The repetitive immunization at multiple sites (RIMMS) protocol capitalizes on the animal's innate immune system, which is genetically preprogrammed to recognize many antigens. By repetitively immunizing the animal, B cells that recognize the antigen are kept continuously expanding until the lymph nodes are harvested for hybridoma generation. This is a good method for making a more diverse repertoire of antibodies or antibodies directed against conformational epitopes.

β-Galactosidase has been used extensively both as a label in enzyme immunoassays and for immunocytochemistry. One good substrate is 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal), which gives an intense blue product. The product is stable and insoluble in alcohol as well as H₂O.

Adeno-associated virus (AAV) recombinants are currently the vector of choice for many gene therapy applications. As experimental therapies progress to clinical trials, the need to characterize recombinant adeno-associated viruses (rAAVs) accurately and reproducibly increases. Accurate determination of rAAV infectious titer is important for determining the activity of each vector lot and for ensuring lot-to-lot consistency. The following protocol developed in our laboratory uses a 96-well TCID₅₀ format and quantitative polymerase chain reaction (qPCR) detection for the determination of rAAV infectious titer.

This protocol is used to determine the concentration of DNase-resistant vector genomes (i.e., packaged in the capsid) in purified recombinant adeno-associated virus (rAAV) preparations. The protocol begins with treatment of the vector stock with DNase I to eliminate unencapsidated AAV DNA or contaminating plasmid DNA. This is followed by a heat treatment to heat-inactivate DNase I, to disrupt the viral capsid, and to release the packaged vector genomes for quantification by real-time polymerase chain reaction (PCR) using a set of standards (linearized plasmid used for vector production) containing known copy numbers. To accomplish high-throughput titration, the primer and probe sets used in real-time PCR are usually designed to target common elements present in most rAAV genomes, such as promoters and poly(A) signals. This strategy significantly reduces the number of PCRs, controls, and turnaround time. Several important controls should be included in the assay as follows: The first two controls should have a known copy number of the rAAV genome plasmid treated or not treated with DNase I. This control tests the effectiveness of DNase treatment. To control for potential cross-contamination between samples during the preparation process, a blank control containing nuclease-free water only should be processed and tested in parallel. A validation vector sample with a known titer should be included in every assay to monitor interassay variability. Finally, for the PCR run, a no-template control (NTC) is included to indicate cross-contamination during PCR setup.

Tobacco smoking is the primary risk factor for lung cancer, driven by the addictive nature of nicotine and the indisputable carcinogenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as well as other compounds. The integration of lung cancer chemoprevention with smoking cessation is one potential approach to reduce this risk and mitigate lung cancer mortality. Experimental data from our group suggest that kava, commonly consumed in the South Pacific Islands as a beverage to promote relaxation, may reduce lung cancer risk by enhancing NNK detoxification and reducing NNK-derived DNA damage. Building upon these observations, we conducted a pilot clinical trial to evaluate the effects of a 7-day course of kava on NNK metabolism in active smokers. The primary objective was to compare urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL plus its glucuronides, major metabolites of NNK) before and after kava administration as an indicator of NNK detoxification. Secondary objectives included determining kava's safety, its effects on DNA damage, tobacco use, and cortisol (a biomarker of stress). Kava increased urinary excretion of total NNAL and reduced urinary 3-methyladenine in participants, suggestive of its ability to reduce the carcinogenicity of NNK. Kava also reduced urinary total nicotine equivalents, indicative of its potential to facilitate tobacco cessation. Plasma cortisol and urinary total cortisol equivalents were reduced upon kava use, which may contribute to reductions in tobacco use. These results demonstrate the potential of kava intake to reduce lung cancer risk among smokers.

