Artificial intelligence-based recurrence prediction outperforms classical histopathological methods in pulmonary adenocarcinoma biopsies.

INTRODUCTION: Between 10 and 50% of early-stage lung adenocarcinoma patients experience local or distant recurrence. Histological parameters such as a solid or micropapillary growth pattern are well-described risk factors for recurrence. However, not every patient presenting with such a pattern will develop recurrence. Designing a model which can more accurately predict recurrence on small biopsy samples can aid the stratification of patients for surgery, (neo-)adjuvant therapy, and follow-up. MATERIAL AND METHODS: In this study, a statistical model on biopsies fed with histological data from early and advanced-stage lung adenocarcinomas was developed to predict recurrence after surgical resection. Additionally, a convolutional neural network (CNN)-based artificial intelligence (AI) classification model, named AI-based Lung Adenocarcinoma Recurrence Predictor (AILARP), was trained to predict recurrence, with an ImageNet pre-trained EfficientNet that was fine-tuned on lung adenocarcinoma biopsies using transfer learning. Both models were validated using the same biopsy dataset to ensure that an accurate comparison was demonstrated. RESULTS: The statistical model had an accuracy of 0.49 for all patients when using histology data only. The AI classification model yielded a test accuracy of 0.70 and 0.82 and an area under the curve (AUC) of 0.74 and 0.87 on patch-wise and patient-wise hematoxylin and eosin (H&E) stained whole slide images (WSIs), respectively. CONCLUSION: AI classification outperformed the traditional clinical approach for recurrence prediction on biopsies by a fair margin. The AI classifier may stratify patients according to their recurrence risk, based only on small biopsies. This model warrants validation in a larger lung biopsy cohort.

The value of prognostic and predictive parameters in early-stage lung adenocarcinomas: A comparison between biopsies and resections.

INTRODUCTION: Since lung adenocarcinoma (LUAD) biopsies are usually small, it is questionable if their prognostic and predictive information is comparable to what is offered by large resection specimens. This study compares LUAD biopsies and resection specimens for their ability to provide prognostic and predictive parameters. METHODS: We selected 187 biopsy specimens with stage I and II LUAD. In 123 cases, subsequent resection specimens were also available. All specimens were evaluated for growth pattern, nuclear grade, fibrosis, inflammation, and genomic alterations. Findings were compared using non-parametric testing for categorical variables. Model performance was assessed using the area under the curve for both biopsies and resection specimens, and overall (OS) and disease-free survival (DFS) was calculated. RESULTS: The overall growth pattern concordance between biopsies and resections was 73.9%. The dominant growth pattern correlated with OS and DFS in resected adenocarcinomas and for high-grade growth pattern in biopsies. Multivariate analysis of biopsy specimens revealed that T2-tumors, N1-status, KRAS mutations and a lack of other driver mutations were associated with poorer survival. Model performance using clinical, histological and genetic data from biopsy specimens for predicting OS and DSF demonstrated an AUC of 0.72 and 0.69, respectively. CONCLUSIONS: Our data demonstrated the prognostic relevance of a high-grade growth pattern in biopsy specimens of LUAD. Combining clinical, histological and genetic information in one model demonstrated a suboptimal performance for DFS prediction and good performance for OS prediction. However, for daily practice, more robust (bio)markers are required to predict prognosis and stratify patients for therapy and follow-up.

Autologous dendritic cells pulsed with allogeneic tumour cell lysate induce tumour-reactive T-cell responses in patients with pancreatic cancer: A phase I study.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is notorious for its poor prognosis even after curative resection. Responses to immunotherapy are rare and related to inadequate T-cell priming. We previously demonstrated the potency of allogeneic lysate-dendritic cell (DC) vaccination in a preclinical model. Here we translate this concept to patients. METHODS: In this phase I study, patients with resected PDAC were included when they demonstrated no radiologic signs of recurrence after standard-of-care treatment. Allogeneic tumour lysate-loaded autologous monocyte-derived DCs were injected at weeks 0, 2, 4 and at months 3 and 6. Objectives are feasibility, safety and immunogenicity of allogeneic tumour-DCs. The presence of tumour antigens shared between the vaccine and patient tumours was investigated. Immunological analyses were performed on peripheral blood, skin and tumour. RESULTS: Ten patients were included. DC production and administration were successful. All patients experienced a grade 1 injection-site and infusion-related reaction. Two patients experienced a grade 2 fever and 1 patient experienced a grade 3 dyspnoea. No vaccine-related serious adverse events were observed. Shared tumour antigens were found between the vaccine and patient tumours. All evaluated patients displayed a vaccine-induced response indicated by increased frequencies of Ki67+ and activated PD-1+ circulating T-cells. In addition, treatment-induced T-cell reactivity to autologous tumour of study patients was detected. Seven out of ten patients have not experienced disease recurrence or progression at a median follow-up of 25 months (15-32 months). CONCLUSION: Allogeneic tumour lysate-DC treatment is feasible, safe and induces immune reactivity to PDAC expressed antigens.

