Targeted next-generation sequencing has incremental value in the diagnostic work-up of patients with suspect pancreatic masses; a multi-center prospective cross sectional study.

BACKGROUND: The diagnostic process of patients with suspect pancreatic lesions is often lengthy and prone to repeated diagnostic procedures due to inconclusive results. Targeted Next-Generation Sequencing (NGS) performed on cytological material obtained with fine needle aspiration (FNA) or biliary duct brushing can speed up this process. Here, we study the incremental value of NGS for establishing the correct diagnosis, and subsequent treatment plan in patients with inconclusive diagnosis after regular diagnostic work-up for suspect pancreatic lesions. METHODS: In this prospective cross-sectional cohort study, patients were screened for inclusion in four hospitals. NGS was performed with AmpliSeq Cancer Hotspot Panel v2 and v4b in patients with inconclusive cytology results or with an uncertain diagnosis. Diagnostic results were evaluated by the oncology pancreatic multidisciplinary team. The added value of NGS was determined by comparing diagnosis (malignancy, cystic lesion or benign condition) and proposed treatment plan (exploration/resection, neoadjuvant chemotherapy, follow-up, palliation or repeated FNA) before and after integration of NGS results. Final histopathological analysis or a 6-month follow-up period were used as the reference standard in case of surgical intervention or non-invasive treatment, respectively. RESULTS: In 50 of the 53 included patients, cytology material was sufficient for NGS analysis. Diagnosis before and after integration of NGS results differed in 24% of the patients. The treatment plan was changed in 32% and the diagnosis was substantiated by the NGS data in 44%. Repetition of FNA/brushing was prevented in 14% of patients. All changes in treatment plan were correctly made after integration of NGS. Integration of NGS increased overall diagnostic accuracy from 68% to 94%. INTERPRETATION: This study demonstrates the incremental diagnostic value of NGS in patients with an initial inconclusive diagnosis. Integration of NGS results can prevent repeated EUS/FNA, and can also rigorously change the final diagnosis and treatment plan.

Cumulative sum learning curves guiding multicenter multidisciplinary quality improvement of EUS-guided tissue acquisition of solid pancreatic lesions.

Background and study aims In this study, we evaluated the performance of community hospitals involved in the Dutch quality in endosonography team regarding yield of endoscopic ultrasound (EUS)-guided tissue acquisition (TA) of solid pancreatic lesions using cumulative sum (CUSUM) learning curves. The aims were to assess trends in quality over time and explore potential benefits of CUSUM as a feedback-tool. Patients and methods All consecutive EUS-guided TA procedures for solid pancreatic lesions were registered in five community hospitals between 2015 and  2018. CUSUM learning curves were plotted for overall performance and for performance per center. The American Society of Gastrointestinal Endoscopy-defined key performance indicators, rate of adequate sample (RAS), and diagnostic yield of malignancy (DYM) were used for this purpose. Feedback regarding performance was provided on multiple occasions at regional interest group meetings during the study period. Results A total of 431 EUS-guided TA procedures in 403 patients were included in this study. The overall and per center CUSUM curves for RAS improved over time. CUSUM curves for DYM revealed gradual improvement, reaching the predefined performance target (70 %) overall, and in three of five contributing centers in 2018. Analysis of a sudden downslope development in the CUSUM curve of DYM in one center revealed temporary absence of a senior cytopathologist to have had a temporary negative impact on performance. Conclusions CUSUM-derived learning curves allow for assessment of best practices by comparison among peers in a multidisciplinary multicenter quality improvement initiative and proved to be a valuable and easy-to-interpret means to evaluate EUS performance over time.

Effect of high salt diet on blood pressure and renal damage during vascular endothelial growth factor inhibition with sunitinib.