Mammographic breast density is a strong risk factor for breast cancer. We comprehensively investigated the associations of body mass index (BMI) change from ages 10, 18, and 30 to age at mammogram with mammographic breast density in postmenopausal women. We used multivariable linear regression models, adjusted for confounders, to investigate the associations of BMI change with volumetric percent density, dense volume, and nondense volume, assessed using Volpara in 367 women. At the time of mammogram, the mean age was 57.9 years. Compared with women who had a BMI gain of 0.1–5 kg/m² from age 10, women who had a BMI gain of 5.1–10 kg/m² had a 24.4% decrease [95% confidence interval (CI), 6.0%–39.2%] in volumetric percent density; women who had a BMI gain of 10.1–15 kg/m² had a 46.1% decrease (95% CI, 33.0%–56.7%) in volumetric percent density; and women who had a BMI gain of >15 kg/m² had a 56.5% decrease (95% CI, 46.0%–65.0%) in volumetric percent density. Similar, but slightly attenuated associations were observed for BMI gain from ages 18 and 30 to age at mammogram and volumetric percent density. BMI gain over the life course was positively associated with nondense volume, but not dense volume. We observed strong associations between BMI change over the life course and mammographic breast density. The inverse associations between early-life adiposity change and volumetric percent density suggest that childhood adiposity may confer long-term protection against postmenopausal breast cancer via its effect of mammographic breast density.

Colorectal cancer is a growing burden in adults less than 50 years old. In 2018, the American Cancer Society published a guideline update recommending a reduction in the colorectal cancer screening start age for average-risk individuals from 50 to 45. Implementing these recommendations would have important implications for public health. However, the approximate number of people impacted by this change, the average-risk population ages 45–49, is not well-described in the literature. Here, we provide methodology to conservatively estimate the average-risk and screening-eligible population in the United States, including those who would be impacted by a lowered colorectal cancer screening start age. Using multiple data sources, we estimated the current average-risk population by subtracting individuals with symptomatic colorectal cancer, with a family history of colorectal cancer, and with inflammatory bowel disease and hereditary nonpolyposis colorectal cancer from the total population. Within this population, we estimated the number of screening-eligible individuals by subtracting those with previous colorectal cancer screening (45- to 49-year-old) or up to date with colorectal cancer screening (50- to 74-year-old). The total average-risk population is estimated between 102.1 and 106.5 million people, of whom 43.4–45.2 million people are eligible for colorectal cancer screening. Lowering the screening age would add roughly 19 million people to the average-risk population and increase the current number of screening-eligible individuals on immediate implementation by over 60% (from 27 to 44 million). Estimating the population size impacted by lowering the recommended colorectal cancer screening start age enables more accurate decision-making for policymakers and epidemiologists focused on cancer prevention.

Women of Latin American origin in the United States are more likely to be diagnosed with advanced breast cancer and have a higher risk of mortality than non-Hispanic White women. Studies in U.S. Latinas and Latin American women have reported a high incidence of HER2 positive (+) tumors; however, the factors contributing to this observation are unknown. Genome-wide genotype data for 1,312 patients from the Peruvian Genetics and Genomics of Breast Cancer Study (PEGEN-BC) were used to estimate genetic ancestry. We tested the association between HER2 status and genetic ancestry using logistic and multinomial logistic regression models. Findings were replicated in 616 samples from Mexico and Colombia. Average Indigenous American (IA) ancestry differed by subtype. In multivariate models, the odds of having an HER2+ tumor increased by a factor of 1.20 with every 10% increase in IA ancestry proportion (95% CI, 1.07–1.35; P = 0.001). The association between HER2 status and IA ancestry was independently replicated in samples from Mexico and Colombia. Results suggest that the high prevalence of HER2+ tumors in Latinas could be due in part to the presence of population-specific genetic variant(s) affecting HER2 expression in breast cancer.Significance:The positive association between Indigenous American genetic ancestry and HER2+ breast cancer suggests that the high incidence of HER2+ subtypes in Latinas might be due to population and subtype-specific genetic risk variants.

Although ample evidence indicates that immune cell homeostasis is an important prognostic outcome determinant in patients with cancer, few studies have examined whether it also determines cancer risk among initially healthy individuals. We performed a case–cohort study including incident cases of breast (n = 207), colorectal (n = 111), lung (n = 70), and prostate (n = 201) cancer as well as a subcohort (n = 465) within the European Prospective Investigation into Cancer and Nutrition-Heidelberg cohort. Relative counts of neutrophils, monocytes, and lymphocyte sublineages were measured by qRT-PCR. HRs and 95% confidence intervals were used to measure the associations between relative counts of immune cell and cancer risks. When relative counts of immune cell types were taken individually, a significant positive association was observed between relative counts of FOXP3+ regulatory T cells (Tregs) and lung cancer risk, and significant inverse associations were observed between relative CD8+ counts and risks of lung and breast cancer (overall and ER+ subtype). Multivariable models with mutual adjustments across immune markers showed further significant positive associations between higher relative FOXP3+ T-cell counts and increased risks of colorectal and breast cancer (overall and ER− subtype). No associations were found between immune cell composition and prostate cancer risk. These results affirm the relevance of elevated FOXP3+ Tregs and lower levels of cytotoxic (CD8+) T cells as risk factors for tumor development.Significance:This epidemiologic study supports a role for both regulatory and cytotoxic T cells in determining cancer risk among healthy individuals.See related commentary by Song and Tworoger, p. 1801

