Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine.

BACKGROUND: Patients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients. METHODS: ccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media. Established cultures were characterized by genomic verification of mutations present in the primary tumors, expression of renal epithelial markers, and transcriptional profiling. RESULTS: The apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping revealed that the majority of cultures contained normal, not cancer cells. ccRCC characteristically shows biallelic loss of the von Hippel Lindau (VHL) gene, leading to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Purification of cells based on expression of carbonic anhydrase IX (CA9), a cell surface HIF target, followed by culture in FBS enabled establishment of ccRCC cell cultures with an efficiency of >80 %. Culture in serum-free conditions selected for growth of normal renal proximal tubule epithelial cells. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures. CONCLUSIONS: The ability to establish primary cultures of ccRCC cells and matched normal kidney epithelial cells from almost every patient provides a resource for future development of novel therapies and personalized medicine for ccRCC patients.

Using machine learning on clinical data to identify unexpected patterns in groups of COVID-19 patients.

As clinicians are faced with a deluge of clinical data, data science can play an important role in highlighting key features driving patient outcomes, aiding in the development of new clinical hypotheses. Insight derived from machine learning can serve as a clinical support tool by connecting care providers with reliable results from big data analysis that identify previously undetected clinical patterns. In this work, we show an example of collaboration between clinicians and data scientists during the COVID-19 pandemic, identifying sub-groups of COVID-19 patients with unanticipated outcomes or who are high-risk for severe disease or death. We apply a random forest classifier model to predict adverse patient outcomes early in the disease course, and we connect our classification results to unsupervised clustering of patient features that may underpin patient risk. The paradigm for using data science for hypothesis generation and clinical decision support, as well as our triaged classification approach and unsupervised clustering methods to determine patient cohorts, are applicable to driving rapid hypothesis generation and iteration in a variety of clinical challenges, including future public health crises.

A Saliva-Based Serological and Behavioral Analysis of SARS-CoV-2 Antibody Prevalence in Howard County, Maryland.

The objective of the study was to estimate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence in the Howard County, Maryland, general population and demographic subpopulations attributable to natural infection or coronavirus disease 2019 (COVID-19) vaccination and to identify self-reported social behaviors that may affect the likelihood of recent or past SARS-CoV-2 infection. A cross-sectional, saliva-based serological study of 2,880 residents of Howard County, Maryland, was carried out from July through September 2021. Natural SARS-CoV-2 infection prevalence was estimated by inferring infections among individuals according to anti-nucleocapsid immunoglobin G levels and calculating averages weighted by sample proportions of various demographics. Antibody levels between BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) recipients were compared. Antibody decay rate was calculated by fitting exponential decay curves to cross-sectional indirect immunoassay data. Regression analysis was carried out to identify demographic factors, social behaviors, and attitudes that may be linked to an increased likelihood of natural infection. The estimated overall prevalence of natural infection in Howard County, Maryland, was 11.9% (95% confidence interval, 9.2% to 15.1%), compared with 7% reported COVID-19 cases. Antibody prevalence indicating natural infection was highest among Hispanic and non-Hispanic Black participants and lowest among non-Hispanic White and non-Hispanic Asian participants. Participants from census tracts with lower average household income also had higher natural infection rates. After accounting for multiple comparisons and correlations between participants, none of the behavior or attitude factors had significant effects on natural infection. At the same time, recipients of the mRNA-1273 vaccine had higher antibody levels than those of BNT162b2 vaccine recipients. Older study participants had overall lower antibody levels compared with younger study participants. The true prevalence of SARS-CoV-2 infection is higher than the number of reported COVID-19 cases in Howard County, Maryland. A disproportionate impact of infection-induced SARS-CoV-2 positivity was observed across different ethnic/racial subpopulations and incomes, and differences in antibody levels across different demographics were identified. Taken together, this information may inform public health policy to protect vulnerable populations. IMPORTANCE We employed a highly innovative noninvasive multiplex oral fluid SARS-CoV-2 IgG assay to ascertain our seroprevalence estimates. This laboratory-developed test has been applied in NCI's SeroNet consortium, possesses high sensitivity and specificity according to FDA Emergency Use Authorization guidelines, correlates strongly with SARS-CoV-2 neutralizing antibody responses, and is Clinical Laboratory Improvement Amendments-approved by the Johns Hopkins Hospital Department of Pathology. It represents a broadly scalable public health tool to improve understanding of recent and past SARS-CoV-2 exposure and infection without drawing any blood. To our knowledge, this is the first application of a high-performance salivary SARS-CoV-2 IgG assay to estimate population-level seroprevalence, including identifying COVID-19 disparities. We also are the first to report differences in SARS-CoV-2 IgG responses by COVID-19 vaccine manufacturers (BNT162b2 [Pfizer-BioNTech] and mRNA-1273 [Moderna]). Our findings demonstrate remarkable consistency with those of blood-based SARS-CoV-2 IgG assays in terms of differences in the magnitude of SARS-CoV-2 IgG responses between COVID-19 vaccines.

