cGAS-STING drives the IL-6-dependent survival of chromosomally instable cancers.

Chromosomal instability (CIN) drives cancer cell evolution, metastasis and therapy resistance, and is associated with poor prognosis1. CIN leads to micronuclei that release DNA into the cytoplasm after rupture, which triggers activation of inflammatory signalling mediated by cGAS and STING2,3. These two proteins are considered to be tumour suppressors as they promote apoptosis and immunosurveillance. However, cGAS and STING are rarely inactivated in cancer4, and, although they have been implicated in metastasis5, it is not known why loss-of-function mutations do not arise in primary tumours4. Here we show that inactivation of cGAS-STING signalling selectively impairs the survival of triple-negative breast cancer cells that display CIN. CIN triggers IL-6-STAT3-mediated signalling, which depends on the cGAS-STING pathway and the non-canonical NF-κB pathway. Blockade of IL-6 signalling by tocilizumab, a clinically used drug that targets the IL-6 receptor (IL-6R), selectively impairs the growth of cultured triple-negative breast cancer cells that exhibit CIN. Moreover, outgrowth of chromosomally instable tumours is significantly delayed compared with tumours that do not display CIN. Notably, this targetable vulnerability is conserved across cancer types that express high levels of IL-6 and/or IL-6R in vitro and in vivo. Together, our work demonstrates pro-tumorigenic traits of cGAS-STING signalling and explains why the cGAS-STING pathway is rarely inactivated in primary tumours. Repurposing tocilizumab could be a strategy to treat cancers with CIN that overexpress IL-6R.

Functional ex vivo DNA fibre assay to measure replication dynamics in breast cancer tissue.

Replication stress (RS) is a key trait of cancer cells, and a potential actionable target in cancer treatment. Accurate methods to measure RS in tumour samples are currently lacking. DNA fibre analysis has been used as a common technique to measure RS in cell lines. Here, we investigated DNA fibre analysis on fresh breast cancer specimens and correlated DNA replication kinetics to known RS markers and genomic alterations. Fresh, treatment-naïve primary breast cancer samples (n = 74) were subjected to ex vivo DNA fibre analysis to measure DNA replication kinetics. Tumour cell proliferation was confirmed by EdU incorporation and cytokeratin AE1/AE3 (CK) staining. The RS markers phospho-S33-RPA and γH2AX and the RS-inducing proto-oncogenes Cyclin E1 and c-Myc were analysed by immunohistochemistry. Copy number variations (CNVs) were assessed from genome-wide single nucleotide polymorphism (SNP) arrays. We found that the majority of proliferating (EdU-positive) cells in each sample were CK-positive and therefore considered to be tumour cells. DNA fibre lengths varied largely in most tumour samples. The median DNA fibre length showed a significant inverse correlation with pRPA expression (r = -0.29, p = 0.033) but was not correlated with Cyclin E1 or c-Myc expression and global CNVs in this study. Nuclear Cyclin E1 expression showed a positive correlation with pRPA levels (r = 0.481, p < 0.0001), while cytoplasmic Cyclin E1 expression exhibited an inverse association with pRPA expression (r = -0.353, p = 0.002) and a positive association with global CNVs (r = 0.318, p = 0.016). In conclusion, DNA fibre analysis performed with fresh primary breast cancer samples is feasible. Fibre lengths were associated with pRPA expression. Cyclin E1 expression was associated with pRPA and the percentage of CNVs. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Presence of crown-like structures in breast adipose tissue; differences between healthy controls, BRCA1/2 gene mutation carriers and breast cancer patients.