BACKGROUND: Antiangiogenic treatment with the multitargeted vascular endothelial growth factor (VEGF) receptor inhibitor sunitinib associates with a blood pressure (BP) rise and glomerular renal injury. Recent evidence indicates that VEGF derived from tubular cells is required for maintenance of the peritubular vasculature. In the present study, we focussed on tubular and glomerular pathology induced by sunitinib and explored whether a high salt (HS) diet augments the BP rise and renal abnormalities. METHODS: Normotensive Wistar Kyoto (WKY) rats were exposed to a normal salt (NS) or HS diet for 2 weeks and subsequently for 8 days to sunitinib or vehicle administration after which the rats were euthanized and kidneys excised. Mean arterial pressure (MAP) was telemetrically measured. Urine was sampled for proteinuria and endothelinuria, and blood for measurement of endothelin-1, creatinine and cystatin C. RESULTS: Compared with the NS diet, MAP rapidly rose by 27 ± 3 mmHg with the HS diet. On sunitinib, MAP rose further by 15 ± 1 with the NS and by 23 ± 4 mmHg with the HS diet (P < 0.05). The HS diet itself had no effect on proteinuria, endothelinuria or the plasma levels of endothelin-1, creatinine and cystatin C. Only with the HS diet, sunitinib administration massively increased proteinuria and endothelinuria and these two parameters were related (r = 0.50, P < 0.01). Likewise, renal glomerular pathology was enhanced during sunitinib with the HS diet, whereas tubulointerstitial injury or reduced peritubular capillary density did not occur. CONCLUSIONS: An HS diet induces a marked BP rise in WKY rats and exacerbates both the magnitude of the BP rise and glomerular injury induced by sunitinib.

Greater Sensitivity of Blood Pressure Than Renal Toxicity to Tyrosine Kinase Receptor Inhibition With Sunitinib.

Hypertension and renal injury are off-target effects of sunitinib, a tyrosine kinase receptor inhibitor used for the treatment of various tumor types. Importantly, these untoward effects are accompanied by activation of the endothelin system. Here, we set up a study to explore the dose dependency of these side effects. Normotensive Wistar Kyoto rats were exposed to 3 different doses of sunitinib or vehicle. After 8 days, rats were euthanized. Telemetrically measured blood pressure rose dose dependently, from 13 to 30 mm Hg. Proteinuria was present at all doses, but a rise in cystatin C occurred only at the intermediate and high doses. Compared with vehicle circulating endothelin-1 increased dose dependently, whereas 24-hour urinary endothelin excretion decreased. Light and electron microscopy revealed glomerular endotheliosis and ischemia with the intermediate and high doses of sunitinib but completely absent histological abnormalities with the low dose. Podocyte number per glomerular circumference did not change. Glomerular nephrin, Neph1, podocin, and endothelin-converting enzyme gene expression were downregulated in a dose-dependent manner. We conclude that the sunitinib-induced rise in blood pressure requires lower doses than its induction of renal function impairment and that functional changes in glomerular filtration barrier contribute to the occurrence of proteinuria, given the lack of histopathologic changes with the low dose of sunitinib.

Treatment of hypertension and renal injury induced by the angiogenesis inhibitor sunitinib: preclinical study.

Common adverse effects of angiogenesis inhibition are hypertension and renal injury. To determine the most optimal way to prevent these adverse effects and to explore their interdependency, the following drugs were investigated in unrestrained Wistar Kyoto rats exposed to the angiogenesis inhibitor sunitinib: the dual endothelin receptor antagonist macitentan; the calcium channel blocker amlodipine; the angiotensin-converting enzyme inhibitor captopril; and the phosphodiesterase type 5 inhibitor sildenafil. Mean arterial pressure was monitored telemetrically. After 8 days, rats were euthanized and blood samples and kidneys were collected. In addition, 24-hour urine samples were collected. After sunitinib start, mean arterial pressure increased rapidly by ≈30 mm Hg. Coadministration of macitentan or amlodipine largely prevented this rise, whereas captopril or sildenafil did not. Macitentan, captopril, and sildenafil diminished the sunitinib-induced proteinuria and endothelinuria and glomerular intraepithelial protein deposition, whereas amlodipine did not. Changes in proteinuria and endothelinuria were unrelated. We conclude that in our experimental model, dual endothelin receptor antagonism and calcium channel blockade are suitable to prevent angiogenesis inhibition-induced hypertension, whereas dual endothelin receptor antagonism, angiotensin-converting enzyme inhibitor, and phosphodiesterase type 5 inhibition can prevent angiogenesis inhibition-induced proteinuria. Moreover, the variable response of hypertension and renal injury to different antihypertensive agents suggests that these side effects are, at least in part, unrelated.