Most renal oncocytomas appear to grow: observations of tumor kinetics with active surveillance.

PURPOSE: We examined the growth of tissue proven renal oncocytoma on serial imaging to improve our understanding of its natural history. MATERIALS AND METHODS: We reviewed the charts of 69 patients with oncocytoma diagnosed by biopsy or surgery between 2004 and 2010. A total of 29 cases were managed by active surveillance for at least 12 months and had 3 or more imaging events. Tumor size was documented and the average tumor growth rate was calculated using a random coefficient model. Interaction terms were used to investigate correlations between variables of interest, including age at diagnosis, gender, symptom status, laterality, initial tumor size, surveillance duration and number of imaging events. RESULTS: At a mean surveillance duration of 40 months 80% of oncocytomas increased in size. Based on the random coefficient model the estimated average growth rate was 0.16 mm monthly (95% CI 0.097-0.228, p <0.0001). We identified no variables that significantly correlated with growth. CONCLUSIONS: Despite its low metastatic potential renal oncocytoma appears to progress locally with a growth rate similar to that of RCC. Thus, absent tumor growth on serial imaging is not a robust prognostic factor for benign histology. Biopsy remains the mainstay of diagnosis. At centers where it can be performed safely and accurately, active surveillance of tissue proven oncocytoma appears to be safe in the short term. Alternative management includes partial nephrectomy and minimally invasive approaches. To our knowledge this is the largest study of oncocytoma natural history.

Renal cell carcinoma in the native and allograft kidneys of renal transplant recipients.

PURPOSE: Renal cell carcinoma develops in renal transplant recipients 30 or more times more commonly than in the general population. We assessed the prevalence, histology and outcome of renal cell carcinoma in a large, single center recipient population. MATERIALS AND METHODS: We examined outcomes in patients who underwent renal transplantation at our center to determine the prevalence, histology and outcome of those in whom renal cell carcinoma developed. RESULTS: A total of 3,568 patients received a renal allograft at our institution between 1966 and 2009. A total of 45 renal cell carcinomas were diagnosed in the native kidney of 39 patients (1.1%) and in 8 (0.2%) renal cell carcinoma developed in the allograft kidney. Mean age at diagnosis was 51.6 and 48.2 years in patients with native kidney and allograft tumors, respectively. The mean interval between transplantation and the native or allograft renal cell carcinoma diagnosis was 10.6 and 12.1 years, respectively. Clear cell renal cell carcinoma was the most common tumor histology in native kidneys, diagnosed in 21 cases, while papillary renal cell carcinoma was diagnosed in 20. Five allograft tumors were papillary renal cell carcinoma and 3 were clear cell renal cell carcinoma. Native kidney tumors were managed by radical nephrectomy in 44 or by observation after biopsy. Allograft tumors were managed by transplant nephrectomy in 3 cases, radio frequency ablation in 3 and partial nephrectomy in 2. At a mean 6.6-year followup 32 patients with native kidney renal cell carcinoma were alive while 7 with allograft tumors were alive at a mean 3.6-year followup. CONCLUSIONS: Renal cell carcinoma is more prevalent in patients with renal transplantation than the general population, although the subtype distribution differs. Excellent survival is seen at more than 6 years after treatment.

