or indicates ongoing liver regeneration after prior injury.

Prostate radiotherapy in the setting of bilateral hip prostheses: All commonly used photon-based radiation approaches are feasible.

The prevalence of hip prostheses is increasing. Prostate radiation delivery in the setting of hip prostheses is complicated by both imaging artifacts that interfere with volume delineation and dosimetric effects that must be addressed in the planning process. We hypothesized that with specialized planning, any photon-based definitive prostate radiotherapy approach may be utilized in patients with bilateral hip prostheses. Imaging data from sequential patients with prostate cancer and bilateral hip prostheses treated definitively with radiation were retrospectively reviewed. Bimodality imaging was used to define targets and organs at risk (OARs) along with specialized MRI sequences and/or orthopedic metal artifact reduction (OMAR) for MRI and CT artifact suppression, respectively. Multiple VMAT plans were generated for each set of patient images to include three fractionation schemes (conventional, hypofractionated, and SBRT), each with hip avoidance and with simulated normal hip. The ability to meet standard dose constraints was assessed for each plan type. Differences in target and OAR dosing between plans accounting for prosthetic hips via avoidance vs plans with simulated absence of prosthetic hip were also assessed. T-tests were used to compare dosimetric parameters. Ten patients with bilateral hip prostheses were identified, and 6 plans were created for each patient for a total of 60 radiation plans. Prosthetic hip avoidance did not result in failure to meet dose constraints for any patient. Hip avoidance resulted in minimal increases in high dose to the rectum and bladder (increases in mean V80%, V90%, and V95% ranged from 0.1% to 2.4%). Larger increases were seen at lower dose levels, with rectal V50% significantly increased in all three plan types with hip avoidance (conventional: 26.0% [standard deviation, SD 13.9] vs 16.9% [SD 10.2, p = 0.003]; hypofractionation: 26.4% [SD 13.3] vs 17.1% [SD 10.1, p = 0.002]; SBRT: 18.3% [SD 10.7] vs 10.5% [SD 6.9, p = 0.008]). Similarly, hip avoidance resulted in increases in bladder V50% to 31.7% (SD 16.8) vs 23.3% (SD 14.0, p = 0.001), 31.3% (SD 17.0) vs 23.3% (SD 13.8, p = 0.002), and 22.7% (SD 12.3) vs 16.5% (SD 12.6, p < 0.001) for conventional, hypofractionated, and SBRT plans, respectively. Hydrogel spacer resulted in reductions in rectal dose. For example, V70% for hip avoidance plans decreased with spacer presence to 8.3% (SD 6.7) vs 21.1% (SD 5.8, p = 0.021), 8.6% (SD 6.5) vs 21% (SD 5.7, p = 0.022), and 3.7% (SD 3.2) vs 15% (SD 8.2, p = 0.010) for conventional, hypofractionated, and SBRT plans, respectively. Any photon-based definitive prostate radiotherapy approach can be used with bimodality imaging for target and OAR definition and planning techniques to avoid dose attenuation effects of hip prostheses. Hydrogel spacer is a useful adjunct.

Prevalence of cannabis use among individuals with a history of cancer in the United States.

BACKGROUND: Patients with cancer have played a key role in advocating for legal access to cannabis, but little is known about links between cancer and cannabis use or cannabis-related beliefs. The authors used data from a national survey to study these relationships. METHODS: Nationally representative data collected by the National Survey on Drug Use and Health from 2015 to 2019 were acquired. Patterns of cannabis use and cancer history were examined and tested within age group subpopulations via domain analysis using survey weights. RESULTS: Data for 214,505 adults, including 4741 individuals (3.8%) with past (>1 year ago) cancer diagnosis and 1518 individuals (1.2%) with recent (≤1 year ago) cancer diagnosis, were examined. Cannabis use was less common in those with past (8.9%; 95% CI, 8.0%-9.8%) or recent (9.9%; 95% CI, 6.9%-11.1%) cancer diagnosis than in those without a history of cancer (15.9%; 95% CI, 15.7%-16.1%). However, when analyses were stratified by age group, those 18 to 34 years of age were more likely to report past cannabis use, and those 35 to 49 years of age were more likely to report past or recent cannabis use if they had a history of cancer. Younger patients felt that cannabis was more accessible and less risky if they had a history of cancer. CONCLUSIONS: Patients with cancer were less likely to report cannabis use, but there were different cannabis perceptions and use patterns by age. Age should be considered in studies of cannabis and cancer, and policy initiatives may be needed to aid provision of quality information on cannabis risk to those with cancer. LAY SUMMARY: Cannabis (marijuana) use is increasing in the United States, but we do not have much information on the relationship between cannabis use and cancer. We studied information from a representative group of people and found that younger patients generally reported more past and/or recent cannabis use if they had been diagnosed with cancer whereas older individuals did not. Beliefs about cannabis risk and accessibility differed by age. Clinical trials to study cannabis should account for patient age, and accurate information about cannabis should be provided to help patients with cancer make decisions about cannabis use.

