Optimizing NK-92 serial killers: gamma irradiation, CD95/Fas-ligation, and NK or LAK attack limit cytotoxic efficacy.

BACKGROUND: The NK cell line NK-92 and its genetically modified variants are receiving attention as immunotherapies to treat a range of malignancies. However, since NK-92 cells are themselves tumors, they require irradiation prior to transfer and are potentially susceptible to attack by patients' immune systems. Here, we investigated NK-92 cell-mediated serial killing for the effects of gamma-irradiation and ligation of the death receptor Fas (CD95), and NK-92 cell susceptibility to attack by activated primary blood NK cells. METHODS: To evaluate serial killing, we used 51Cr-release assays with low NK-92 effector cell to target Raji, Daudi or K562 tumor cell (E:T) ratios to determine killing frequencies at 2-, 4-, 6-, and 8-h. RESULTS: NK-92 cells were able to kill up to 14 Raji cells per NK-92 cell in 8 h. NK-92 cells retained high cytotoxic activity immediately after irradiation with 10 Gy but the cells surviving irradiation lost > 50% activity 1 day after irradiation. Despite high expression of CD95, NK-92 cells maintained their viability following overnight Fas/CD95-ligation but lost some cytotoxic activity. However, 1 day after irradiation, NK-92 cells were more susceptible to Fas ligation, resulting in decreased cytotoxic activity of the cells surviving irradiation. Irradiated NK-92 cells were also susceptible to killing by both unstimulated and IL-2 activated primary NK cells (LAK). In contrast, non-irradiated NK-92 cells were more resistant to attack by NK and LAK cells. CONCLUSIONS: Irradiation is deleterious to both the survival and cytotoxicity mediated by NK-92 cells and renders the NK-92 cells susceptible to Fas-initiated death and death initiated by primary blood NK cells. Therefore, replacement of irradiation as an antiproliferative pretreatment and genetic deletion of Fas and/or NK activation ligands from adoptively transferred cell lines are indicated as new approaches to increase therapeutic efficacy.

Cancer of unknown primary: Incidence rates, risk factors and survival among adolescents and young adults.

Cancer of unknown primary (CUP) is a clinical challenge especially when it occurs in adolescents and young adults (AYA), aged 15-39 years, due to the sparse data in this population. The available data has not described the population-based epidemiological features of CUP among AYA. Therefore, we collected patient information from the Surveillance, Epidemiology and End Results (SEER) registry, 1990-2015. Age, gender, ethnic, five pathological classification groups were assessed along with an aggregate level socioeconomic status (SES) index and population density at the county level. Incidence rates, modeled relative risks and survival of AYA patients with CUP were assessed. Among 2,480 AYA patients, 907 met the definition of standard pathology classifications. The majority of AYA patients with CUP had a neuroendocrine, squamous cell and poorly differentiated carcinomas with 0.4 cases per 1,000,000 population. AYA living in areas with the highest SES level had the highest risks of CUP; adjusted relative risks (ARR) of 1.17 (95% CI 1.0-1.4) and 1.99 (95% CI 1.5-2.6), respectively. AYA living in nonmetropolitan areas had a lower risk of CUP (ARR = 0.16; 95% CI 0.1-0.2). The incidence of differentiated neoplasms has been decreasing slower than undifferentiated neoplasms since the early 1990s. The median overall survival (OS) was 11 months (95% CI 9-13 months) with squamous CUP having the longest median OS 16 years (95% CI 3-24 years). In conclusion, this analysis answers several gaps in the knowledge of CUP among AYA and provides a platform to better understand this disease and its management within this group.

Lifestyle and Risk of Hypertension: Follow-Up of a Young Pre-Hypertensive Cohort.