Results of high intensity focused ultrasound treatment of prostate cancer: early Canadian experience at a single center.

INTRODUCTION: High intensity focused ultrasound (HIFU) is a non-invasive technique that uses focused ultrasound waves to ablate tissue. This retrospective study evaluates the early HIFU experience at a single Canadian center. MATERIALS AND METHODS: Ninety-five patients were treated between March 2006 and December 2007 using the Sonablate-500 device (Focus Surgery, Indianapolis, IN, USA). Follow up occurred at 3 month intervals and included serial prostate-specific antigen (PSA) measurements, assessments of erectile function and continence rates with the international index of erectile function (IIEF) and expanded prostate cancer index composite (EPIC) questionnaires respectively. Early and late complications were also studied. RESULTS: There were 95 patients treated by five urologists. The mean age of patients was 64 years (range 46-91). The majority of men treated had Gleason 6 (n = 53) or Gleason 7 (n = 35) disease. The remainder had Gleason 8 (n = 5) and Gleason 9 (n = 2) prostate cancer. Prostate volume in the pre-treatment group was 30.5 cc (range 14.4 cc-73 cc). Cytoreductive androgen deprivation therapy prior to treatment was administered to 10 men. Post-HIFU with a minimum 6 months follow up (mean 10.62 months), 2% (1/59) of men had de novo moderate to severe erectile dysfunction (IIEF ≤ 11). With a minimum of 6 months follow up (mean 8.85 months), 17% (7/41) of the men had significant incontinence according to their EPIC scores. Early complications included catheter-related problems (n = 10), retention (n = 16), and urosepsis (n = 1). Late complications included need for cystoscopy (n = 25), TURP (n = 6), VIU/dilatation for stricture or bladder neck contracture (n = 13) and self-catheterization (n = 1). Prostatorectal fistula occurred in one patient who had prior radiotherapy. Salvage HIFU following radiation failure was performed in seven men. Recurrence of cancer following HIFU was diagnosed in seven men. Salvage treatment included radical prostatectomy (n = 3), radiation therapy (n = 2), repeat HIFU (n = 1), hormone therapy (n = 1). CONCLUSIONS: In our early experience HIFU treatment for prostate cancer was associated with a moderate rate of complications and failure. Further studies are required to examine long term outcomes with HIFU.

Cancer stem cells are underestimated by standard experimental methods in clear cell renal cell carcinoma.

Rare cancer stem cells (CSC) are proposed to be responsible for tumour propagation and re-initiation and are functionally defined by identifying tumour-initiating cells (TICs) using the xenotransplantation limiting dilution assay (LDA). While TICs in clear cell renal cell carcinoma (ccRCC) appeared rare in NOD/SCID/IL2Rγ(-/-) (NSG) mice, xenografts formed more efficiently from small tumour fragments, indicating the LDA underestimated ccRCC TIC frequency. Mechanistic interrogation of the LDA identified multiple steps that influence ccRCC TIC quantitation. For example, tissue disaggregation destroys most ccRCC cells, common assays significantly overestimate tumour cell viability, and microenvironmental supplementation with human extracellular factors or pharmacological inhibition of anoikis increase clonogenicity and tumourigenicity of ccRCC cell lines and primary tumour cells. Identification of these previously uncharacterized concerns that cumulatively lead to substantial underestimation of TICs in ccRCC provides a framework for development of more accurate TIC assays in the future, both for this disease and for other cancers.