PURPOSE: Crown-like structures (CLS) in breast adipose tissue are associated with inflammation and a potential factor in breast cancer behaviour. Whether this effect varies between breast cancer subtypes and is influenced by BMI and BRCA mutation status is presently unknown. Therefore, we compared CLS presence between adipose tissue of healthy controls, BRCA1/2 gene mutation carriers and breast cancer patients, and assessed the relation of CLS with clinical outcome in breast cancer patients. METHODS: Immunohistochemical staining for CD68 was performed on breast adipose tissue sections of 48 healthy controls, 78 BRCA1/2 gene mutation carriers and 259 breast cancer patients. CLS presence and index (CLS/cm2) were correlated with BMI, BRCA status, tumour presence, intrinsic tumour subtype and tumour characteristics. Associations with clinical outcome were assessed. RESULTS: CLS were more often present in breast cancer patients compared to BRCA carriers and healthy controls. CLS presence was associated with the presence of breast cancer and high BMI. CLS were more often present in Luminal-B-like tumours compared to the other subtypes. No correlations between CLS and BRCA status or age was found. In TNBC, CLS were related to lymphovascular invasion. No association with survival was found. CONCLUSION: In conclusion, CLS were more frequently present in breast adipose tissue of breast cancer patients compared to BRCA1/2 gene mutation carriers and healthy controls. Furthermore, our study provides evidence of the association between obesity and presence of CLS. The prognostic significance and impact on clinical outcome of differences in CLS numbers should be further assessed in prospective studies.

Histopathological comparison of Sjögren-related features between paired labial and parotid salivary gland biopsies of sicca patients.

OBJECTIVES: To compare focus score (FS) and other histopathological features between paired labial and parotid salivary gland biopsies in a diagnostic cohort of suspected Sjögren's disease (SjD) patients. METHODS: Labial and parotid salivary gland biopsies were simultaneously obtained from patients with sicca complaints, suspected of having SjD. Biopsies were formalin fixed and paraffin embedded. Sections were stained with haematoxylin & eosin (H&E) and for CD3, CD20, CD45, cytokeratin, CD21, Bcl6, activation induced deaminase (AID), and IgA/IgG. FS and other histopathological features characteristic for SjD were analysed. RESULTS: Based on the expert opinion of three experienced rheumatologists, 36 patients were diagnosed as SjD and 63 as non-SjD sicca patients. When taking all patients together, absolute agreement of various histopathological features between labial and parotid biopsies was high and varied between 80% (FS) and 93% ((pre-)lymphoepithelial lesions (LELs)). More labial gland biopsies had a FS ≥ 1 compared with their parotid counterpart. Accordingly, the area of infiltrate was larger in labial gland biopsies. When considering only SjD patients, labial glands contained significantly less B-lymphocytes, GCs/mm2 and less severe LELs compared with parotid glands. CONCLUSION: Labial and parotid glands from SjD patients contain similar histopathological key features, and thus both glands can be used for diagnosis and classification of SjD. However, parotid salivary glands reveal more evident B-lymphocyte related features, while labial glands exhibit more inflammation, which may be partially unrelated to SjD.

Considerable interlaboratory variation in PD-L1 positivity for head and neck squamous cell carcinoma in the Netherlands- A nationwide evaluation study.

AIMS: Patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) are eligible for first-line immune checkpoint inhibition if their tumour is positive for programmed death ligand 1 (PD-L1) determined by the combined positive score (CPS). This nationwide study, using real-world data, investigated the developing PD-L1 testing landscape in the first 3 years after introduction of the test in HNSCC and examined interlaboratory variation in PD-L1 positivity rates. METHODS: Pathology reports of HNSCC patients mentioning PD-L1 were extracted from the Dutch Pathology Registry (Palga). Tumour and PD-L1 testing characteristics were analysed per year and interlaboratory variation in PD-L1 positivity rates was assessed using funnel plots with 95% confidence limits around the overall mean. RESULTS: A total of 817 PD-L1 tests were reported in 702 patients among 19 laboratories; 85.2% of the tests on histological material were stated to be positive. The national PD-L1 positivity rate differed significantly per year during the study period (79.7-89.9%). The use of the recommended 22C3 antibody increased from 59.9 to 74.3%. A total of 673 PD-L1 tests on histological material from 12 laboratories were analysed to investigate interlaboratory variation. Four (33%) deviated significantly from the national mean of PD-L1-positive cases using CPS ≥ 1 cut-off, while two (17%) deviated significantly for CPS ≥ 20 cut-off. CONCLUSION: In the first 3 years of PD-L1 assessment in HNSCC, the testing landscape became more uniform. However, interlaboratory variation in PD-L1 positivity rates between Dutch laboratories was substantial. This implies that there is a need for further test standardisation to reduce this variation.

Quantitative comparison of immunohistochemical HER2-low detection in an interlaboratory study.