OBJECTIVES: To determine whether healthy lifestyle decreases the risk of developing hypertension in pre-hypertensive patients. STUDY DESIGN: A longitudinal study. SETTING & PARTICIPANTS: Randomly selected pre-hypertensive young adults 20-45 years old without any vascular disease such as stroke or diabetes. PREDICTORS: Four lifestyle factors (a body mass index [BMI] of 18.5-24.9 kg/m2, regular physical activity, no alcohol use and 6-8 h of sleep per day), individually and in combination. OUTCOMES: Hypertension was defined as a systolic blood pressure (SBP) ≥ 140 mmHg, or a diastolic BP (DBP) ≥ 90 mmHg or self-reported hypertension. MEASUREMENTS: Multivariate adjusted Cox proportional hazards. RESULTS: During a median follow-up of 4.7 years, 1009 patients were enrolled in our study, and 182 patients developed hypertension. Compared with a BMI of 18.5-24.9 kg/m2, a BMI of 25-30 kg/m2 and a BMI of >30 kg/m2 were associated with an increased risk of hypertension occurrence (hazard ratio [HR], 1.83; 95% confidence interval [CI], 1.19-2.84 and HR, 2.62; 95% CI, 1.01-6.80, respectively). Compared with sleep duration of >8 h/day, 6-8 h/day of sleep was associated with a lower risk of hypertension occurrence (HR, 0.40; 95% CI, 0.18-0.86). There were no statistically significant associations between physical activity or alcohol use and hypertension occurrence (P>0.05). LIMITATION: All lifestyle factors were measured only once. CONCLUSION: Healthy BMI (18.5-24.9 kg/m(2)) and sleep duration (6-8 h/day) were associated with a lower risk of the occurrence of hypertension in pre-hypertension patients.

Cancer of unknown primary: time trends in incidence, United States.

PURPOSE: To describe the epidemiological features and trends of cancer of unknown primary (CUP) in a large and diverse US population. METHODS: The Surveillance Epidemiology and End Results registry was used to examine incidence rates, adjusted to the World Segi 1960 population, by demographic and tumor characteristics among patients diagnosed with CUP between 1973 and 2010. Annual percent changes in incidence rates were estimated using Joinpoint regression. RESULTS: The incidence rate of pathologically investigated CUP was 4.1 per 100,000 and is consistent with reports from other countries. In the USA, CUP incidence rates have been decreasing since the early 1980s, 3.6 % per year in the last two decades. The USA experienced decreases earlier than other countries. US males and African Americans had the highest rates of CUP. The rates of non-microscopically confirmed CUP have dropped 2.6 % per year since 1973, but 24 % of CUP patients do not receive microscopic confirmation and 21 % of those with microscopically investigated cancer receive a vague histology (i.e., epithelial) diagnosis. Twenty percent of patients with pathological investigation receive radiation. Patients were twice as likely to be diagnosed with a non-pathologically investigated CUP if they were living in areas with the lowest income quartile relative to areas with the highest income quartile. CONCLUSION: Although the incidence of CUP is decreasing, we document CUP that may be due to insufficient diagnostic inquiry. Questions raised by the findings in this data provide hypotheses for further epidemiological and biological studies in the elucidation of CUP incidence and treatment.

The association of state law to breastfeeding practices in the US.

We assessed the relationship between breastfeeding initiation and duration with laws supportive of breastfeeding enacted at the state level. We analyzed breastfeeding practices using the 2003-2010 National Health and Nutrition Examination Survey. We evaluated three measures of breastfeeding practices: Mother's reported breastfeeding initiation, a proxy report of infants ever being breastfeed, and a proxy report of infants being breastfeed for at least 6 months. Survey data were linked to eight laws supportive of breastfeeding enacted at the state level. The most robust laws associated with increased infant breastfeeding at 6 months were an enforcement provision for workplace pumping laws [OR (95 % CI) 2.0 (1.6, 2.6)] and a jury duty exemption for breastfeeding mothers [OR (95 % CI) 1.7 (1.3, 2.1)]. Having a private area in the workplace to express breast milk [OR (95 % CI) 1.3 (1.1, 1.7)] and having break time to breastfeed or pump [OR (95 % CI) 1.2 (1.0, 1.5)] were also important for infant breastfeeding at 6 months. This research responds to breastfeeding advocates' calls for evidence-based data to generate the necessary political action to enact legislation and laws to protect, promote, and support breastfeeding. We identify the laws with the greatest potential to reach the Healthy People 2020 targets for breastfeeding initiation and duration.