Psychosocial impact of renal donation.

PURPOSE: To determine the psychosocial effects of donor nephrectomy on a sample of Canadian donors. MATERIALS AND METHODS: Patients donating one of their kidneys for transplantation at the Toronto Hospital between 1991-1996 were asked to complete a 170-item questionnaire designed to assess their psychosocial well-being and the impact of renal donation on various aspects of their lives. Of the 153 donors contacted, 104 (68.0%) have responded to date. RESULTS: Less than 5% of donors complained of renal donation severely affecting any aspect of their life. Most donors (84%) were able to perform their normal daily activities within 12 weeks of nephrectomy, and 75% had recovered their pre-donation level of work function by this time. Almost one third of donors lost wages because of their donation, and half incurred significant transportation costs. Very few donors (< 10%) complained of other costs. Almost 90% of donors felt that donating a kidney had positively impacted their relationship with the recipient, and donors felt that their relationships with the recipient were significantly more positive at follow-up (p<.003). CONCLUSIONS: Donating a kidney results in a moderate psychosocial impact on the donor and appears to strengthen the bond between donor and recipient. Recovery times to daily activities and work may be longer than anticipated in a large proportion of donors.

Living donor nephrectomy: follow-up renal function, blood pressure, and urine protein excretion.

PURPOSE: To assess the effect of donor nephrectomy on blood pressure, 24-hour urine protein excretion, and renal function. MATERIALS AND METHODS: Of the 198 individuals who donated a kidney between 1991-1996, 101 had their blood pressure, 24-hour urine protein excretion, and serum creatinine concentration levels measured. The mean duration of follow-up was 3.2 +/- 1.6 years (range: 8.5 months to 6.5 years). RESULTS: Serum creatinine concentration was significantly higher (p<.001) at follow-up (107 +/- 20 umol/L) compared to before donation (86 +/- 18 umol/L). When follow-up serum creatinine concentrations were expressed as percentages of their pre-operative values, a gradual decline was observed with time (R= -.380). Diastolic blood pressures (p<.05) and 24-hour urine protein levels (p<.001) were significantly higher at follow-up, however, neither increased with time. The prevalence of hypertension and proteinuria in our donors was no different from that of the general population. CONCLUSIONS: Donor nephrectomy does not impair renal function or result in a progressive rise in blood pressure or urine protein excretion up to 6.5 years after nephrectomy.

Delay in surgical therapy for clinically localized prostate cancer and biochemical recurrence after radical prostatectomy.

BACKGROUND: In Canada, waiting times for cancer care have been increasing, particularly for patients with genitourinary malignancies. We examined whether delay from diagnosis for patients undergoing surgery for clinically localized prostate cancer affects cancer cure rates. METHODS: We conducted a historical cohort study among 645 patients who underwent radical prostatectomy between 1987 and 1997, using biochemical recurrence (PSA elevation) and metastasis as endpoints. We examined whether patients who underwent surgery >/= months (delayed surgery group) from the date of diagnosis had reduced recurrence-free survival, compared to patients who had surgery <3 months (early surgery group) from the date of diagnosis, adjusting for grade, stage and PSA level at diagnosis. RESULTS: The crude 10-year recurrence-free and metastasis-free survival rates for all patients were 71.1% (95% C.I.: 64.9% - 77.3%) and 95.3% (95% C.I.: 91.3% - 99.3%), respectively. Of the 645 patients, 189 (29.3%) had surgery >/= months after diagnosis. The median time from the date of diagnosis to surgery was 68 days (range 15 to 951 days). The 10-year recurrence-free survival was higher for patients who underwent early surgery (74.6%, 95% C.I.: 67.9% - 81.4%) compared to patients in the delayed surgery group (61.3%, 95% C.I.: 46.7% - 76.0%, p=0.05). The crude and adjusted hazard ratios for developing biochemical recurrence for patients in the delayed surgery group were 1.58 (95% C.I.: 1.0 - 2.4, p=0.04) and 1.46 (95% C.I.: 0.9 - 2.3, p=0.09), respectively, compared to patients who underwent early surgery. CONCLUSIONS: There may exist a possible relationship between delays from diagnosis for radical prostatectomy and prostate cancer cure rates. These findings may have many biases that could not be properly accounted in this retrospective analysis and larger cohort analyses will be required to confirm these findings.