Differences between cancer patients and others who use medicinal Cannabis.

BACKGROUND: Cancer patients have been at the forefront of policy discussions leading to legalization of medical Cannabis (marijuana). Unfortunately, Cannabis use among those with cancer is poorly understood. METHODS: A diverse group of patients seeking certification for medical Cannabis in the state of Michigan were surveyed at the time of their presentation to medical dispensaries. The survey assessed demographics, employment/disability, pain, physical functioning, mental health, mode of Cannabis use, and frequency/amount of Cannabis use. Chi-square and t-tests were performed to compare those who did and did not endorse cancer diagnosis. RESULTS: Analysis of data from 1485 adults pursuing medical Cannabis certification, including 72 (4.8%) reporting a cancer diagnosis, indicated that those with cancer were older [mean age 53.4 years (SD = 10.5) vs. 44.7 years (SD = 13.0); p<0.001] than those without cancer. They also differed regarding employment status (p<0.001; working: 20.8% vs. 46.2%; disabled: 44.4% vs. 26.5% for those with vs. those without cancer, respectively). Those with cancer used less Cannabis (p = 0.033 for quantity used) and used Cannabis less often (p = 0.032 for frequency of use); they less frequently endorsed smoking Cannabis (80% vs 91%; p = 0.015). There was a non-significant trend to increased edible use in those with cancer (57% vs. 44%; p = 0.052). CONCLUSIONS: Patients with cancer who are seeking medical Cannabis are different from those seeking medical Cannabis without cancer, and they report using Cannabis differently. Further research to characterize the patterns and consequences of Cannabis use in cancer patients is needed.

TNFR1 and the TNFα axis as a targetable mediator of liver injury from stereotactic body radiation therapy.

INTRODUCTION: Radiation therapy for the management of intrahepatic malignancies can adversely affect liver function. Liver damage has been associated with increased levels of inflammatory cytokines, including tumor necrosis factor alpha (TNFα). We hypothesized that an inflammatory state, characterized by increased soluble TNFα receptor (sTNFR1), mediates sensitivity of the liver to radiation. MATERIALS/METHODS: Plasma samples collected during 3 trials of liver radiation for liver malignancies were assayed for sTNFR1 level via enzyme-linked immunosorbent assay (ELISA). Univariate and multivariate logistic regression and longitudinal models were used to characterize associations between liver toxicity (defined as a ≥2-point increase in Child-Pugh [CP] score within 6 months of radiation treatment) and sTNFR1 levels, ALBI score, biocorrected mean liver dose (MLD), age, and baseline laboratory values. RESULTS: Samples from 78 patients given liver stereotactic body radiation therapy [SBRT] (92%) or hypofractionated radiation were examined. There was a significant association between liver toxicity and sTNFR1 levels, and higher values were associated with increased toxicity over a range of mean liver doses. When ALBI score and biocorrected dose were included in the model with sTNFR1, baseline ALBI score and change in ALBI (ΔALBI) were significantly associated with toxicity, but sTNFR1 was not. Baseline aminotransferase levels also predicted toxicity but not independently of ALBI score. CONCLUSIONS: Elevated plasma sTNFR1 levels are associated with liver injury after liver radiation, suggesting that elevated inflammatory cytokine activity is a predictor of radiation-induced liver dysfunction. Future studies should determine whether administration of agents that decrease inflammation prior to treatment is warranted.

A comparison of two measures of HIV diversity in multi-assay algorithms for HIV incidence estimation.