Patient characteristics associated with definitive diagnosis of metastatic pancreatic cancer in those initially diagnosed with cancer of unknown primary.

Cancer of unknown primary (CUP) and pancreatic cancer (PC) are malignancies associated with poor prognosis. CUP is the fourth most common cause of cancer mortality in the US, and median survival time is 3-4 months. PC is the third most common cause of cancer mortality in the US, and median survival time for patients with stage 3 or 4 PC is 2-3 months. The present study aimed to understand the patient characteristics of those initially misdiagnosed with CUP who ultimately received a diagnosis of PC. The present study used 2010-2015 Surveillance, Epidemiology, and End Results-Medicare data, a US population-based cancer registry linked to Medicare health insurance claims. Odds ratios (ORs) and 95% confidence intervals were calculated using two binary logistic regression models to compare the characteristics of patients who received definitive diagnosis between the CUP-PC group (those with an initial diagnosis of CUP who eventually received a stage 3 or 4 PC diagnosis) and the PC group (those diagnosed with stage 3 or 4 PC only). Approximately 26% of patients who received a definitive diagnosis of metastatic PC started with an initial diagnosis of CUP (n=17,565). The odds of definitive PC diagnosis in patients with CUP were lower for those with a comorbidity score of 0 [OR, 0.85 (95% CI: 0.79, 0.91)] and epithelial/unspecified histology [OR, 0.76 (95% CI: 0.71, 0.82)]. The odds of definitive PC diagnosis in patients with CUP were higher for patients of other race [OR, 1.27 (95% CI: 1.13, 1.43)] compared with white patients. Definitive diagnosis of PC in patients with CUP was lower in patients who were older with fewer or no comorbidities and unspecified histology. The complexity of CUP diagnosis and patient performance status may influence delays in diagnosis to a known primary site.

Detection of Antibody-Dependent Cell-Mediated Cytotoxicity-Supporting Antibodies by NK-92-CD16A Cell Externalization of CD107a: Recognition of Antibody Afucosylation and Assay Optimization.

Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes eliminates cells infected with viruses. Anti-viral ADCC requires three components: (1) antibody; (2) effector lymphocytes with the Fc-IgG receptor CD16A; and (3) viral proteins in infected cell membranes. Fc-afucosylated antibodies bind with greater affinity to CD16A than fucosylated antibodies; individuals' variation in afucosylation contributes to differences in ADCC. Current assays for afucosylated antibodies involve expensive methods. We report an improved bioassay for antibodies that supports ADCC, which encompasses afucosylation. This assay utilizes the externalization of CD107a by NK-92-CD16A cells after antibody recognition. We used anti-CD20 monoclonal antibodies, GA101 WT or glycoengineered (GE), 10% or ~50% afucosylated, and CD20-positive Raji target cells. CD107a increased detection 7-fold compared to flow cytometry to detect Raji-bound antibodies. WT and GE antibody effective concentrations (EC50s) for CD107a externalization differed by 20-fold, with afucosylated GA101-GE more detectable. The EC50s for CD107a externalization vs. 51Cr cell death were similar for NK-92-CD16A and blood NK cells. Notably, the % CD107a-positive cells were negatively correlated with dead Raji cells and were nearly undetectable at high NK:Raji ratios required for cytotoxicity. This bioassay is very sensitive and adaptable to assess anti-viral antibodies but unsuitable as a surrogate assay to monitor cell death after ADCC.

Patient Characteristics Associated with Definitive Diagnosis of Metastatic Pancreatic Cancer in Those Initially Diagnosed with Cancer of Unknown Primary.