Oncologic outcomes following radical prostatectomy in the active surveillance era.

OBJECTIVE: In this study, we examine the oncologic outcomes of men with low, intermediate and high preoperative risk for prostate cancer treated with radical prostatectomy prior to and during the active surveillance era. METHODS: We analyzed records from patients who underwent radical prostatectomy at our Canadian tertiary care facility from 2000 to 2012. Patients were stratified by D'Amico preoperative risk category and by year of treatment. Biochemical recurrence-free survival was estimated using the Kaplan-Meier method. RESULTS: We included 2643 consecutive patients in our analysis. The proportion of men with low-risk disease undergoing radical prostatectomy decreased from 2007 onwards coincident with the implementation of an active surveillance strategy in our institution. Men with low-risk and high-risk disease showed significantly worse biochemical outcomes from 2007 to 2012 compared to 2000 to 2006 (p < 0.05), while men with intermediate-risk prostate cancer showed no significant differences (p = 0.27). Within the low-risk cohort, the later treatment group displayed significantly lower age, pre-treatment prostate specific antigen and tumour volume and significantly higher testosterone and body mass index. CONCLUSIONS: The time period corresponding with the implementation of active surveillance at our institution corresponded with significant deterioration of biochemical outcomes in the low- and high-risk groups. This suggests that the men with most favourable disease deferred treatment, whereas men with worse preoperative disease characteristics were increasingly treated with radical prostatectomy in the past 6 years perhaps to their benefit.

Effect of healthcare provider characteristics on nodal yield at radical cystectomy.

OBJECTIVES: Current evidence suggests that patient outcomes after radical cystectomy are dependent on nodal yield, even in cases of node-negative disease. We hypothesized that the final lymph node (LN) counts would be associated with the service provider (surgeon, pathologist) and the level of experience of the provider's assistant. METHODS: We reviewed a series of 89 consecutive patients who had undergone cystectomy from 2001 to 2005 for the effect of provider factors on LN counts. The effect of the surgeon was assessed on an individual basis and the effect of the pathologist was determined according to the uropathologic subspecialization. Provider assistant experience was classified according to the training level of the surgical assistant and the caseload volume of the pathology assistant. Multivariate linear regression analysis, controlling for patient factors, number of nodal packets, and margin status, was used to determine the provider factors associated with the final nodal counts. RESULTS: The median number of LNs harvested was 14 per patient. On univariate analyses, the individual surgeon and the number of nodal packets submitted were significantly associated with nodal yield (P <0.001 for both). Surgical margin status was also significant (P = 0.003), with fewer LNs collected from those with margin-positive disease. On multivariate linear regression analysis, only surgeon (P = 0.01) and number of packets (P < 0.001; with more LNs taken with more packets) remained statistically significant. CONCLUSIONS: The results of our study have shown that the nodal yield from radical cystectomy is dependent on surgeon-specific factors (the number of packets submitted and the operating surgeon). The patient or pathology service providers did not influence the ultimate LN counts.

Transplant renal artery stenosis: outcome after percutaneous intervention.