A novel nicastrin mutation in a three-generation Dutch family with hidradenitis suppurativa: a search for functional significance.

BACKGROUND: Mutations in the γ-secretase enzyme subunits have been described in multiple kindreds with familial hidradenitis suppurativa (HS). OBJECTIVE: In this study, we report a novel nicastrin (NCSTN) mutation causing HS in a Dutch family. We sought to explore the immunobiological function of NCSTN mutations using data of the Immunological Genome Project. METHODS: Blood samples of three affected and two unaffected family members were collected. Whole-genome sequencing was performed using genomic DNA isolated from peripheral blood leucocytes. Sanger sequencing was done to confirm the causative NCSTN variant and the familial segregation. The microarray data set of the Immunological Genome Project was used for thorough dissection of the expression and function of wildtype NCSTN in the immune system. RESULTS: In a family consisting of 23 members, we found an autosomal dominant inheritance pattern of HS and detected a novel splice site mutation (c.1912_1915delCAGT) in the NCSTN gene resulting in a frameshift and subsequent premature stop. All affected individuals had HS lesions on non-flexural and atypical locations. Wildtype NCSTN appears to be upregulated in myeloid cells like monocytes and macrophages, and in mesenchymal cells such as fibroblastic reticular cells and fibroblasts. In addition, within the 25 highest co-expressed genes with NCSTN we identified CAPNS1, ARNT and PPARD. CONCLUSION: This study reports the identification a novel NCSTN gene splice site mutation which causes familial HS. The associated immunobiological functions of NCSTN and its co-expressed genes ARNT and PPARD link genetics to the most common environmental and metabolic HS risk factors which are smoking and obesity.

Exhaled breath profiling using broadband quantum cascade laser-based spectroscopy in healthy children and children with asthma and cystic fibrosis.

Exhaled breath analysis is a potential non-invasive tool for diagnosing and monitoring airway diseases. Gas chromatography-mass spectrometry and electrochemical sensor arrays are the main techniques to detect volatile organic compounds (VOC) in exhaled breath. We developed a broadband quantum cascade laser spectroscopy technique for VOC detection and identification. The objective of this study was to assess the repeatability of exhaled breath profiling with broadband quantum cascade laser-based spectroscopy and to explore the clinical applicability by comparing exhaled breath samples from healthy children with those from children with asthma or cystic fibrosis (CF). Healthy children and children with stable asthma or stable CF, aged 6-18 years, were included. Two to four exhaled breath samples were collected in Tedlar bags and analyzed by quantum cascade laser spectroscopy to detect VOCs with an absorption profile in the wavenumber region between 832 and 1262.55 cm(-1). We included 35 healthy children, 39 children with asthma and 15 with CF. Exhaled breath VOC profiles showed poor repeatability (Spearman's rho = 0.36 to 0.46) and agreement of the complete profiles. However, we were able to discriminate healthy children from children with stable asthma or stable CF and identified VOCs that were responsible for this discrimination. Broadband quantum cascade laser-based spectroscopy detected differences in VOC profiles in exhaled breath samples between healthy children and children with asthma or CF. The combination of a relatively easy and fast method and the possibility of molecule identification makes broadband quantum cascade laser-based spectroscopy attractive to investigate the diagnostic and prognostic potential of volatiles in exhaled breath.

SMAD4 exerts a tumor-promoting role in hepatocellular carcinoma.

Further understanding of the molecular biology and pathogenesis of hepatocellular carcinoma (HCC) is crucial for future therapeutic development. SMAD4, recognized as an important tumor suppressor, is a central mediator of transforming growth factor beta (TGFB) and bone morphogenetic protein (BMP) signaling. This study investigated the role of SMAD4 in HCC. Nuclear localization of SMAD4 was observed in a cohort of 140 HCC patients using tissue microarray. HCC cell lines were used for functional assay in vitro and in immune-deficient mice. Nuclear SMAD4 levels were significantly increased in patient HCC tumors as compared with adjacent tissues. Knockdown of SMAD4 significantly reduced the efficiency of colony formation and migratory capacity of HCC cells in vitro and was incompatible with HCC tumor initiation and growth in mice. Knockdown of SMAD4 partially conferred resistance to the anti-growth effects of BMP ligand in HCC cells. Importantly, simultaneous elevation of SMAD4 and phosphorylated SMAD2/3 is significantly associated with poor patient outcome after surgery. Although high levels of SMAD4 can also mediate an antitumor function by coupling with phosphorylated SMAD1/5/8, this signaling, however, is absent in majority of our HCC patients. In conclusion, this study revealed a highly non-canonical tumor-promoting function of SMAD4 in HCC. The drastic elevation of nuclear SMAD4 in sub-population of HCC tumors highlights its potential as an outcome predictor for patient stratification and a target for personalized therapeutic development.

