Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform.

Genetic interactions mediate the emergence of phenotype from genotype, but technologies for combinatorial genetic perturbation in mammalian cells are challenging to scale. Here, we identify background-independent paralog synthetic lethals from previous CRISPR genetic interaction screens, and find that the Cas12a platform provides superior sensitivity and assay replicability. We develop the in4mer Cas12a platform that uses arrays of four independent guide RNAs targeting the same or different genes. We construct a genome-scale library, Inzolia, that is ~30% smaller than a typical CRISPR/Cas9 library while also targeting ~4000 paralog pairs. Screens in cancer cells demonstrate discrimination of core and context-dependent essential genes similar to that of CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes. Importantly, the in4mer platform offers a fivefold reduction in library size compared to other genetic interaction methods, substantially reducing the cost and effort required for these assays.

Efficient gene knockout and genetic interactions: the IN4MER CRISPR/Cas12a multiplex knockout platform.

Genetic interactions mediate the emergence of phenotype from genotype, but initial technologies for combinatorial genetic perturbation in mammalian cells suffer from inefficiency and are challenging to scale. Recent focus on paralog synthetic lethality in cancer cells offers an opportunity to evaluate different approaches and improve on the state of the art. Here we report a meta-analysis of CRISPR genetic interactions screens, identifying a candidate set of background-independent paralog synthetic lethals, and find that the Cas12a platform provides superior sensitivity and assay replicability. We demonstrate that Cas12a can independently target up to four genes from a single guide array, and we build on this knowledge by constructing a genome-scale library that expresses arrays of four guides per clone, a platform we call 'in4mer'. Our genome-scale human library, with only 49k clones, is substantially smaller than a typical CRISPR/Cas9 monogenic library while also targeting more than four thousand paralog pairs, triples, and quads. Proof of concept screens in four cell lines demonstrate discrimination of core and context-dependent essential genes similar to that of state-of-the-art CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes, a capability not offered by any extant library. Importantly, the in4mer platform offers a fivefold reduction in the number of clones required to assay genetic interactions, dramatically improving the cost and effort required for these studies.

Color or money?: The impact of socioeconomic status and race/ethnicity on breast cancer mortality.

BACKGROUND: Although the incidence of breast cancer is highest in White women, Black women die at a higher rate. Our aim was to compare the relative association between race/ethnicity and socioeconomic status on breast cancer mortality. METHODS: We identified female breast cancer patients diagnosed between 2007 - 2011 and followed through 2016 in the SEER database. Patients were grouped into socioeconomic quartiles by a prosperity index. The primary outcome of interest was 5-year cancer-specific survival. RESULTS: A total of 286,520 patients were included. Five-year survival was worst for Black women compared to other races/ethnicities in each socioeconomic quartile. When compared to White women in the lowest quartile, Black women in the lowest quartile, 2nd quartile, and 3rd quartile experienced the lowest 5-year survival rates (Hazard ratio 1.33, 1.23, 1.20; P < 0.01). CONCLUSION: Regarding cancer mortality, only in the most prosperous quartile do Black women achieve a similar outcome to the poorest quartile White women.

Application and Match Rates in the Complex General Surgical Oncology Match.

BACKGROUND: The Society of Surgical Oncology collaborates with the National Resident Matching Program (NRMP) to facilitate the Complex General Surgical Oncology (CGSO) Match. OBJECTIVE: The purpose of this study was to understand trends in CGSO Match outcomes. We hypothesized that (1) match rates would increase with time; (2) US allopathic graduates would have higher match rates than non-US allopathic graduates; and (3) most applicants would match at one of their top three ranked choices. METHODS: The NRMP provided applicant and program data from the CGSO Match (2014-2021). Chi-square tests elucidated temporal trends and match rates by applicant archetype. RESULTS: The annual number of applicants decreased from 103 to 90 (13% decrease), while the annual number of fellowship positions increased from 56 to 67 (20% increase) from 2014-2021. The annual percentage of applicants who did not match decreased from 46% to 26% (p < 0.05). Annual match rates increased from 54% to 74% (p < 0.05). US allopathic graduates had higher match rates than non-US allopathic graduates but this disparity narrowed over time (84% vs. 55% in 2021; p < 0.001). Approximately half of all applicants matched at one of their top three choices (first, 29%; second, 12%; third, 8%). Applicants matching at one of their top three choices increased from 36% to 50% (p < 0.05). CONCLUSIONS: CGSO Match rates have increased over the past decade, thus primarily benefiting non-US allopathic graduates. Most applicants match at one of their top three choices. More research is needed to understand disparities in match rates by applicant and residency program characteristics.