AIMS: Recently, human epidermal growth factor 2 (HER2)-low (i.e. HER2 score 1+ or 2+ without amplification) breast cancer patients became eligible for trastuzumab-deruxtecan treatment. To improve assay standardisation and detection of HER2-low in a quantitative manner, we conducted an external quality assessment-like study in the Netherlands. Dynamic range cell lines and immunohistochemistry (IHC) calibrators were used to quantify HER2 expression and to assess interlaboratory variability. METHODS AND RESULTS: Three blank slides with a dynamic range cell line and an IHC calibrator were stained with routine HER2 assays by 35 laboratories. Four different antibody clones were used: 19 (54.3%) 4B5, six (17.1%) A0485, five (14.3%) DG44 (HercepTest) and five (14.3%) SP3. Laboratories used two different detection kits for 4B5 assays: 14 (73.7%) ultraView and five (26.3%) OptiView. Variability of HER2 expression in cell lines, measured with artificial intelligence software, was median (min-max) = negative core 0.5% (0.0-57.0), 1+ core 4.3% (1.6-71.3), 2+ core 42.8% (30.4-92.6) and 3+ core 96.2% (91.8-98.8). The calibrators DG44 and 4B5 OptiView had the highest analytical sensitivity, closely followed by 4B5 ultraView. SP3 was the least sensitive. Calibrators of A0485 assays were not analysable due to background staining. CONCLUSIONS: As assays were validated for detecting HER2-amplified tumours, not all assays and antibodies proved suitable for HER2-low detection. Some tests showed distinct expression in the negative cell line. Dynamic range cell line controls and quantitative analysis using calibrators demonstrated more interlaboratory variability than commonly appreciated. Revalidation of HER2 tests by laboratories is needed to ensure clinical applicable HER2-low assays.

Advancing Ki67 hotspot detection in breast cancer: a comparative analysis of automated digital image analysis algorithms.

AIM: Manual detection and scoring of Ki67 hotspots is difficult and prone to variability, limiting its clinical utility. Automated hotspot detection and scoring by digital image analysis (DIA) could improve the assessment of the Ki67 hotspot proliferation index (PI). This study compared the clinical performance of Ki67 hotspot detection and scoring DIA algorithms based on virtual dual staining (VDS) and deep learning (DL) with manual Ki67 hotspot PI assessment. METHODS: Tissue sections of 135 consecutive invasive breast carcinomas were immunohistochemically stained for Ki67. Two DIA algorithms, based on VDS and DL, automatically determined the Ki67 hotspot PI. For manual assessment; two independent observers detected hotspots and calculated scores using a validated scoring protocol. RESULTS: Automated hotspot detection and assessment by VDS and DL could be performed in 73% and 100% of the cases, respectively. Automated hotspot detection by VDS and DL led to higher Ki67 hotspot PIs (mean 39.6% and 38.3%, respectively) compared to manual consensus Ki67 PIs (mean 28.8%). Comparing manual consensus Ki67 PIs with VDS Ki67 PIs revealed substantial correlation (r = 0.90), while manual consensus versus DL Ki67 PIs demonstrated high correlation (r = 0.95). CONCLUSION: Automated Ki67 hotspot detection and analysis correlated strongly with manual Ki67 assessment and provided higher PIs compared to manual assessment. The DL-based algorithm outperformed the VDS-based algorithm in clinical applicability, because it did not depend on virtual alignment of slides and correlated stronger with manual scores. Use of a DL-based algorithm may allow clearer Ki67 PI cutoff values, thereby improving the clinical usability of Ki67.

Increased Interferon Signaling in Vaginal Tissue of Patients With Primary Sjögren Syndrome.