BACKGROUND: Multi-assay algorithms (MAAs) can be used to estimate HIV incidence in cross-sectional surveys. We compared the performance of two MAAs that use HIV diversity as one of four biomarkers for analysis of HIV incidence. METHODS: Both MAAs included two serologic assays (LAg-Avidity assay and BioRad-Avidity assay), HIV viral load, and an HIV diversity assay. HIV diversity was quantified using either a high resolution melting (HRM) diversity assay that does not require HIV sequencing (HRM score for a 239 base pair env region) or sequence ambiguity (the percentage of ambiguous bases in a 1,302 base pair pol region). Samples were classified as MAA positive (likely from individuals with recent HIV infection) if they met the criteria for all of the assays in the MAA. The following performance characteristics were assessed: (1) the proportion of samples classified as MAA positive as a function of duration of infection, (2) the mean window period, (3) the shadow (the time period before sample collection that is being assessed by the MAA), and (4) the accuracy of cross-sectional incidence estimates for three cohort studies. RESULTS: The proportion of samples classified as MAA positive as a function of duration of infection was nearly identical for the two MAAs. The mean window period was 141 days for the HRM-based MAA and 131 days for the sequence ambiguity-based MAA. The shadows for both MAAs were <1 year. Both MAAs provided cross-sectional HIV incidence estimates that were very similar to longitudinal incidence estimates based on HIV seroconversion. CONCLUSIONS: MAAs that include the LAg-Avidity assay, the BioRad-Avidity assay, HIV viral load, and HIV diversity can provide accurate HIV incidence estimates. Sequence ambiguity measures obtained using a commercially-available HIV genotyping system can be used as an alternative to HRM scores in MAAs for cross-sectional HIV incidence estimation.

HIV diversity as a biomarker for HIV incidence estimation: including a high-resolution melting diversity assay in a multiassay algorithm.

Multiassay algorithms (MAAs) can be used to estimate cross-sectional HIV incidence. We previously identified a robust MAA that includes the BED capture enzyme immunoassay (BED-CEIA), the Bio-Rad Avidity assay, viral load, and CD4 cell count. In this report, we evaluated MAAs that include a high-resolution melting (HRM) diversity assay that does not require sequencing. HRM scores were determined for eight regions of the HIV genome (2 in gag, 1 in pol, and 5 in env). The MAAs that were evaluated included the BED-CEIA, the Bio-Rad Avidity assay, viral load, and the HRM diversity assay, using HRM scores from different regions and a range of region-specific HRM diversity assay cutoffs. The performance characteristics based on the proportion of samples that were classified as MAA positive by duration of infection were determined for each MAA, including the mean window period. The cross-sectional incidence estimates obtained using optimized MAAs were compared to longitudinal incidence estimates for three cohorts in the United States. The performance of the HRM-based MAA was nearly identical to that of the MAA that included CD4 cell count. The HRM-based MAA had a mean window period of 154 days and provided cross-sectional incidence estimates that were similar to those based on cohort follow-up. HIV diversity is a useful biomarker for estimating HIV incidence. MAAs that include the HRM diversity assay can provide accurate HIV incidence estimates using stored blood plasma or serum samples without a requirement for CD4 cell count data.

Performance of a limiting-antigen avidity enzyme immunoassay for cross-sectional estimation of HIV incidence in the United States.

BACKGROUND: A limiting antigen avidity enzyme immunoassay (HIV-1 LAg-Avidity assay) was recently developed for cross-sectional HIV incidence estimation. We evaluated the performance of the LAg-Avidity assay alone and in multi-assay algorithms (MAAs) that included other biomarkers. METHODS AND FINDINGS: Performance of testing algorithms was evaluated using 2,282 samples from individuals in the United States collected 1 month to >8 years after HIV seroconversion. The capacity of selected testing algorithms to accurately estimate incidence was evaluated in three longitudinal cohorts. When used in a single-assay format, the LAg-Avidity assay classified some individuals infected >5 years as assay positive and failed to provide reliable incidence estimates in cohorts that included individuals with long-term infections. We evaluated >500,000 testing algorithms, that included the LAg-Avidity assay alone and MAAs with other biomarkers (BED capture immunoassay [BED-CEIA], BioRad-Avidity assay, HIV viral load, CD4 cell count), varying the assays and assay cutoffs. We identified an optimized 2-assay MAA that included the LAg-Avidity and BioRad-Avidity assays, and an optimized 4-assay MAA that included those assays, as well as HIV viral load and CD4 cell count. The two optimized MAAs classified all 845 samples from individuals infected >5 years as MAA negative and estimated incidence within a year of sample collection. These two MAAs produced incidence estimates that were consistent with those from longitudinal follow-up of cohorts. A comparison of the laboratory assay costs of the MAAs was also performed, and we found that the costs associated with the optimal two assay MAA were substantially less than with the four assay MAA. CONCLUSIONS: The LAg-Avidity assay did not perform well in a single-assay format, regardless of the assay cutoff. MAAs that include the LAg-Avidity and BioRad-Avidity assays, with or without viral load and CD4 cell count, provide accurate incidence estimates.

HIV incidence determination in the United States: a multiassay approach.