PURPOSE: To assess the outcome of percutaneous transluminal angioplasty (PTA) and stent placement as the primary treatment for transplant renal artery stenosis (TRAS). MATERIALS AND METHODS: A retrospective review of PTA and stent placement procedures performed for TRAS from April 1997 to July 2003 was conducted. Reviewed parameters included technical success, date of transplantation, dates of percutaneous intervention, mean arterial blood pressure, number of blood pressure medications, and serum creatinine level before and after intervention. Twenty-one interventions were performed in 18 allografts. The primary clinical indication for imaging and treatment was increased creatinine level in 12 allografts and hypertension in six allografts. Patency rates were estimated with use of the Kaplan-Meier method. RESULTS: The technical success rate of PTA/stent placement was 100% and the clinical success rate was 94% (17 of 18 allografts). Thirteen interventions involved PTA alone, with eight combined PTA and stent insertions. The mean preintervention serum creatinine level among 12 allografts presenting with elevated creatinine levels was 2.8 mg/dL +/- 1.4 (SD), compared with a 1-month postintervention mean of 2.2 mg/dL +/- 0.7 (P = .03). Of six allografts that presented with hypertension, significant improvement was seen between the preintervention and 1-month postintervention mean systolic (174 mm Hg vs 135 mm Hg, P = .003) and diastolic (99 mm Hg vs 82 mm Hg, P = .02) pressures. These patients required a mean of 2.3 medications for blood pressure control before intervention, compared with a mean of 1.0 medications at 1 month after intervention (P = .002). Primary patency rates at 3, 6, and 12 months (+/-95% CI) were 94% +/- 6%, 72% +/- 12%, and 72% +/- 12%, respectively. Secondary patency rates at 3, 6, and 12 months (+/-95 CI) were 100%, 85% +/- 10%, and 85% +/- 10%, respectively. Mean follow-up time was 27 months. Of the eight allografts that underwent stent placement, all eight remained patent at last follow-up (mean, 18.3 months +/- 9.2). One major complication of a puncture site pseudoaneurysm occurred (5%). CONCLUSION: Primary treatment of TRAS with PTA with or without stent placement has good intermediate-term patency and is associated with significant early improvement in blood pressure and creatinine level.

The uncertainty of radio frequency treatment of renal cell carcinoma: findings at immediate and delayed nephrectomy.

PURPOSE: Radio frequency thermal therapy for the ablation of renal cell carcinoma has been reported. Outcomes are usually measured by imaging alone. We have performed ex vivo and in vivo experiments using radio frequency in porcine models in our laboratory. We now report our early experience in the treatment of renal cell carcinoma in patients who underwent post-radio frequency radical or partial nephrectomy. MATERIALS AND METHODS: We treated 10 patients diagnosed with small renal masses with radio frequency. All masses were biopsied before treatment. In 4 patients 5 renal cell carcinomas were treated with radio frequency after surgical exposure of the tumor followed immediately by partial or radical nephrectomy (acute group). Six other patients were treated percutaneously with ultrasound or computerized tomography guided radio frequency under local anesthesia and intravenous sedation 7 days before partial or radical nephrectomy (delayed group). A median of 2 radio frequency cycles was applied. Mean total heating time was 17 minutes 15 seconds. Specimens were analyzed grossly and histologically. Triphasic contrast-enhanced computerized tomography and/or magnetic resonance imaging was performed before and 7 days after radio frequency treatment in the delayed group. RESULTS: Mean radiological largest diameter of all 11 masses was 2.4 cm. and mean gross diameter was 2.2 cm. Pathological examination demonstrated residual viable tumor in approximately 5% of the volume in 4 of the 5 tumors in the acute group and in 3 of the 6 masses of the delayed group. In 1 delayed case the viable tumor appeared to be in contact with the renal vein. No significant complications were observed in 9 of the 10 patients. In 1 delayed case, a subcapsular hepatic hematoma, biliary fistula and pneumonia developed and resolved. CONCLUSIONS: Based on our experience, we continue to consider percutaneous radio frequency for the treatment of small renal cell carcinomas as a potentially curative therapy. However, complete tumor cell death appears to be difficult to achieve with our current treatment protocol. More phase II testing is indicated to ensure that this technique is an effective and reproducible treatment alternative.