Inflammatory conditions affect gene expression and function of human adipose tissue-derived mesenchymal stem cells.

There is emerging interest in the application of mesenchymal stem cells (MSC) for the prevention and treatment of autoimmune diseases, graft-versus-host disease and allograft rejection. It is, however, unknown how inflammatory conditions affect phenotype and function of MSC. Adipose tissue-derived mesenchymal stem cells (ASC) were cultured with alloactivated peripheral blood mononuclear cells (PBMC) (mixed lymphocyte reaction: MLR), with proinflammatory cytokines [interferon (IFN)-γ, tumour necrosis factor (TNF)-α and interleukin (IL)-6] or under control conditions, and their full genome expression and function examined. Proinflammatory cytokines mainly increased indoleamine-2,3-dioxygenase expression, whereas ASC cultured with MLR showed increased expression of COX-2, involved in prostaglandin E(2) production. Both conditions had a stimulatory, but differential, effect on the expression of proinflammatory cytokines and chemokines, while the expression of fibrotic factors was decreased only in response to proinflammatory cytokines. Functional analysis demonstrated that inflammatory conditions affected morphology and proliferation of ASC, while their differentiation capacity and production of trophic factors was unaffected. The immunosuppressive capacity of ASC was enhanced strongly under inflammatory conditions. In conclusion, ASC showed enhanced immunosuppressive capacity under inflammatory conditions, while their differentiation capacity was preserved. Therefore, in vitro preconditioning provides ASC with improved properties for immediate clinical immune therapy.

Human MUC1 mucin: a potent glandular morphogen.

Human MUC1 mucin is a high-molecular-weight transmembrane glycoprotein, which is apically expressed in the majority of glandular epithelia. During embryonic development, changes in the pattern of MUC1 mucin expression coincide with the onset of glandular differentiation. This mucin is also frequently overexpressed and aberrantly glycosylated in carcinomas. To investigate the potential role of MUC1 mucin in morphogenesis, a full length MUC1 cDNA was transfected into murine mammary adenocarcinoma (410.4) and Madin-Darby canine kidney (MDCK) cells. This generated four clonal cell lines. Western blotting, FACS analysis, and immunohistochemistry confirmed expression of MUC1. All four MUC1-expressing clones demonstrated altered morphogenesis when cultured in three-dimensional type I collagen gels. While parental and vector control 410.4 cells formed compact spherical structures, the MUC1-expressing clones formed complex branching structures. Similarly, while parental and vector control MDCK cells formed small circumscribed colonies with a central lumen, the MUC1-expressing clones formed elongated tubules. MUC1 expression was also associated with reduced cellular cohesion and enhanced migration on type I collagen-coated surfaces for all except one of the clones, which expressed only low levels of MUC1 on the cell surface. These results show that MUC1 expression stimulates morphogenetic changes in two distinct epithelial cell lines. Taken together with previous observations on MUC1 expression in embryonic development and carcinomas, this finding suggests that MUC1 may induce changes in tissue architecture in both normal development and cancer.

Differentially expressed genes in hormone refractory prostate cancer: association with chromosomal regions involved with genetic aberrations.

Differential gene expression between the androgen sensitive human prostate cancer cell line LNCaP and an insensitive clonal variant, LNCaP-r, was demonstrated by suppression subtractive hybridization. Twenty-one sequences were identified of which 9 are homologous to known genes, 11 are represented by expressed sequence tags (ESTs), and 1 is novel. We present data for 5 of 7 sequences confirmed to be differentially expressed by Northern blot analysis and semiquantitative RT-PCR. Only one gene, fibronectin (FN), was highly overexpressed (>60-fold) in LNCaP-r cells, consistent with previously reported overexpression of FN in prostate cancer. Four sequences were down-regulated in LNCaP-r cells, including an inactive variant of the E2 ubiquitin conjugating enzyme (UEV-1), a novel metalloproteinase-related collagenase (PM5), and a potential tumor suppressor gene (breast basic conserved gene, BBC1). UEV-1 is multifunctional, regulates the cell cycle via cdk1, has homology to MMS2 and likewise functions as a DNA protection protein, and also has homology to TSG101. Aberrant splice variants of TSG101 occur frequently in both breast and prostate cancer, but its mechanism of action is unknown. FN, BBC1, and UEV-1 localize to regions of chromosomal aberration (2q3.4, 16q24.3, and 20q13.2, respectively) associated with advanced prostate cancer and thus may be highly relevant to disease progression.

Second messenger up-regulation of androgen receptor gene transcription is absent in androgen insensitive human prostatic carcinoma cell lines, PC-3 and DU-145.