Purpose: Cancer of unknown primary (CUP) is the fourth most common cause of cancer mortality in the U.S. Median survival after CUP diagnosis is 3-4 months. As CUP and metastatic pancreatic cancer (PC) are comparable in prevalence and survival, PC diagnosis is a useful endpoint to assess patient characteristics associated with definitive diagnosis in older patients who initially present with CUP. Methods: This study used 2010-2015 SEER-Medicare data. Logistic regression models compared patient characteristics who received definitive diagnosis in two subsets: CUP-PC and PC only. Results: Approximately 26% of patients who received a definitive diagnosis of metastatic pancreatic cancer started with an initial diagnosis of CUP (n=17,565). The odds of definitive diagnosis in CUP-PC were lower for those with a comorbidity score of 0 (OR 0.85 [0.79, 0.91]) and epithelial/unspecified histology (OR 0.76 [0.71, 0.82]). The odds of definitive diagnosis in CUP-PC were higher for patients of Other race (OR 1.27 [1.13, 1.43]) compared to White patients. Conclusion: Definitive diagnosis of CUP-PC was favorable in patients in the Other race category with fewer or no comorbidities. Unfavorable characteristics included older patients and those with epithelial/unspecified histology. Future studies will focus on patterns of care and survival in patients with CUP-PC.

Systematic review of the CUP trials characteristics and perspectives for next-generation studies.

BACKGROUND: Research on therapeutic strategies for patients with unknown primary cancer (CUP) has been underwhelming. This paper summarized and evaluated the CUP therapeutic research over the previous five years. Based on this evaluation, recommendations for clinical trial designs are made to improve the impact of CUP research on patients. METHODS: Published and ongoing research were evaluated. PubMed was searched from January 1, 2015, to November 1, 2021. The start date of 2015 was chosen to identify research published after ESMO issued new diagnostic and therapeutic guidelines. The US National Library of Medicine indexed ongoing clinical trials. FINDINGS: Of the 244 CUP studies indexed in PubMed, 11.9% were prospective studies, and 4.9% were clinical trials. The review protocol deemed 65 publications eligible for full-text review. Eleven studies evaluating therapeutic regimens were retained. The two prospective studies and non-randomized trials showed promising outcomes for site-specific treatments. Randomized clinical trials were less promising; however, the trials had recruitment challenges resulting in biased accrual and the inability to keep pace with advancing diagnostics and therapeutics. Most of the 35 ongoing studies were phase II single-arm trials assessing immune checkpoint inhibitors (ICI) or site-specific therapies among CUP patients with suspected favorable prognoses. CONCLUSION: Our evaluation suggests two prospective clinical trial designs that addressed recent study design and recruitment challenges. A visionary approach uses a multi-arm, multistage randomized trial to address rapid advancements in diagnosis and therapy. A pragmatic approach utilizes a single-arm trial with historical controls to overcome comparison group and recruitment challenges.

The outcome of patients with serous papillary peritoneal cancer, fallopian tube cancer, and epithelial ovarian cancer by treatment eras: 27 years data from the SEER registry.

AIM: To determine the differential effect of the treatment periods on the survival of patients with stage IV serous papillary peritoneal carcinoma (SPPC), fallopian tube cancers, and epithelial ovarian cancers (EOC). METHODS: This was an exploratory, population-based observational study of all patients with stage IV SPPC, fallopian tube cancers, and EOC collected from the SEER Research Data 1973-2017. The study period was divided into three time-periods: platinum combinations before the taxane era (1990-1995), platinum plus taxane chemotherapy era (1996-2013), and bevacizumab era (2014-2017). RESULTS: A total of 9828 patients were eligible for analyses: SPPC (3898 patients; 39.7%), fallopian tube cancers (1290 patients; 13.1%) and EOC (4640 patients, 47.2%). In the 1990-1995 era, the 3-year cause-specific survival was 40% for SPPC, 53% for fallopian tube cancers, and 40% for POC. In the following era 1993-2013, the 3-year cause-specific survival increased to 55% for SPPC, 74% for fallopian tube cancers, and 45% for POC. The last era 2014-2017 showed a 3-year cause-specific survival of 64%, 67%, and 45% for patients with SPPC, fallopian tube cancers, and POC, respectively. The differences in cause-specific survival were statistically significant for patients with SPPC (p=0.004). Multivariable analysis showed that the treatment eras and age at diagnosis were associated with cause-specific survival. CONCLUSION: The results of this study are hypothesis-generating and cannot be considered conclusive given the inherent limitations of registry analysis. Subgroup analyses of the phase III randomized controlled trials, by tumor subset (EOC, fallopian tube cancer, and SPPC) would shed more light on the differential effects of novel therapies.