OBJECTIVE: Vaginal dryness is an important factor influencing sexual function in women with primary Sjögren syndrome (pSS). Previous studies showed a higher degree of inflammation in vaginal biopsies from patients with pSS compared to non-pSS controls. However, the molecular pathways that drive this inflammation remain unclear. Therefore, the aim of this study was to investigate inflammatory pathway activity in the vaginal tissue of patients with pSS. METHODS: Vaginal biopsies of 8 premenopausal patients with pSS experiencing vaginal dryness and 7 age-matched non-pSS controls were included. Expression of genes involved in inflammation and tissue homeostasis was measured using NanoString technology and validated using TaqMan Real-Time PCR. Vaginal tissue sections were stained by immunohistochemistry for myxovirus resistance protein 1 (MxA) and CD123 (plasmacytoid dendritic cells [pDCs]). RESULTS: The most enriched pathway in vaginal biopsies from patients with pSS compared to non-pSS controls was the interferon (IFN) signaling pathway (P < 0.01). Pathway scores for Janus kinase and signal transducer and activator of transcription (JAK-STAT) and Notch signaling were also higher (P < 0.01 for both pathways). Conversely, transforming growth factor-ß signaling and angiogenesis pathway scores were lower in pSS (P = 0.02 and P = 0.04, respectively). Differences in IFN signaling between patients with pSS and non-pSS controls were confirmed by PCR and MxA tissue staining. No CD123+ pDCs were detected in vaginal biopsies. IFN-stimulated gene expression levels correlated positively with CD45+ cell numbers in vaginal biopsies and serum anti-SSA/Ro positivity. CONCLUSION: Upregulation of IFN signaling in vaginal tissue of women with pSS, along with its association with tissue pathology, suggests that IFNs contribute to inflammation of the vaginal wall and potentially also to clinical symptomatology (ie, vaginal dryness).

Diagnosis of laryngopharyngeal carcinoma through office-based flexible laryngoscopy as a reliable alternative for biopsies under general anesthesia: Faster diagnostics with equal oncological outcome.

PURPOSE: Diagnostic endoscopy with biopsy under general anesthesia (DE-GA) is still considered as the established standard to assess laryngopharyngeal cancer patients. Office-based flexible laryngoscopic biopsy (FLB) offers an alternative, but the effect on oncological outcome remains uncertain. Therefore, the diagnostic process and survival of patients undergoing FLB, compared to those undergoing DE-GA were evaluated. METHODS: Patients suspected of laryngopharyngeal cancer who underwent FLB were evaluated. Patients with FLB-confirmed squamous cell carcinoma (SCC) were matched with DE-GA patients based on tumor site, T-classification, N-classification, age, and p16 overexpression. Time from first visit to diagnosis (FVD), time to treatment interval (TTI), disease-specific survival (DSS) and overall survival (OS) were analyzed. RESULTS: FLB yielded a definitive diagnosis in 155/164 (95 %) patients. No complications were observed. Ninety-eight of the 124 patients in which FLB revealed a SCC received curative treatment and were compared with 98 matched patients who underwent DE-GA. Median FVD interval was 6 days after FLB and 15 days after DE-GA (p < 0.001). Median TTI interval (FLB: 28 days, DE-GA: 28 days) was equal (p = 0.91). Oncological outcomes were comparable (p > 0.05) between FLB (OS: 2-yr: 76 %, 5-yr: 42 %; DSS: 2-yr: 86 %, 5-yr: 85 %) and DE-GA groups (OS: 2-yr: 76 %, 5-yr: 50 %; DSS: 2-yr: 81 %, 5-yr: 79 %). CONCLUSION: FLB in the outpatient setting demonstrates a high diagnostic accuracy, is safe, accelerates the diagnostic process and has no negative effects on clinical outcome compared to DE-GA. Therefore, FLB should be considered as the standard diagnostic procedure in patients suspected of laryngopharyngeal cancer.

Potential imaging targets in primary head and neck squamous cell carcinoma and lymph node metastases.