Determining mechanisms of resistance to αPD-1/PD-L1 immune-checkpoint immunotherapy is key to developing new treatment strategies. Cancer-associated fibroblasts (CAF) have many tumor-promoting functions and promote immune evasion through multiple mechanisms, but as yet, no CAF-specific inhibitors are clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, and 4T1) to investigate how CAFs influence the immune microenvironment and affect response to different immunotherapy modalities [anticancer vaccination, TC1 (HPV E7 DNA vaccine), αPD-1, and MC38] and found that CAFs broadly suppressed response by specifically excluding CD8+ T cells from tumors (not CD4+ T cells or macrophages); CD8+ T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8+ T cells from CAF-rich murine tumors and immunochemistry analysis of human tumors identified significant upregulation of CTLA-4 in the absence of other exhaustion markers; inhibiting CTLA-4 with a nondepleting antibody overcame the CD8+ T-cell exclusion effect without affecting Tregs. We then examined the potential for CAF targeting, focusing on the ROS-producing enzyme NOX4, which is upregulated by CAF in many human cancers, and compared this with TGFβ1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacologic inhibition [GKT137831 (Setanaxib)] of NOX4 “normalized” CAF to a quiescent phenotype and promoted intratumoral CD8+ T-cell infiltration, overcoming the exclusion effect; TGFβ1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAF-rich tumors. These findings demonstrate that CAF-mediated immunotherapy resistance can be effectively overcome through NOX4 inhibition and could improve outcome in a broad range of cancers.Significance:NOX4 is critical for maintaining the immune-suppressive CAF phenotype in tumors. Pharmacologic inhibition of NOX4 potentiates immunotherapy by overcoming CAF-mediated CD8+ T-cell exclusion.Graphical Abstract:http://cancerres.aacrjournals.org/content/canres/80/9/1846/F1.large.jpg.See related commentary by Hayward, p. 1799

RING-finger E3 ligases are instrumental in the regulation of inflammatory cascades, apoptosis, and cancer. However, their roles are relatively unknown in TGFβ/SMAD signaling. SMAD3 and its adaptors, such as β2SP, are important mediators of TGFβ signaling and regulate gene expression to suppress stem cell–like phenotypes in diverse cancers, including hepatocellular carcinoma (HCC). Here, PJA1, an E3 ligase, promoted ubiquitination and degradation of phosphorylated SMAD3 and impaired a SMAD3/β2SP-dependent tumor-suppressing pathway in multiple HCC cell lines. In mice deficient for SMAD3 (Smad3+/−), PJA1 overexpression promoted the transformation of liver stem cells. Analysis of genes regulated by PJA1 knockdown and TGFβ1 signaling revealed 1,584 co-upregulated genes and 1,280 co-downregulated genes, including many implicated in cancer. The E3 ligase inhibitor RTA405 enhanced SMAD3-regulated gene expression and reduced growth of HCC cells in culture and xenografts of HCC tumors, suggesting that inhibition of PJA1 may be beneficial in treating HCC or preventing HCC development in at-risk patients.Significance: These findings provide a novel mechanism regulating the tumor suppressor function of TGFβ in liver carcinogenesis.

