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.

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.

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.

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.

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.

Strong association of fascin expression with triple negative breast cancer and basal-like phenotype in African-American women.

BACKGROUND: Fascin, an actin bundling protein, plays a critical role in cell motility due to formation of actin rich protrusions called filopodia, important in cell migration, invasion and metastatic spread. Fascin overexpression has been associated with epithelial to mesenchymal transition and correlates with progression and unfavourable prognosis in breast carcinoma. OBJECTIVE: To evaluate fascin expression by immunohistochemistry and correlate the expression pattern with clinicopathological parameters in breast cancer in African-American (AA) women, in whom triple negative breast cancer (TNBC), an aggressive subtype, is more prevalent. METHODS: Tissue microarrays were constructed from formalin-fixed, paraffin-embedded blocks of tumour tissue from primary breast carcinomas in 202 AA women. Immunohistochemical detection of fascin was correlated with four major subtypes of breast carcinoma (luminal A, luminal B, human epidermal growth factor receptor 2 and triple negative (TN)) and other clinicopathological factors, including age, grade, tumour size, stage, regional lymph node status and survival. RESULTS: We observed a significant association between fascin expression and TN subtype, oestrogen receptor (ER) negativity, progesterone receptor (PR) negativity, Elston-Nottingham (EN) grade 3 and decreased overall survival. There was also a significant association between expression of CK 5/6, a marker of basal-like phenotype, and fascin expression. CONCLUSION: These results suggest that fascin is a marker for TN subtype having a basal-like phenotype and decreased overall survival. Fascin may represent a target for therapy in TNBC in AA women.

Characterizing lobular carcinoma of the male breast using the SEER database.

BACKGROUND: Lobular carcinoma of the male breast is rare. We sought to investigate the clinical characteristics, treatment, and outcomes of men and women with lobular breast cancer, using a population-based database. METHODS: We reviewed the Surveillance, Epidemiology, and End Results database 1988-2008 and identified patients with a lobular breast cancer diagnosis (invasive lobular carcinoma [ILC] and lobular carcinoma in situ [LCIS]) using the "International Classification of Diseases for Oncology, Third Edition" codes. Bivariate analyses compared the male and female patients on demographics, clinical characteristics, and treatment modalities. Multivariate logistic regression analysis determined the risk-adjusted likelihood of receiving treatment. Survival analysis was done and hazard ratios were obtained using Cox proportional models. RESULTS: Overall, 133,339 patients were identified, including 133,168 women (99.9%) and 171 men (0.1%). Most had ILC (82.08%). The median age was 66 ± 20 y for the men and 61 ± 21 y for the women. The men with ILC were more likely to have poorly differentiated tumors (26.45% versus 15.61%; P < 0.001) and stage IV disease (9.03% versus 4.18%; P = 0.005) than were the women. The cancer-specific 5-year survival rates for ILC were 82.9% for the men and 91.9% for the women. Adjusted survival was better for patients with ILC receiving surgery plus radiotherapy than patients receiving neither (hazard ratio 0.52, 95% confidence interval 0.49-0.56). Women with ILC had a 55% increased odds of receiving surgery plus radiotherapy compared with men (odds ratio 1.55, 95% confidence interval 1.08-2.22). CONCLUSIONS: ILC presents at a higher grade and stage in men. The difference in disease characteristics and survival rates suggests that the treatment of men with lobular breast cancer should be adjusted to improve their outcomes.

Epithelial Na⁺ sodium channels in magnocellular cells of the rat supraoptic and paraventricular nuclei.

The epithelial Na⁺ channels (ENaCs) are present in kidney and contribute to Na⁺ and water homeostasis. All three ENaC subunits (α, ß, and γ) were demonstrated in the cardiovascular regulatory centers of the rat brain, including the magnocellular neurons (MNCs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). However, the functional significance of ENaCs in vasopressin (VP) and oxytocin (OT) synthesizing MNCs is completely unknown. In this study, we show with immunocytochemical double-labeling that the α-ENaC is colocalized with either VP or OT in MNCs in the SON and PVN. In addition, parvocellular neurons in the dorsal, ventrolateral, and posterior subregions of the PVN (not immunoreactive to VP or OT) are also immunoreactive for α-ENaC. In contrast, immunoreactivity to ß- and γ-ENaC is colocalized with VP alone within the MNCs. Furthermore, immunoreactivity for a known target for ENaC expression, the mineralcorticoid receptor (MR), is colocalized with both VP and OT in MNCs. Using single-cell RT-PCR, we detected mRNA for all three ENaC subunits and MR in cDNA libraries derived from single MNCs. In whole cell voltage clamp recordings, application of the ENaC blocker benzamil reversibly reduced a steady-state inward current and decreased cell membrane conductance approximately twofold. Finally, benzamil caused membrane hyperpolarization in a majority of VP and about one-half of OT neurons in both spontaneously firing and quiet cells. These results strongly suggest the presence of functional ENaCs that may affect the firing patterns of MNCs, which ultimately control the secretion of VP and OT.