Sunitinib-induced hypothyroidism is due to induction of type 3 deiodinase activity and thyroidal capillary regression.

CONTEXT: Anticancer treatment with the tyrosine kinase inhibitor sunitinib causes thyroid dysfunction. OBJECTIVE: Our objective was to investigate the time course and underlying mechanisms of sunitinib-induced thyroid dysfunction. DESIGN: Thyroid function tests of 83 patients on sunitinib were collected retrospectively for their total treatment duration between January 2006 and November 2009 and prospectively in 15 patients on sunitinib for 10 wk. Additionally, thyroid function and histology were assessed in rats on sunitinib (8 d; n = 10) and after sunitinib withdrawal (11 d; n = 7) and compared with controls (n = 7). SETTING: Patients were seen at a university outpatient oncology clinic. Patients and Animals: Patients with metastatic renal cell carcinoma or gastrointestinal stromal tumors participated in the clinical study and Wistar Kyoto rats were used in the rat study. INTERVENTION: Sunitinib was taken according to a 4 wk "on," 2 wk "off" treatment regimen. Blood samples for measurement of thyroid function were collected at baseline and at wk 4 and 10. In rats, blood, liver, and thyroid were collected to assess thyroid hormones, deiodinase activity, and thyroid histology. MAIN OUTCOME MEASURES: TSH and free T(4) levels, deiodinase activity, and thyroid histology were assessed. RESULTS: Forty-two percent of patients in the retrospective study developed elevated TSH levels. Prospective analysis showed increased TSH levels within 10 wk of treatment, accompanied by a decreased T(3)/rT(3) ratio. In rats, serum T(4) and T(3) decreased, hepatic type 3 deiodinase activity increased, and thyroid histology showed marked capillary regression, which all but thyroid hormones reversed after sunitinib withdrawal. CONCLUSION: Sunitinib induces hypothyroidism due to alterations in T(4)/T(3) metabolism as well as thyroid capillary regression.

The vascular endothelial growth factor receptor inhibitor sunitinib causes a preeclampsia-like syndrome with activation of the endothelin system.

Angiogenesis inhibition is an established treatment for several tumor types. Unfortunately, this therapy is associated with adverse effects, including hypertension and renal toxicity, referred to as "preeclampsia." Recently, we demonstrated in patients and in rats that the multitarget tyrosine kinase inhibitor sunitinib induces a rise in blood pressure (BP), renal dysfunction, and proteinuria associated with activation of the endothelin system. In the current study we investigated the effects of sunitinib on rat renal histology, including the resemblance with preeclampsia, as well as the roles of endothelin 1, decreased nitric oxide (NO) bioavailability, and increased oxidative stress in the development of sunitinib-induced hypertension and renal toxicity. In rats on sunitinib, light and electron microscopic examination revealed marked glomerular endotheliosis, a characteristic histological feature of preeclampsia, which was partly reversible after sunitinib discontinuation. The histological abnormalities were accompanied by an increase in urinary excretion of endothelin 1 and diminished NO metabolite excretion. In rats on sunitinib alone, BP increased (ΔBP: 31.6±0.9 mm Hg). This rise could largely be prevented with the endothelin receptor antagonist macitentan (ΔBP: 12.3±1.5 mm Hg) and only mildly with Tempol, a superoxide dismutase mimetic (ΔBP: 25.9±2.3 mm Hg). Both compounds could not prevent the sunitinib-induced rise in serum creatinine or renal histological abnormalities and had no effect on urine nitrates but decreased proteinuria and urinary endothelin 1 excretion. Our findings indicate that both the endothelin system and oxidative stress play important roles in the development of sunitinib-induced proteinuria and that the endothelin system rather than oxidative stress is important for the development of sunitinib-induced hypertension.

Bioengineering of living renal membranes consisting of hierarchical, bioactive supramolecular meshes and human tubular cells.