While immuno-oncology has made significant advances in activating local tumor immune responses, leading to improved outcomes, the role of systemic immunity in cancer incidence remains poorly understood. Le Cornet and colleagues prospectively studied circulating immune cells quantified by DNA methylation markers in relation to incidence of breast, colorectal, lung, and prostate cancer among initially healthy individuals. A positive association with cancer risk was observed for higher FOXP3+ T-cell–mediated immune tolerance and lower CD8+ T-cell–mediated cytotoxicity. Further studies of systemic immunity in cancer development are crucial to identify novel prediction markers and interventional targets for cancer immunoprevention.See related article by Le Cornet et al., p. 1885

Metastatic disease is the leading cause of death in patients with solid cancers. The progression to metastasis is a multistep process that involves detachment of tumor cells from their constraining basement membrane at the primary site, migration and intravasation into the circulation, survival in the circulation, extravasation into the secondary organ, and survival and growth at the secondary site. During these steps, tumor and immune cells interact and influence each other both within the tumor microenvironment and systemically. In particular, myeloid cells such as monocytes, macrophages, neutrophils, and myeloid-derived suppressor cells (myeloid regulatory cells) have been shown to play important roles in the metastatic process. These interactions open new avenues for targeting cancer metastasis, especially given the increasing interest in development of cancer immunotherapies. In this review, we describe the currently reported pathways and mechanisms involved in myeloid cell enhancement of the metastatic cascade.

Brain metastases are associated with poor prognosis irrespective of the primary tumor they originate from. Current treatments for brain metastases are palliative, and patients with symptomatic brain metastasis have a one-year survival of <20%. Lung cancer, breast cancer, and melanoma have higher incidences of brain metastases compared with other types of cancers. However, it is not very clear why some cancers metastasize to the brain more frequently than others. Studies thus far suggest that brain-specific tropism of certain types of cancers is defined by a winning combination of the following factors: unique genetic subtypes of primary tumors or its subclones enabling detachment, dissemination, blood–brain barrier penetration, plus proliferation and survival in hypoxic low-glucose microenvironment; specific transcriptomic and epigenetic changes of colony-forming metastatic cells, allowing their outgrowth; favorable metastasis-permissive microenvironment of the brain created by interactions of cancer cells and cells in the brain through triggering inflammation, recruiting myeloid-derived suppressor cells, and promoting metabolic adaptation; immunosuppression resulting in the failure of adaptive immune response to recognize or kill cancer cells in the brain. Here, we briefly review recent advances in understanding brain metastasis organotropism and outline directions for future research.

Consistently, the field of genetic counseling has advocated that parents be advised to defer elective genetic testing of minors until adulthood to prevent a range of potential harms, including stigma, discrimination, and the loss of the child's ability to decide for him- or herself as an adult. However, consensus around the policy of "defer-when-possible" obscures the extent to which this norm is currently under siege. Increasingly, routine use of full or partial genome sequencing challenges our ability to control what is discovered in childhood or, when applied in a prenatal context, even before birth. The expansion of consumer-initiated genetic testing services challenges our ability to restrict what is available to minors. As the barriers to access crumble, medical professionals should proceed with caution, bearing in mind potential risks and continuing to assess the impact of genetic testing on this vulnerable population.

BACKGROUND AND PURPOSE:

Diffuse intrinsic pontine glioma is a lethal childhood brain cancer with dismal prognosis and MR imaging is the primary methodology used for diagnosis and monitoring. Our aim was to determine whether advanced diffusion, perfusion, and permeability MR imaging metrics predict survival and pseudoprogression in children with newly diagnosed diffuse intrinsic pontine glioma.

BACKGROUND AND PURPOSE:

Not all tandem occlusions diagnosed on traditional vascular imaging modalities, such as MRA, represent actual complete ICA occlusion. This study aimed to explore the utility of high-resolution vessel wall imaging in identifying true ICA tandem occlusions and screening patients for their suitability for endovascular recanalization.

BACKGROUND AND PURPOSE:

Conventional nonadhesive liquid embolic agents currently are the criterion standard for endovascular embolization of cerebral AVMs. However, inadequate distal penetration into the nidus and unstable proximal plug formation are the major limitations of this approach and of the currently available embolic materials. The aim of this study was to evaluate the hypothetic efficacy of combining liquid embolic agents with different properties and viscosities for use in endovascular embolization of cerebral AVMs.

BACKGROUND AND PURPOSE:

Endovascular embolization only has been advocated for treatment of brain arteriovenous malformations in recent trials. Our aim was to evaluate the results of embolization only in a cohort of patients who were enrolled in the A Randomized Trial of Unruptured Brain Arteriovenous Malformations (ARUBA) study at 39 clinical sites in 9 countries.

BACKGROUND AND PURPOSE:

Flow-diverter treatment for previously stented aneurysms has been reported to be less effective and prone to complications. In this study, we evaluated the effectiveness and safety of flow diverters for recurrent aneurysms after stent-assisted coiling.