PURPOSE: To investigate glycoprotein nonmetastatic melanoma protein B (GPNMB) and vascular endothelial growth factor (VEGF) as potential fluorescent imaging markers by comparing their protein expression to epidermal growth factor receptor (EGFR). MATERIALS AND METHODS: Thirty-eight paired samples of untreated head and neck squamous cell carcinoma (HNSCC) primary tumours (PT) and corresponding synchronous lymph node metastases (LNM) were selected. After immunohistochemical staining, expression was assessed and compared by the percentage of positive tumour cells. Data were analysed using the Mann-Whitney test, effect sizes (ESr) and Spearman's correlation coefficient (r). RESULTS: GPNMB expression was observed in 100 % of PT, and median 80 % (range 5-100 %) of tumour cells, VEGF in 92 % and 60 % (0-100 %), EGFR in 87 % and 60 % (0-100 %) respectively. In corresponding LNM, GPNMB expression was observed in 100 % of LNM and median 90 % (20-100 %) of tumour cells, VEGF in 87 % and 65 % (0-100 %), and EGFR in 84 % and 35 % (0-100 %). A positive correlation was found between expression in PT and LNM for GPNMB (r = 0.548) and EGFR (r = 0.618) (p < 0.001), but not for VEGF (r = -0.020; p = 0.905). GPNMB expression was present in a higher percentage of tumour cells compared to EGFR in PT (p = 0.015, ESr = -0.320) and in LNM (p < 0.001, ESr = -0.478), while VEGF was not (p = 1.00, ESr = -0.109 and - 0.152, respectively). CONCLUSION: GPNMB expression is higher than EGFR in untreated HNSCC PT and corresponding LNM, while VEGF expression is comparable to EGFR. GPNMB is a promising target for fluorescent imaging in HNSCC.

Narrow band imaging reveals field cancerisation undetected by conventional white light: Optical diagnosis versus histopathology.

OBJECTIVE: To assess whether narrow band imaging (NBI) detects fields of cancerisation around suspicious lesions in the upper aerodigestive tract, which were undetected by white light imaging (WLI). METHODS: In 96 patients with laryngeal and pharyngeal lesions suspicious for malignancy, 206 biopsies were taken during laryngoscopy: 96 biopsies of suspicious lesions detected by both WLI and NBI (WLI+/NBI+), 60 biopsies adjacent mucosa only suspicious with NBI (WLI-/NBI+), and 46 biopsies of NBI and WLI unsuspicious mucosa (WLI-/NBI-) as negative controls. Optical diagnosis according to the Ni-classification was compared with histopathology. RESULTS: Signs of (pre)malignancy were found in 88% of WLI+/NBI+ biopsies, 32% of WLI-/NBI+ biopsies and 0% in WLI-/NBI- (p < .001). In 58% of the WLI-/NBI+ mucosa any form of dysplasia or carcinoma was detected. CONCLUSION: The use of additional NBI led to the detection of (pre)malignancy in 32% of the cases, that would have otherwise remained undetected with WLI alone. This highlights the potential of NBI as a valuable adjunct to WLI in the identification of suspicious lesions in the upper aerodigestive tract.

Assessing the role of tumour-associated macrophage subsets in breast cancer subtypes using digital image analysis.

PURPOSE: The number of M1-like and M2-like tumour-associated macrophages (TAMs) and their ratio can play a role in breast cancer development and progression. Early clinical trials using macrophage targeting compounds are currently ongoing. However, the most optimal detection method of M1-like and M2-like macrophage subsets and their clinical relevance in breast cancer is still unclear. We aimed to optimize the assessment of TAM subsets in different breast cancer subtypes, and therefore related TAM subset numbers and ratio to clinicopathological characteristics and clinical outcome. METHODS: Tissue microarrays of 347 consecutive primary Luminal-A, Luminal-B, HER2-positive and triple-negative tumours of patients with early-stage breast cancer were serially sectioned and immunohistochemically stained for the pan-macrophage marker CD68 and the M2-like macrophage markers CD163, CSF-1R and CD206. TAM numbers were quantified using a digital image analysis algorithm. M1-like macrophage numbers were calculated by subtracting M2-like TAM numbers from the total TAM number. RESULTS: M2-like markers CD163 and CSF-1R showed a moderate positive association with each other and with CD68 (r ≥ 0.47), but only weakly with CD206 (r ≤ 0.06). CD68 + , CD163 + and CSF-1R + macrophages correlated with tumour grade in Luminal-B tumours (P < 0.001). Total or subset TAM numbers did not correlate with disease outcome in any breast cancer subtype. CONCLUSION: In conclusion, macrophages and their subsets can be detected by means of a panel of TAM markers and are related to unfavourable clinicopathological characteristics in Luminal-B breast cancer. However, their impact on outcome remains unclear. Preferably, this should be determined in prospective series.