Maintenance of polarisation of epithelial cells and preservation of their specialized phenotype are great challenges for bioengineering of epithelial tissues. Mimicking the basement membrane and underlying extracellular matrix (ECM) with respect to its hierarchical fiber-like morphology and display of bioactive signals is prerequisite for optimal epithelial cell function in vitro. We report here on a bottom-up approach based on hydrogen-bonded supramolecular polymers and ECM-peptides to make an electro-spun, bioactive supramolecular mesh which can be applied as synthetic basement membrane. The supramolecular polymers used, self-assembled into nano-meter scale fibers, while at micro-meter scale fibers were formed by electro-spinning. We introduced bioactivity into these nano-fibers by intercalation of different ECM-peptides designed for stable binding. Living kidney membranes were shown to be bioengineered through culture of primary human renal tubular epithelial cells on these bioactive meshes. Even after a long-term culturing period of 19 days, we found that the cells on bioactive membranes formed tight monolayers, while cells on non-active membranes lost their monolayer integrity. Furthermore, the bioactive membranes helped to support and maintain renal epithelial phenotype and function. Thus, incorporation of ECM-peptides into electro-spun meshes via a hierarchical, supramolecular method is a promising approach to engineer bioactive synthetic membranes with an unprecedented structure. This approach may in future be applied to produce living bioactive membranes for a bio-artificial kidney.

The use of fibrous, supramolecular membranes and human tubular cells for renal epithelial tissue engineering: towards a suitable membrane for a bioartificial kidney.

A bioartificial kidney, which is composed of a membrane cartridge with renal epithelial cells, can substitute important kidney functions in patients with renal failure. A particular challenge is the maintenance of monolayer integrity and specialized renal epithelial cell functions ex vivo. We hypothesized that this can be improved by electro-spun, supramolecular polymer membranes which show clear benefits in ease of processability. We found that after 7 d, in comparison to conventional microporous membranes, renal tubular cells cultured on top of our fibrous supramolecular membranes formed polarized monolayers, which is prerequisite for a well-functioning bioartificial kidney. In future, these supramolecular membranes allow for incorporation of peptides that may increase cell function even further.

Distribution pattern and marker profile show two subpopulations of reserve cells in the endocervical canal.

SUMMARY: A previous immunophenotyping study in the fetal uterine cervix provided evidence for the existence of 2 subpopulations of reserve cells, one giving rise to glandular epithelium and the other to squamous epithelium (5). In this study, we investigated whether the adult uterine cervix also harbors different populations of reserve cells on the basis of their marker profile and distribution pattern. Sagittal sections from 10 normal uteri, comprising the region from ectocervix to lower uterine cavity, were histologically examined and immunostained for p63, bcl-2 and cytokeratins (CKs) 5, 7, 8, and 17. The endocervical canal consists of three regions, that is, a part lined with squamous epithelium, a part lined with endocervical cells and a part lined with tubal type epithelial cells. Histologically, we found reserve cells in all 10 investigated cervices, with an abundancy in the area beneath the endocervical columnar epithelium close to the squamo-columnar junction, and high in the endocervical canal where the invaginations consist of tubal type epithelium. In between, an area lined with endocervical columnar cells without reserve cells was identified. No reserve cells were detected in the endometrial epithelium. We defined the end of the endocervix as the point where the surface of the cervical canal and the invaginations are completely lined with tubal type epithelium. From this point, reserve cells were no longer found. Reserve cells show strong expression for p63, CKs 5 and 7, and moderate expression for bcl-2. CK17 is strongly expressed in the reserve cells at the squamo-columnar junction and to a lesser extent in the reserve cells close to the endometrium. Endocervical columnar cells usually express CKs 7 and 8 and sporadically also p63 and CK5. CK17 was only found in endocervical cells in the vicinity of CK17-positive subcolumnar reserve cells. Tubal-type epithelium was present in all samples and contained bcl-2, along with CKs 5, 7, and 8. As a result, bcl-2 and CK5 expression distinguishes tubal epithelium from endocervical columnar cells. We conclude that reserve cells are present in all investigated cervices along the entire cervical canal. The concentration of subglandular reserve cells is highest close to the squamo-columnar junction and in the upper third of the cervix. The marker profile of reserve cells is the same in all parts of the cervix, except for CK17, which shows a decreasing gradient from distal to proximal, indicating a subpopulation of distal reserve cells as progenitor for squamous and columnar epithelium, and proximal reserve cells that can serve as progenitor cells for columnar epithelium.

Severe cervical glandular cell lesions and severe cervical combined lesions: predictive value of the papanicolaou smear.