A theoretical pathway of transcriptional regulation of the androgen receptor (AR) gene is via a cAMP response element (CRE) present in its promoter region (-508 to -501). After 20 h of stimulation with 8-bromo-cAMP, AR mRNA was upregulated in LNCaP but not in either PC-3 or DU-145 cell lines. We have demonstrated that the level of CRE binding protein (CREB) was the same in all cell lines and that the putative AR-CRE forms specific and compatible protein interactions with CREB. The ability to regulate AR gene transcription via the second messenger pathway is lost in the PC-3 and DU-145 cell lines. This may be an important primary mechanism of androgen insensitivity in prostate cancer.

Measurement of androgen receptor expression in adult liver, fetal liver, and Hep-G2 cells by the polymerase chain reaction.

Hepatocellular carcinoma is the most commonly fatal malignant tumour worldwide. The role of androgen receptors, which have been found in hepatocellular carcinoma, is controversial. Sequence specific polymerase chain reaction (PCR) was used to quantify, for the first time, the expression of androgen receptor in four adult liver biopsy specimens (HL-A to HL-D), fetal liver, and Hep-G2 cells. The measurement of androgen receptor is expressed as a ratio (androgen receptor: beta-actin) of the value of androgen receptor to the value of a control gene, beta-actin. The value of the androgen receptor: beta-actin ratios for HL-A, HL-B, HL-C, HL-D, fetal liver, and Hep-G2 were 0.37, 0.86, 0.37, 0.44, 0.87, and 0.66 respectively. To verify sequence specific amplification of the androgen receptor, the PCR androgen receptor fragment was sequenced. The resultant sequence data for both strands of the double stranded PCR androgen receptor fragment had 100% similarity with the published androgen receptor mRNA sequence (complete codons).

Isocratic high-performance liquid chromatographic measurement of optimal 5 alpha-steroid reductase activity in Hep-G2 cells.

Measurement of 5 alpha-reductase activity usually involves quantitation of the radiolabelled products of [3H]testosterone. Recently, however, it has been claimed that the activity of 5 alpha-reductase is masked by the activities of 17 beta-hydroxysteroid dehydrogenase and 3-ketosteroid reductase. Therefore in determining 5 alpha-reductase activity in Hep-G2 cells, we have monitored the concentration of androstenedione to ensure that the conditions for measurement of optimum enzyme activity are maintained. Using a polar (cyano) bonded-phase column and hexane-isopropanol (9:1, v/v) as eluent, the ratio of relative retention times (methyl lithocholate used as the reference standard) of the closest peaks, dihydrotestosterone and estradiol, was 1.2, whilst the highest inter-assay coefficient of variation was 2.7%. Therefore this technique appears suitable for the evaluation of 5 alpha-reductase in cell and tissue samples.

Estrogen and androgen regulation of sex hormone binding globulin secretion by a human liver cell line.

Both estrogens and androgens have been shown to stimulate sex hormone binding globulin (SHBG) secretion in vitro in the hepatocellular carcinoma cell line, Hep G2, in contrast to the expected inhibition by androgens from in vivo studies. However, such in vitro stimulation was only demonstrated at high steroid doses, generally in serum-containing medium, with added Phenol Red. In the present study, Hep G2 cells were grown in serum-free medium, without Phenol Red, under the influence of testosterone (T) (0, 0.5-500 nM) and ethinyl estradiol (EE2) (0, 50 pM-500 nM). Levels of secreted SHBG and albumin were correlated with androgen receptors in cytosolic (ARc) and nuclear (ARn) fractions and with DNA levels. In the presence of increasing T levels, SHBG levels fell to 39% of control values at 5 nM T (P = 0.047), rising to 97% of control at 500 nM. Conversely, incubation with EE2 produced a rise in SHBG secretion of more than 100% at 0.5 nM (P less than 0.02) which was sustained to 50 nM (P less than 0.005). DNA levels did not change with the addition of testosterone or EE2, with the exception of a 15% reduction at 5 nM EE2 (P less than 0.05). Albumin levels in the medium were not significantly altered by either steroid. However, in response to T, androgen receptor (AR) levels were reduced in cytosolic (42% of control) and nuclear (22%) fractions at 5 nM, and these changes in ARc and ARn correlated with SHBG levels over the range of T concentrations (P = 0.04 and P = 0.017, respectively). Nuclear estrogen receptor (ER) increased over 10-fold at 5 and 50 pM EE2 (P less than 0.001) and maintained 50 nM (P less than 0.001). Cytosolic ER was reduced at 0.5 and 5 nM but recovered at 50 nM, correlating with SHBG levels (P less than 0.001). These findings are consistent with the hypothesis that estrogens and androgens regulate SHBG synthesis in man by direct, specific, probably receptor-mediated effects on hepatocytes. Hep G2 cells grown in serum-free medium are a suitable experimental system for further study of this phenomenon.