Interobserver Variation in the Assessment of Immunohistochemistry Expression Levels in HER2-Negative Breast Cancer: Can We Improve the Identification of Low Levels of HER2 Expression by Adjusting the Criteria? An International Interobserver Study.

The classification of human epidermal growth factor receptor 2 (HER2) expression is optimized to detect HER2-amplified breast cancer (BC). However, novel HER2-targeting agents are also effective for BCs with low levels of HER2. This raises the question whether the current guidelines for HER2 testing are sufficiently reproducible to identify HER2-low BC. The aim of this multicenter international study was to assess the interobserver agreement of specific HER2 immunohistochemistry scores in cases with negative HER2 results (0, 1+, or 2+/in situ hybridization negative) according to the current American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. Furthermore, we evaluated whether the agreement improved by redefining immunohistochemistry (IHC) scoring criteria or by adding fluorescent in situ hybridization (FISH). We conducted a 2-round study of 105 nonamplified BCs. During the first assessment, 16 pathologists used the latest version of the ASCO/CAP guidelines. After a consensus meeting, the same pathologists scored the same digital slides using modified IHC scoring criteria based on the 2007 ASCO/CAP guidelines, and an extra "ultralow" category was added. Overall, the interobserver agreement was limited (4.7% of cases with 100% agreement) in the first round, but this was improved by clustering IHC categories. In the second round, the highest reproducibility was observed when comparing IHC 0 with the ultralow/1+/2+ grouped cluster (74.3% of cases with 100% agreement). The FISH results were not statistically different between HER2-0 and HER2-low cases, regardless of the IHC criteria used. In conclusion, our study suggests that the modified 2007 ASCO/CAP criteria were more reproducible in distinguishing HER2-0 from HER2-low cases than the 2018 ASCO/CAP criteria. However, the reproducibility was still moderate, which was not improved by adding FISH. This could lead to a suboptimal selection of patients eligible for novel HER2-targeting agents. If the threshold between HER2 IHC 0 and 1+ is to be clinically actionable, there is a need for clearer, more reproducible IHC definitions, training, and/or development of more accurate methods to detect this subtle difference in protein expression levels.

FDG-PET/CT discriminates between patients with and without lymphomas in primary Sjögren's syndrome.

OBJECTIVES: To assess the usefulness of [18F]-fluorodeoxyglucose (FDG)-PET/CT (i) to discriminate between primary SS (pSS) patients with and without lymphomas and (ii) to evaluate systemic disease activity in pSS. METHODS: ACR-EULAR-positive pSS patients who underwent FDG-PET/CT were included. Scans were visually evaluated and quantitative analysis was performed by measuring standardized uptake values (SUV) of salivary and lacrimal glands and systemic regions. Receiver operating characteristic curve analyses were performed to find SUV cut-off values to discriminate between lymphoma and non-lymphoma. RESULTS: Of the 70 included patients, 26 were diagnosed with a pSS-associated lymphoma, mostly of the mucosa-associated lymphoid tissue type (23/26). Lymphoma patients showed higher FDG uptake in the parotid and submandibular glands, and more frequently showed presence of nodular lung lesions, compared with non-lymphoma patients. The accuracy of the maximum SUV (SUVmax) in the parotid and submandibular gland to predict lymphoma diagnosis was good, with optimal cut-off points of 3.1 and 2.9. After combining these three visual and quantitative findings (nodular lung lesions, parotid SUVmax > 3.1 and submandibular SUVmax > 2.9), sensitivity was 92% when at least one of the three features were present, and specificity was 91% in case at least two features were present. Furthermore, FDG-PET/CT was able to detect systemic manifestations in pSS patients, mostly involving lymph nodes, entheses and lungs. CONCLUSIONS: FDG-PET/CT can assist in excluding pSS-associated lymphomas in patients without PET abnormalities, possibly leading to a decrease of invasive biopsies in suspected lymphoma patients. Furthermore, FDG-PET/CT is able to detect systemic manifestations in pSS and can guide to the best biopsy location.

Urinary albumin excretion and cancer risk: the PREVEND cohort study.