BACKGROUND: The purpose of the current study was to determine the accuracy of routinely screened cervical smears to predict a glandular cell lesion in histologically confirmed cases of cervical adenocarcinoma in situ (AIS), invasive adenocarcinoma (ADCA), adenosquamous carcinoma (ADSQCA), and severe combined glandular and squamous cell lesions. METHODS: Between 1989-2000, a total of 1,141 women with a histologic diagnosis of cervical AIS, ADCA, ADSQCA, and combined lesions (glandular cell lesion with a coexistent squamous cell lesion) were registered in the Dutch National Pathology Archive (PALGA). In 1054 of these 1,141 histologic cases, an additional conventional Papanicolaou (Pap) smear diagnosis was registered from the same patient. Material was evaluated with regard to the accuracy of cytologic diagnosis, the percentage of combined lesions, the mean age of the patients, and the time interval between AIS and ADCA. RESULTS: Of 1,141 registered histologic cases, 57.5% were registered as having an "intraepithelial" lesion, whereas 42.5% were registered as having an "invasive" process. A combined process was diagnosed in 63.2% of cases. From the same patients, a cytologic diagnosis of a severe cervical epithelial lesion was registered in PALGA for 91.2% (n = 961) of 1054 cases. A cytologic registration of a severe glandular cell lesion (with or without a squamous cell component) was made in 547 cases (51.9%). Prediction of a severe glandular cell lesion on the Pap smear was found to be more accurate in cases of histologically confirmed pure glandular cell abnormalities than in cases with a histologic diagnosis of a combined lesion. The cytologic prediction was found to be correct in 75.2% of cases of pure AIS and 47.3% of cases of AIS with coexistent high-grade squamous intraepithelial lesion (HGSIL) (cervical intraepithelial lesion [CIN] type 2 [CIN 2] or CIN 3). The mean ages of the patients with AIS and AIS + HGSIL were 37.3 years and 34 years, respectively, whereas the mean age of the patients with ADCA and ADCA + HGSIL was 41.9 years and 38.1 years, respectively. The interval between the average ages of patients with AIS and ADCA and those with AIS + HGSIL and ADCA + HGSIL was 4.6 years and 4.1 years, respectively. CONCLUSIONS: On the basis of a data search of the PALGA registry, it can be concluded that in a relatively large number of cases a severe cervical glandular cell lesion was not diagnosed on the Pap smear. Furthermore, data demonstrated that the prediction of a glandular abnormality is less accurate in cases of combined squamoglandular cell lesions than in pure glandular cell lesions.

Severe cervical glandular cell lesions with coexisting squamous cell lesions.

BACKGROUND: In the current report, the authors present the results of a reevaluation of cytologic smears and histologic specimens obtained from patients with severe cervical glandular cell lesions (adenocarcinoma in situ [AIS] or adenocarcinoma [ADCA] of the cervix) and coexisting Grade 1, Grade 2, or Grade 3 cervical intraepithelial neoplasia or squamous cell carcinoma. The goal of the current study was to assess whether knowledge of the specific cytologic characteristics of the cervical glandular cell lesions could have made the cytologic diagnosis of these combined neoplasms more accurate. METHODS: Cytologic smears and histologic specimens obtained from 36 patients with combined severe cervical lesions were evaluated for the presence of a range of microscopic cytologic and histologic features that were considered indicative of glandular cell changes. RESULTS: The findings of the current study suggest that the proper identification of characteristic cytomorphologic features of cervical glandular lesions would have resulted in more accurate diagnoses of combined severe cervical lesions. In the set of samples reevaluated by the authors, consideration of these features would have increased the accuracy of cytologic diagnosis from 55.6% to 75.0%. The presence of AIS was predicted in the majority of cytologic specimens, and in most cases, the identity of the predominant subtype of AIS could also be predicted. CONCLUSIONS: The current analysis revealed that consideration of specific cytomorphologic features of glandular lesions of the cervix increased the authors' accuracy in diagnosing combined severe lesions of the cervix. More accurate identification of intraepithelial glandular cell lesions may eventually lead to decreases in cervical adenocarcinoma incidence, just as increases in diagnostic accuracy have led to decreases in the incidence of squamous intraepithelial lesions and invasive squamous carcinoma of the cervix.