BACKGROUND AND PURPOSE:

Flow diversion for the posterior circulation remains a promising treatment option for selected posterior circulation aneurysms. The Flow-Redirection Intraluminal Device (FRED) system has not been previously assessed in a large cohort of patients with posterior circulation aneurysms. The purpose of the present study was to assess safety and efficacy of FRED in this location.

BACKGROUND AND PURPOSE:

Detailed insight into the composition of thrombi retrieved from patients with ischemic stroke by mechanical thrombectomy might improve pathophysiologic understanding and therapy. Thus, this study searched for links between histologic thrombus composition and stroke subtypes and mechanical thrombectomy results.

SUMMARY:

Use of mechanical thrombectomy for stroke has increased since the publication of trials describing outcome improvement when used in the anterior circulation. These results, however, cannot be directly translated to the posterior circulation. While a high NIHSS score has demonstrated an association with poor outcomes in posterior stroke, the NIHSS is weighted toward hemispheric disease, and complex scores potentially delay definitive imaging diagnosis. We performed a retrospective analysis to ascertain whether any rapidly obtainable demographic or clinical and imaging data have a correlation with patient outcome postthrombectomy. Seventy-three cases were audited between September 2010 and October 2017. Presenting with a Glasgow Coma Scale score of >13 meant that the odds of reaching the primary end point of functional independence (defined as a 90-day modified Rankin Scale score of 0–2) were 5.70 times greater; similarly, presenting with a posterior circulation ASPECTS of >9 resulted in the odds of reaching the primary end point being 4.03 times greater. Older age correlated to a lower odds of independence (0.97, p = .04).

BACKGROUND AND PURPOSE:

It is currently not completely clear how well radiologists perform in evaluating large-vessel occlusion on CTA in acute ischemic stroke. The purpose of this study was to investigate potential factors associated with diagnostic error.

BACKGROUND AND PURPOSE:

Anoxic brain injury is a result of prolonged hypoxia. We sought to describe the nonquantitative arterial spin-labeling perfusion imaging patterns of anoxic brain injury, characterize the relationship of arterial spin-labeling and DWI, and evaluate the normalized diffusion-to-perfusion ratio to differentiate patients with anoxic brain injury from healthy controls.

BACKGROUND AND PURPOSE:

NeuroQuant is an FDA-approved software that performs automated MR imaging quantitative volumetric analysis. This study aimed to compare the accuracy of NeuroQuant analysis with visual MR imaging analysis by neuroradiologists with expertise in epilepsy in identifying hippocampal sclerosis.

BACKGROUND AND PURPOSE:

Accurate differentiation between glioblastoma and solitary brain metastasis is of vital importance clinically. This study aimed to investigate the potential value of the inflow-based vascular-space-occupancy MR imaging technique, which has no need for an exogenous contrast agent, in differentiating glioblastoma and solitary brain metastasis and to compare it with DSC MR imaging.

BACKGROUND AND PURPOSE:

Brain metastases are a common finding on brain MRI. However, the factors that dictate their size and distribution are incompletely understood. Our aim was to discover a statistical model that can account for the size distribution of parenchymal metastases in the brain as measured on contrast-enhanced MR imaging.

SUMMARY:

The remarkable temperature sensitivity of the brain is widely recognized and has been studied for its role in the potentiation of ischemic and other neurologic injuries. Pyrexia frequently complicates large-vessel acute ischemic stroke and develops commonly in critically ill neurologic patients; the profound sensitivity of the brain even to minor intraischemic temperature changes, together with the discovery of brain-to-systemic as well as intracerebral temperature gradients, has thus compelled the exploration of cerebral thermoregulation and uncovered its immutable dependence on cerebral blood flow. A lack of pragmatic and noninvasive tools for spatially and temporally resolved brain thermometry has historically restricted empiric study of cerebral temperature homeostasis; however, MR thermometry (MRT) leveraging temperature-sensitive nuclear magnetic resonance phenomena is well-suited to bridging this long-standing gap. This review aims to introduce the reader to the following: 1) fundamental aspects of cerebral thermoregulation, 2) the physical basis of noninvasive MRT, and 3) the physiologic interdependence of cerebral temperature, perfusion, metabolism, and viability.