BACKGROUND: Chronic kidney disease (CKD) is believed to be associated with an increased risk for cancer, especially urinary tract cancer. However, previous studies predominantly focused on the association of decreased estimated glomerular filtration rate (eGFR) with cancer. In this study, we investigated the association of albuminuria with cancer incidence, adjusted for eGFR. METHODS: We included 8490 subjects in the Prevention of Renal and Vascular End-stage Disease (PREVEND) observational study. Urinary albumin excretion (UAE) was measured in two 24-hour urine specimens at baseline. Primary outcomes were the incidence of overall and urinary tract cancer. Secondary outcomes were the incidence of other site-specific cancers, and mortality due to overall, urinary tract, and other site-specific cancers. RESULTS: Median baseline UAE was 9.4 (IQR, 6.3-17.8) mg/24 h. During a median follow-up of 17.7 years, 1341 subjects developed cancer (of which 177 were urinary tract cancers). After multivariable adjustment including eGFR, every doubling of UAE was associated with a 6% (hazard ratios (HR), 1.06, 95% confidence intervals (CI), 1.02-1.10), and 14% (HR, 1.14, 95% CI, 1.04-1.24) higher risk of overall and urinary tract cancer incidence, respectively. Except for lung and hematological cancer, no associations were found between UAE and the incidence of other site-specific cancer. Doubling of UAE was also associated with a higher risk of mortality due to overall and lung cancer. CONCLUSIONS: Higher albuminuria is associated with a higher incidence of overall, urinary tract, lung, and hematological cancer, and with a higher risk of mortality due to overall and lung cancers, independent of baseline eGFR.

Chronic Use of Hydrochlorothiazide and Risk of Skin Cancer in Caucasian Adults: A PharmLines Initiative Inception Cohort Study.

Photosensitizing properties of hydrochlorothiazide may increase skin cancer risk. To date, study findings on the association between hydrochlorothiazide use and skin cancer risk are inconsistent, notably regarding confounding and dose-response. The aim of this study was to investigate the association between hydrochlorothiazide use and incidence of skin cancer in a cohort of unselected Caucasian adults, taking dosing into account. As part of the PharmLines Initiative, which links data from the Lifelines Cohort Study and prescription database IADB.nl, patients aged ≥ 40 years were included from Lifelines, a prospective population-based cohort study in the north of the Netherlands. Skin cancer incidence was compared between subjects starting hydrochlorothiazide treatment (n = 608), subjects starting treatment with other antihypertensives (n = 508), and non-antihypertensive long-term medication users (n = 1,710). Cox regression analyses were performed to obtain hazard ratios, adjusted for potential confounders. The risk of any skin cancer, keratinocyte carcinoma, basal cell carcinoma and squamous cell carcinoma was not significantly increased in general hydrochlorothiazide users. A clear association was observed between high cumulative hydrochlorothiazide use (≥ 5,000 defined daily dose; ≥ 125,000 mg) and the risk of any skin cancer (adjusted hazard ratio 5.32, 95% confidence interval (95% CI) 2.40-11.81), keratinocyte carcinoma (adjusted hazard ratio 7.31, 95% CI 3.12-17.13), basal cell carcinoma (adjusted hazard ratio 7.72, 95% CI 3.11-19.16) and squamous cell carcinoma (adjusted hazard ratio 19.63, 95% CI 3.12-123.56). These findings should lead to awareness with high use of hydrochlorothiazide in Caucasian adults.

Impact of health-related behavioral factors on participation in a cervical cancer screening program: the lifelines population-based cohort.

BACKGROUND: Regular participation in cervical cancer screening is critical to reducing mortality. Although certain sociodemographic factors are known to be associated with one-time participation in screening, little is known about other factors that could be related to regular participation. Therefore, this study evaluated the association between health-related behavioral factors and regular participation in cervical cancer screening. METHODS: The Lifelines population-based cohort was linked to data for cervical cancer screening from the Dutch Nationwide Pathology Databank. We included women eligible for all four screening rounds between 2000 and 2019, classifying them as regular (4 attendances), irregular (1-3 attendances), and never participants. Multinomial logistic regression was performed to evaluate the association between behavioral factors and participation regularity, with adjustment made for sociodemographic factors. RESULTS: Of the 48,325 included women, 55.9%, 35.1%, and 9% were regular, irregular, and never screening participants. After adjustment for sociodemographic factors, the likelihood of irregular or never screening participation was increased by smoking, obesity, marginal or inadequate sleep duration, alcohol consumption and low physical activity, while it was decreased by hormonal contraception use. CONCLUSION: An association exists between unhealthy behavioral factors and never or irregular participation in cervical cancer screening.