Prevalence and prognostic role of triple-negative breast cancer by race: a surveillance study.

INTRODUCTION: Emerging research suggests a substantially greater prevalence of the adverse triple-negative (TN) subtype (human epidermal growth factor receptor [HER]2(-), estrogen receptor [ER](-), and progesterone receptor [PR])(-)) among black patients with breast cancer. No reports however have been generated from a statewide cancer registry. PATIENTS AND METHODS: The study consisted of all black patients (N = 643) and a random sample of white patients (n = 719) diagnosed with primary invasive breast cancer (2000-2003) listed in the National Cancer Institute-Surveillance Epidemiology and End Results (NCI-SEER) Connecticut Tumor Registry (CTR). HER2 status was obtained from pathology reports submitted to the registry. Remaining data were obtained from the registry database. RESULTS: TN tumors were more prevalent in black compared with white patients (30.8% vs. 11.2%, respectively; P < .001.) There was a 2-fold greater frequency of ER(-) and PR(-) phenotypes among black patients, but HER2 status did not differ by race. Patients with lobular cancer were less likely to have TN breast cancer compared with patients with ductal tumors (odds ratio [OR] = 0.23; 95% confidence interval [CI], 0.10-0.58). Among patients with regional disease, black patients exhibited increased risk of death (relative risk [RR] = 2.71; 95% CI, 1.48-4.97) independent of TN status. No survival disparity was found among patients with local disease. DISCUSSION: These registry-based data corroborate reports that TN breast cancer varies substantially by race and histologic subtype. A survival disparity among patients with advanced disease, but not local disease, casts some doubt on TN status as an explanation for differences. CONCLUSION: More research is warranted to understand why black patients with advanced breast cancer may be at increased risk for death whether or not their tumors express the TN phenotype.

Sentinel lymph node biopsy from the vantage point of an oncologic surgeon.

Sentinel lymph node biopsy has greatly influenced the surgical management of clinically localized primary melanoma. Lymphatic mapping and sentinel lymph node biopsy have been used for the selective management of the draining regional lymph node basin of primary cutaneous melanoma. Oncologic surgeons have adopted this procedure to selectively identify occult nodal status in melanoma patients who are at a higher risk of regional metastasis. The current standard of treatment of tumor-positive sentinel lymph node metastasis is immediate completion lymphadenectomy, but considerable debate surrounds the utility of this procedure. This contribution reviews development, technical aspects, selective management of the lymph node basin, and sentinel lymph node biopsy techniques.

Direct tissue proteomics in human diseases: potential applications to melanoma research.

Although the rates of cancer are stabilizing, the number of new invasive melanoma continues to rise. Melanoma represents only 4% of all skin cancers, but nearly 80% of skin cancer deaths. In loss of potential productive life-years, it is second only to adult leukemia. Once melanoma spreads to regional and distant sites, the chance of cure decreases significantly. Unfortunately, current diagnostic and prognostic methods are often inadequate. More precise staging and disease characterization will lead to new and more rational approaches to treatment. Proteomics is a fast-growing discipline in biomedicine that can be defined as the global characterization and differential expression of the entire protein complement of a cell, tissue or organism. Despite major advances in molecular approaches to the diagnosis and prognostication of human diseases such as melanoma, there remain significant obstacles in applying the proteomic technologies to clinical samples to extract important biological information. The application of a shotgun-based technique termed direct tissue proteomics with improved extraction protocol of proteins from formalin-fixed paraffin-embedded tissue would enable retrospective biomarker investigations of the vast archive of pathologically characterized clinical samples that exist worldwide. Combination of this direct tissue proteomics method with laser-capture microdissection may assist in the discovery of new biomarkers and may lead to new diagnostic tests, risk assessment and staging tools as well as improvement in therapeutics. In addition, these tools can provide a molecular characterization of melanoma, which may enable individualized molecular therapy.

Sentinel lymph node mapping for primary breast cancer.

The tumor status of the axillary lymph nodes is the single most important predictor of survival for patients with primary breast cancer. Because of its essential role in staging, regional control, and perhaps survival, axillary lymph node dissection (ALND) has long been the standard of care for patients with operable breast cancer. During the past decade, the introduction and development of sentinel lymph node dissection (SLND) for primary breast cancer have allowed surgeons to determine the tumor status of the axilla without a standard level I and II ALND. Several well-designed studies have documented that SLND is an effective way of assessing axillary nodal status with minimal morbidity and high accuracy. We address the current status and future directions of SLND for primary breast cancer.