An Analysis of Preliminary General Surgery Residents' Matriculation into Categorical Positions.

OBJECTIVE: To analyze the effects of a pipeline program for preliminary general surgery (GS) residents to optimize their future enrollment into categorical positions. DESIGN: Retrospective review of non-designated preliminary (NDP) GS residents between 2014 and 2020 was conducted. Preliminary conversion rates (CRs) were analyzed for residents who matriculated to categorical GS residency or non-GS residency positions. SETTING: Howard University Hospital, Department of Surgery; tertiary academic hospital. PARTICIPANTS: PGY-1 (n = 14) and PGY-2 (n = 26) NDP GS residents RESULTS: Forty NDP GS residents studied (14 PGY-1 and 26 PGY-2). CR for the total cohort was 67.5% (n = 27), with 59.3% (n = 16) acquiring categorical GS positions and 40.7% (n = 13) obtaining categorical positions in other specialties. CR for PGY-1 residents into categorical GS position was 50% (n = 7), while PGY-2 residents had a CR of 34.6% (n = 9). No significant difference was observed between residents successfully matriculating into GS residency as a preliminary PGY-1 or PGY-2 (p = 0.34). Twelve preliminary residents secured categorical GS positions at this institution with 58.3% (n = 7) obtaining a PGY-1 position, 16.7% (n = 2) obtaining a PGY-2, and 25.0% (n = 3) obtaining a PGY-3 position. 7.1% (n = 1) of preliminary PGY-1 and 46.2% (n = 12) of preliminary PGY-2 residents went unmatched as of 2021. CONCLUSIONS: 67.5% of preliminary residents enrolled in categorical positions. Success rates were highest during the PGY-1 year. A residency program committed to uniform clinical curriculum, and standardized, metric-based decisions may have increased CR for preliminary GS residents. Public sharing of preliminary CRs to applicants may influence residency selection decisions, both for applicants and programs.

Persistence of Disparity in Thyroid Cancer Survival After Adjustments for Socioeconomic Status and Access.

INTRODUCTION: Thyroid cancer incidence has increased substantially in the past 4 decades, estimated at 3.5% annually. Incidence is highest in white patients, yet black patients have the worst survival. Racial/ethnic differences in presentation and outcomes are hypothesized to be a result of differences in access to care. Analyses delineating the relative contribution of access to racial/ethnic survival disparities are scarce. We aimed to explore the association of delay in access to care and early/increased detection with racial/ethnic disparities in thyroid cancer survival. METHODS: The Surveillance, Epidemiology, and End Results (SEER) database was queried from 2007 to 2011 for patients with a first primary thyroid cancer diagnosis and up to 5 years of follow-up. Composite scores were generated from county-level variables to capture socioeconomic status and screening habits. Kaplan-Meier analysis and Cox proportional hazards models were utilized for survival analysis. RESULTS: We identified 46,970 patients (67% white, 7% black, 15% Hispanic, 10% Asian or Pacific Islander, and 1% unknown/other). Compared to white patients, black, Hispanic, and Asian or Pacific Islander patients were more likely to present with distant disease (3% vs 5%, 5%, and 6%, respectively; P < .001). After adjusting for sex, age, stage, subtype, tumor size, surgery, radiation, socioeconomics, and screening habits, black patients were the only race/ethnicity found to have increased odds of 5-year mortality compared to white patients (24%, P < .001). CONCLUSION: Thyroid cancer survival is worst for black patients regardless of socioeconomic status or screening habits. Racial/ethnic disparities in survival are not attributable to early detection alone.

Breast Cancer in Black Women: Racial/Ethnic Disparities Affecting Survival.

Breast cancer is the most common noncutaneous malignancy affecting women in the United States, with >245,000 cases diagnosed annually. Breast cancer mortality rates have continued to trend down in the past three decades, yet racial/ethnic disparities persist, with the worst mortality rates seen in Black women. Of note, when compared by race, this downward trend is also trailing in Black women. Survival after breast cancer is mainly driven by factors related to early detection and effective therapy. These factors can be grouped into "biological" such as age, genetic mutations, tumor characteristics; and "social" such as education, income, access to care. There have been studies attributing racial disparities solely to biological factors, and there are those attributing the disparities to social factors alone. Although the exact mechanism is unclear, a relationship between both factors as relates to racial disparities in breast cancer outcomes has been demonstrated. In this report, we review factors contributing to the increased morbidity and mortality for breast cancer in Black women and explore sociological relationships. Facing the worst poverty rates compared with other races, Black women are inevitably more likely to be uninsured, have limited access to quality education, and have fewer financial resources. The goal of this review was to elucidate the complex interplay between biological and social factors contributing to racial disparities in breast cancer outcomes. We conclude by emphasizing the need for interventions made at both local and national levels.

Metastatic Acral lentiginous melanoma: A case report and review.

Of the four subtypes of cutaneous melanoma, acral lentiginous melanoma (ALM) is atypical in its presentation. ALM is a rare melanoma subtype that presents on the volar surfaces of the hand and foot. The difficulty of making an early diagnosis of ALM is highlighted by the case seen in our institution. The dire prognosis associated with ALM is postulated to be not only related to its destructive nature, but also due to a lack of patient awareness and vigilance, inadequate physician awareness, and disparity in healthcare access. We present this as a unique account of an ALM lesion in a 76 year old African-American male presenting originally in the left foot that went misdiagnosed for several years. The original lesion was considered to be an ulcerating left great toe lesion with signs typical of osteomyelitis. These clinical findings were corroborated by radiological x-ray evidence. Upon amputation and biopsy for suspected worsening osteomyelitis five years later, the pathological diagnosis of melanoma was finally made.

An Age-Based Analysis of Pediatric Melanoma: Staging, Surgery, and Mortality in the Surveillance, Epidemiology, and End Results Database.

The pediatric melanoma population is not well described, and current guidelines for their management are not well defined. Our study aims to identify this population, treatment modalities, and outcomes using a national population-based database. We reviewed the Surveillance, Epidemiology, and End Results database (2004-2008). Patients ≤21 years old with melanoma were included and grouped into ≤12 years of age, 13 to 18 years, and 19 to 21 years. Clinical characteristics were analyzed across the groups. A total of 1255 patients were included: 52.7 per cent were 19 to 21 years of age, 36.3 per cent were 13 to 18 years of age, and 11.0 per cent were ≤12 years of age. The 19- to 21-year-olds had the highest proportion of stage I (50.5%) versus ≤12 years of age (31.9%); the ≤12-year-olds had the highest proportion of stage IV (3.6%) versus 19 to 21 years of age (0.9%), P < 0.001. The 19- to 21-year-olds had the highest proportion receiving wide local excisions only (34.8%) versus ≤12 years of age (26.4%); the ≤12-year-olds had the highest proportion of patients without any surgeries (16.0%) versus 13 to 18 years of age (9.4%), P = 0.169. On adjusted analysis, the 19- to 21-year-olds had worse survival compared with ≤12 years of age (hazard ratio: 5.26, P = 0.017, 95% confidence interval 1.34-20.65). Disparities were found in the ≤12-year-old melanoma population, as they had later stage melanomas, less invasive surgery, and lower survival. Clearer prognostic factors are needed to better elucidate their management.

Readmissions After Complex Cancer Surgery: Analysis of the Nationwide Readmissions Database.

PURPOSE: Hospital readmissions after surgery are a focus of quality improvement efforts. Although some reflect appropriate care, others are potentially preventable readmissions (PPRs). We aim to describe the burden, timing, and factors associated with readmissions after complex cancer surgery. METHODS: The Nationwide Readmissions Database (2013) was used to select patients undergoing a complex oncologic resection, which was defined as esophagectomy/gastrectomy, hepatectomy, pancreatectomy, colorectal resection, lung resection, or cystectomy. Readmissions within 30 days from discharge were analyzed. International Classification of Diseases (9th revision) primary diagnosis codes were reviewed to identify PPRs. Multivariable logistic regression analyses identified demographic, clinical, and hospital factors associated with readmissions. RESULTS: Of the 59,493 eligible patients, 14% experienced a 30-day readmission, and 82% of these were deemed PPRs. Half of the readmissions occurred within the first 8 days of discharge. Infections (26%), GI complications (17%), and respiratory conditions (10%) accounted for most readmissions. Factors independently associated with an increased likelihood of readmission included Medicaid versus private insurance (odds ratio [OR], 1.32; 95% CI, 1.17 to 1.48), higher comorbidity score (OR, 1.5; 95% CI, 1.33 to 1.63), discharge to a facility (OR, 1.39; 95% CI, 1.29 to 1.51), prolonged length of stay (OR, 1.42; 95% CI, 1.32 to 1.52), and occurrence of a major in-hospital complication (OR, 1.24; 95% CI, 1.16 to 1.34). CONCLUSION: One in seven patients undergoing complex cancer surgery suffered a readmission within 30 days. We identified common causes of these and identified patients at high risk for such an event. These data can be used by physicians, administrators, and policymakers to develop strategies to decrease readmissions.

Comparison of Rates and Outcomes of Readmission to Index vs Nonindex Hospitals After Major Cancer Surgery.

Importance: Increasing regionalization of cancer surgery has the inadvertent potential to lead to fragmentation of care if readmissions occur at a facility other than the index hospital. The magnitude and adverse effects of readmission to a facility other than the one where the surgery was performed are unclear. Objectives: To assess rates of readmission to nonindex hospitals after major cancer surgery and to compare outcomes between index and nonindex hospital readmissions. Design, Setting, and Participants: In this multicenter, population-based, nationally representative study of adult patients undergoing a major cancer operation (defined as esophagectomies or gastrectomies, hepaticobiliary resections, pancreatectomies, colorectal resections, or cystectomies), retrospective analyses were performed using the Nationwide Readmissions Database (admissions from January 1 through September 30, 2013). Descriptive analyses were performed to determine 90-day readmission characteristics, including timing, cost, and outcomes. Adjusting for clustering by facility, the study used multivariate logistic regression to identify factors associated with nonindex vs index readmissions. The study also used regression models to identify differences in mortality, major complications, and subsequent readmissions between the 2 groups. Data analysis was performed from January 1 through December 31, 2013. Exposures: Readmission to index vs nonindex hospitals (defined as any hospital other than the hospital where the major cancer operation was performed). Main Outcomes and Measures: Proportion of 90-day readmissions and nonindex readmissions after major cancer surgery, factors associated with nonindex readmissions, and difference between in-hospital mortality, hospital costs, and subsequent readmissions for patients admitted to index vs nonindex hospitals. Results: A total of 60 970 patients were included in the study (mean [SD] age, 67 [13] years; 7619 [55.6%] male and 6075 [44.4%] female). The 90-day readmission rate was 23.0%. Of the 13 695 first readmissions, 20.1% were to a nonindex hospital. Independent factors associated with readmission to a nonindex hospital included type of procedure, comorbidities (OR, 1.40; 95% CI, 1.15-1.70), elective admission (OR, 1.21; 95% CI, 1.06-1.37), discharge to a nursing facility (OR, 1.20; 95% CI, 1.07-1.36), and surgery at a teaching hospital (OR, 1.16; 95% CI, 1.00-1.34) (all P < .05). After risk adjustment, patients readmitted to nonindex hospitals had 31.2% higher odds of mortality (odds ratio, 1.31; 95% CI, 1.05-1.64) and 27.3% higher odds of having a major complication (odds ratio, 1.27; 95% CI, 1.14-1.42). Subsequent readmissions and hospital costs were not different between the 2 groups. Conclusions and Relevance: Approximately one-fifth of readmissions were to a nonindex hospital and were associated with higher mortality and morbidity than readmission to index hospitals. Factors that influence nonindex readmissions have been identified to target interventions.

How does a concurrent diagnosis of cancer influence outcomes in emergency general surgery patients?

BACKGROUND: A significant proportion of hospital admissions in the US are secondary to emergency general surgery (EGS). The aim of this study is to quantify outcomes for EGS patients with cancer. METHODS: The Nationwide Inpatient Sample (2007 to 2011) was queried for patients with a diagnosis of an EGS condition as determined by the American Association for the Surgery of Trauma. Of these, patients with a diagnosis of malignant cancers (ICD-9-CM diagnosis codes; 140-208.9, 238.4, 289.8) were identified. Patients with and without cancer were matched across baseline characteristics using propensity-scores. Outcome measures included all-cause mortality, complications, failure-to-rescue, length of stay, and cost. Multivariable logistic regression analyses further adjusted for hospital characteristics and volume. RESULTS: Analysis of 3,625,906 EGS patients revealed an 8.9% prevalence of concurrent malignancies. The most common EGS conditions in cancer patients included gastro-intestinal bleeding (24.8%), intestinal obstruction (13.5%), and peritonitis (10.7%). EGS patients with cancer universally had higher odds of complications (odds ratio [OR] 95% confidence interval [CI]: 1.20 [1.19 to 1.21]), mortality (OR [95% CI]: 2.00 [1.96 to 2.04]), failure-to-rescue (OR [95% CI]: 1.52 [1.48 to 1.56]), and prolonged hospital stay (OR [95% CI]: 1.69 [1.67 to 1.70]). CONCLUSIONS: EGS patients with concurrent cancer have worse outcomes compared with patients without cancer after risk-adjustment.

Particle Size, Surface Area, and Amorphous Content as Predictors of Solubility and Bioavailability for Five Commercial Sources of Ferric Orthophosphate in Ready-To-Eat Cereal.

Ferric orthophosphate (FePO4) has had limited use as an iron fortificant in ready-to-eat (RTE) cereal because of its variable bioavailability, the mechanism of which is poorly understood. Even though FePO4 has desirable sensory properties as compared to other affordable iron fortificants, few published studies have well-characterized its physicochemical properties. Semi-crystalline materials such as FePO4 have varying degrees of molecular disorder, referred to as amorphous content, which is hypothesized to be an important factor in bioavailability. The objective of this study was to systematically measure the physicochemical factors of particle size, surface area, amorphous content, and solubility underlying the variation in FePO4 bioavailability. Five commercial FePO4 sources and ferrous sulfate were added to individual batches of RTE cereal. The relative bioavailability value (RBV) of each iron source, determined using the AOAC Rat Hemoglobin Repletion Bioassay, ranged from 51% to 99% (p < 0.05), which is higher than typically reported. Solubility in dilute HCl accurately predicted RBV (R² = 0.93, p = 0.008). Amorphous content measured by Dynamic Vapor Sorption ranged from 1.7% to 23.8% and was a better determinant of solubility (R² = 0.91; p = 0.0002) than surface area (R² = 0.83; p = 0.002) and median particle size (R² = 0.59; p = 0.12). The results indicate that while solubility of FePO4 is highly predictive of RBV, solubility, in turn, is strongly linked to amorphous content and surface area. This information may prove useful for the production of FePO4 with the desired RBV.

Predictors of Self-Reported Family Health History of Breast Cancer.

The objective of this study was to identify predictors of self-reported family health history of breast cancer in an ethnically diverse population of women participating in a breast cancer screening program. Participants completed a self-administered questionnaire about their demography, health, breast health and family health history of breast cancer. The association between family health history of breast cancer and categorical variables were analyzed using the T test, chi square, and multi-nominal logistic regression. Those who were least likely to report a family history of cancer were African Americans (p = 0.02), and immigrant women from South America (p < 0.001) and Africa (p = 0.04). However, 34.4 % reported having a second-degree maternal relative with breast cancer compared to 6.9 % who reported having a second degree paternal relative with breast cancer. Therefore, there is a need to increase efforts to educate families about the importance of collecting and sharing one's family health history.

C-reactive protein response to a vegan lifestyle intervention.

This brief lifestyle intervention, including a vegan diet rich in fresh fruits and vegetables, whole grains and various legumes, nuts and seeds, significantly improved health risk factors and reduced systemic inflammation as measured by circulating CRP. The degree of improvement was associated with baseline CRP such that higher levels predicted greater decreases. The interaction between gender and baseline CRP was significant and showed that males with higher baseline CRP levels appeared to have a more robust decrease in CRP due to the intervention than did their female counterparts. It is likely that the vegetable and high fiber content of a vegan diet reduces CRP in the presences of obesity. Neither the quantity of exercise nor the length of stay was significant predictors of CRP reduction. Additionally, those participants who had a vegan diet prior to the intervention had the lowest CRP risk coming into the program. Direct measure of body fat composition, estrogen and other inflammatory mediators such as IL-6 and TNF-alpha would enhance current understanding of the specific mechanisms of CRP reduction related to lifestyle interventions.

Effects of exercise on biobehavioral outcomes of fatigue during cancer treatment: results of a feasibility study.

UNLABELLED: Cancer treatment is associated with decreased hemoglobin (Hb) concentration and aerobic fitness (VO2 max), which may contribute to cancer-related fatigue (CRF) and decreased quality of life (QoL). Endurance exercise may attenuate CRF and improve QoL, but the mechanisms have not been thoroughly investigated. Objectives. To (a) determine the feasibility of conducting an exercise intervention among women receiving treatment for breast cancer; (b) examine the effects of exercise on Hb and VO2 max and determine their association with changes in CRF and QoL; and (c) investigate changes in selected inflammatory markers. Methods. Fourteen women receiving chemotherapy for Stages I-II breast cancer were randomly assigned to exercise (n = 7) or usual care (n = 7). Women in the exercise group performed supervised, individualized treadmill exercise 2-3 times/week for the duration of chemotherapy (9-12 weeks). Data were collected 4 times over 15-16 weeks. Results. Recruitment rate was 45.7%. Sixteen women consented and 14 completed the trial, for a retention rate of 87.5%. Adherence to exercise protocol was 95-97%, and completion of data collection was 87.5-100%. Exercise was well tolerated. VO2 max was maintained at prechemotherapy levels in exercisers but declined in the usual-care group (p < .05). Hb decreased (p < .001) in all participants as they progressed through chemotherapy. Exercise did not have significant effects on CRF or QoL. Changes in inflammatory markers favored the exercise group. CONCLUSIONS: Exercise during chemotherapy may protect against chemotherapy-induced decline in VO2 max but not Hb concentration.

Large-scale proteomic characterization of melanoma expressed proteins reveals nestin and vimentin as biomarkers that can potentially distinguish melanoma subtypes.

Melanoma is an aggressive type of skin cancer, which accounts for only 4% of skin cancer cases but causes around 75% of skin cancer deaths. Currently, there is a limited set of protein biomarkers that can distinguish melanoma subtypes and provide an accurate prognosis of melanoma. Thus, we have selected and profiled the proteomes of five different melanoma cell lines from different stages of progression in comparison with a normal melanocytes using tandem mass spectrometry. We also profiled the proteome of a solid metastatic melanoma tumor. This resulted in the identification of 4758 unique proteins, among which ∼200-300 differentially expressed proteins from each set were found by quantitative proteomics. Correlating protein expression with aggressiveness of each melanoma cell line and literature mining resulted in the final selection of six proteins: vimentin, nestin, fibronectin, annexin A1, dipeptidyl peptidase IV, and histone H2A1B. Validation of nestin and vimentin using 40 melanoma samples revealed pattern of protein expression can help predict melanoma aggressiveness in different subgroups of melanoma. These results, together with the combined list of 4758 expressed proteins, provide a valuable resource for selecting melanoma biomarkers in the future for the clinical and research community.