The relation between hypoxia and proliferation biomarkers with radiosensitivity in locally advanced laryngeal cancer.

PURPOSE: Treatment decision-making in advanced-stage laryngeal squamous cell carcinoma (LSCC) is difficult due to the high recurrence rates and the desire to preserve laryngeal functions. New predictive markers for radiosensitivity are needed to facilitate treatment choices. In early stage glottic LSCC treated with primary radiotherapy, expression of hypoxia (HIF-1α and CA-IX) and proliferation (Ki-67) tumour markers showed prognostic value for local control. The objective of this study is to examine the prognostic value of tumour markers for hypoxia and proliferation on locoregional recurrent disease and disease-specific mortality in a well-defined cohort of patients with locally advanced LSCC treated with primary, curatively intended radiotherapy. METHODS: In pre-treatment biopsy tissues from a homogeneous cohort of 61 patients with advanced stage (T3-T4, M0) LSCC primarily treated with radiotherapy, expression of HIF-1α, CA-IX and Ki-67 was evaluated with immunohistochemistry. Demographic data (age and sex) and clinical data (T- and N-status) were retrospectively collected from the medical records. Cox regression analysis was performed to assess the relation between marker expression, demographic and clinical data, and locoregional recurrence and disease-specific mortality. RESULTS: Patients with high expression of HIF-1α developed significantly more often a locoregional recurrence (39%) compared to patients with a low expression (21%) (p = 0.002). The expression of CA-IX and Ki-67 showed no association with locoregional recurrent disease. HIF-1α, CA-IX and Ki-67 were not significantly related to disease-specific mortality. Clinical N-status was an independent predictor of recurrent disease (p < 0.001) and disease-specific mortality (p = 0.003). Age, sex and T-status were not related to locoregional recurrent disease or disease-specific mortality. CONCLUSION: HIF-1α overexpression and the presence of regional lymph node metastases at diagnosis were independent predictors of locoregional recurrent disease after primary treatment with curatively intended radiotherapy in patients with locally advanced LSCC.

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study.

Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

Digital image analysis of intraepithelial B-lymphocytes to assess lymphoepithelial lesions in salivary glands of Sjögren's syndrome patients.

OBJECTIVE: Salivary glands of primary SS (pSS) patients characteristically harbour periductal infiltrates, in which lymphoepithelial lesions (LELs) can develop. LELs are composed of hyperplastic ductal epithelium with infiltrating lymphocytes and may assist in the challenging diagnostic process of pSS. As manual identification of LELs remains difficult, we aimed to identify LELs by using an objective digital image analysis (DIA) algorithm that detects intraepithelial lymphocytes. METHODS: A virtual triple-staining technique developed for this study was used to count intraepithelial lymphocytes in consecutive slides stained for CD3 (T-lymphocytes), high-molecular-weight cytokeratin (hmwCK) (striated ducts) and CD20 (B-lymphocytes) in labial and parotid gland biopsies in a diagnostic cohort of 109 sicca patients. Patients were classified as having pSS or non-SS according to the ACR-EULAR classification criteria. RESULTS: T-lymphocytes were detected in almost all analysed ducts of pSS and non-SS sicca patients, whereas intraepithelial B-lymphocytes were present in 59-68% of labial and parotid gland biopsies of pSS patients, against only 2-3% of patients classified as non-SS. Intraepithelial B-lymphocytes were found in almost all striated ducts with hyperplasia (LELs). Remarkably, ∼25% of analysed striated ducts without hyperplasia of pSS patients also contained B-lymphocytes (precursor-LELs). Furthermore, presence of intraepithelial B-lymphocytes was associated with clinical parameters of pSS (i.e. serology). CONCLUSION: The presence of intraepithelial B-lymphocytes in salivary gland biopsies of sicca patients is a clear indicator of pSS and can be used as an objective alternative to LEL scoring. Therefore, identification of B-lymphocyte-containing ducts should be added to the diagnostic histopathological work-up of patients suspected of pSS.