BACKGROUND: Accurate testing algorithms are needed for estimating human immunodeficiency virus (HIV) incidence from cross-sectional surveys. METHODS: We developed a multiassay algorithm (MAA) for HIV incidence that includes the BED capture enzyme immunoassay (BED-CEIA), an antibody avidity assay, HIV load, and CD4(+) T-cell count. We analyzed 1782 samples from 709 individuals in the United States who had a known duration of HIV infection (range, 0 to >8 years). Logistic regression with cubic splines was used to compare the performance of the MAA to the BED-CEIA and to determine the window period of the MAA. We compared the annual incidence estimated with the MAA to the annual incidence based on HIV seroconversion in a longitudinal cohort. RESULTS: The MAA had a window period of 141 days (95% confidence interval [CI], 94-150) and a very low false-recent misclassification rate (only 0.4% of 1474 samples from subjects infected for >1 year were misclassified as indicative of recent infection). In a cohort study, annual incidence based on HIV seroconversion was 1.04% (95% CI, .70%-1.55%). The incidence estimate obtained using the MAA was essentially identical: 0.97% (95% CI, .51%-1.71%). CONCLUSIONS: The MAA is as sensitive for detecting recent HIV infection as the BED-CEIA and has a very low rate of false-recent misclassification. It provides a powerful tool for cross-sectional HIV incidence determination.

Impact of mutation type and amplicon characteristics on genetic diversity measures generated using a high-resolution melting diversity assay.

We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology.

Analysis of HIV using a high resolution melting (HRM) diversity assay: automation of HRM data analysis enhances the utility of the assay for analysis of HIV incidence.

BACKGROUND: HIV diversity may be a useful biomarker for discriminating between recent and non-recent HIV infection. The high resolution melting (HRM) diversity assay was developed to quantify HIV diversity in viral populations without sequencing. In this assay, HIV diversity is expressed as a single numeric HRM score that represents the width of a melting peak. HRM scores are highly associated with diversity measures obtained with next generation sequencing. In this report, a software package, the HRM Diversity Assay Analysis Tool (DivMelt), was developed to automate calculation of HRM scores from melting curve data. METHODS: DivMelt uses computational algorithms to calculate HRM scores by identifying the start (T1) and end (T2) melting temperatures for a DNA sample and subtracting them (T2 - T1 =  HRM score). DivMelt contains many user-supplied analysis parameters to allow analyses to be tailored to different contexts. DivMelt analysis options were optimized to discriminate between recent and non-recent HIV infection and to maximize HRM score reproducibility. HRM scores calculated using DivMelt were compared to HRM scores obtained using a manual method that is based on visual inspection of DNA melting curves. RESULTS: HRM scores generated with DivMelt agreed with manually generated HRM scores obtained from the same DNA melting data. Optimal parameters for discriminating between recent and non-recent HIV infection were identified. DivMelt provided greater discrimination between recent and non-recent HIV infection than the manual method. CONCLUSION: DivMelt provides a rapid, accurate method of determining HRM scores from melting curve data, facilitating use of the HRM diversity assay for large-scale studies.

Use of a high resolution melting assay to analyze HIV diversity in HIV-infected Ugandan children.

BACKGROUND: We used a novel high resolution melting (HRM) diversity assay to analyze HIV diversity in Ugandan children (age 0.6-12.4 years) who were enrolled in an observational study of antiretroviral treatment (ART). Children were maintained on ART if they were clinically and immunologically stable. METHODS: HIV diversity was measured before ART (baseline) in 76 children and after 48 or 96 weeks of ART in 14 children who were not virally suppressed. HIV diversity (expressed as HRM scores) was measured in 6 regions of the HIV genome (2 in gag, 1 in pol, 3 in env). RESULTS: Higher baseline HRM scores were significantly associated with older age (≥2 years, P ≤ 0.001 for all 6 regions). HRM scores from different regions were weakly correlated. Higher baseline HRM scores in 3 regions (1 in gag, 2 in env) were associated with ART failure. HIV diversity was lower in 4 regions (2 in gag, 1 in pol, 1 in env) after 48-96 weeks of nonsuppressive ART compared with baseline. CONCLUSIONS: Higher levels of HIV diversity were observed in older children before ART, and higher levels of diversity in some regions of the HIV genome were associated with ART failure. Prolonged exposure to nonsuppressive ART was associated with a significant decrease in viral diversity in selected regions of the HIV genome.

Comparison of a high-resolution melting assay to next-generation sequencing for analysis of HIV diversity.

Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution.