Precision oncology using organoids of a secretory carcinoma of the salivary gland treated with TRK-inhibitors.

The use of anticancer drugs targeting specific molecular tumor characteristics is rapidly increasing in clinical practice, but selecting patients to benefit from these remains a challenge. It has been suggested that organoid cultures would be ideally suited to test drug responses in vitro. Here we describe and characterize in depth a case of ETV6-NTRK3 gene fusion-positive secretory carcinoma of the salivary glands and corresponding organoid cultures that responded and subsequently acquired resistance to TRK targeting therapy with larotrectinib. This case-culture-characterization illustrates the advances made in precision oncology, but also exposes important caveats in using organoids to predict treatment response.

Development and characterization of patient-derived salivary gland cancer organoid cultures.

OBJECTIVE: Three-dimensional organoid cell cultures have been established for a variety of human cancers. For most rare cancers, including salivary gland cancer (SGC), these models are lacking, despite the great unmet need to study cancer biology in these diseases. Therefore, we aimed to develop patient-derived organoid (PDO) models for different subtypes of SGC. METHODS: Tumor samples of SGC patients were processed and embedded in Matrigel. Successful PDOs (expandable > 1*106 cells) were phenotypically characterized using immunohistochemistry (IHC) and genotypically by gene fusion analysis and by targeted and whole-exome sequencing. Successfully established PDOs were subjected to small-scale drug screening. RESULTS: Out of 37 attempts, 7 viable short-term PDOs were established (19 % success rate; 3 salivary duct carcinoma, 3 adenoid cystic carcinoma and 1 mucoepidermoid carcinoma). Each PDO showed close phenotypical mimicry to parental tissue. Genotypic characterization revealed that in each PDO > 97.6 % of all COSMIC annotated variants and all MYB, MYBL1 and NFIB gene rearrangements were retained. Drug screening was proven feasible in all PDOs. CONCLUSION: We present the first comprehensively characterized short-term SGC PDO models for three subtypes of SGC with close phenotypic and genotypic resemblance to parental tissue, which can be used for drug screening applications.

Molecular Phenotyping of AR Signaling for Predicting Targeted Therapy in Castration Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) is defined by resistance of the tumor to androgen deprivation therapy (ADT). Several molecular changes, particularly in the AR signaling cascade, have been described that may explain ADT resistance. The variety of changes may also explain why the response to novel therapies varies between patients. Testing the specific molecular changes may be a major step towards personalized treatment of CRPC patients. The aim of our study was to evaluate the molecular changes in the AR signaling cascade in CRPC patients. We have developed and validated several methods which are easy to use, and require little tissue material, for exploring AR signaling pathway changes simultaneously. We found that the AR signaling pathway is still active in the majority of our CRPC patients, due to molecular changes in AR signaling components. There was heterogeneity in the molecular changes observed, but we could classify the patients into 4 major subgroups which are: AR mutation, AR amplification, active intratumoral steroidogenesis, and combination of AR amplification and active intratumoral steroidogenesis. We suggest characterizing the AR signaling pathway in CRPC patients before beginning any new treatment, and a recent fresh tissue sample from the prostate or a metastatic site should be obtained for the purpose of this characterization.

Partial Adrenalectomy Carries a Considerable Risk of Incomplete Cure in Primary Aldosteronism.

PURPOSE: Laparoscopic adrenalectomy is standard treatment for patients with unilateral aldosterone-producing adenomas, but surgeons are increasingly tempted to perform partial adrenalectomy, disregarding potential multinodularity of the adrenal. We assess the diagnostic value of endoscopic ultrasound for differentiating solitary adenomas from multinodularity by examining in-depth adrenal pathology with ex vivo 11.7 T magnetic resonance imaging and immunohistochemistry. MATERIALS AND METHODS: In 15 primary aldosteronism patients, we performed intraoperative endoscopic ultrasound, ex vivo magnetic resonance imaging and histopathological examination. Every adrenal was intraoperatively and postoperatively assessed for solitary adenomas or multinodular hyperplasia. After unblinding for ex vivo magnetic resonance imaging results a second detailed histopathological examination, including immunohistochemistry analysis with CYP11B2 (aldosterone synthase) and chemokine receptor 4 (CXCR4), a new marker for aldosterone-producing adenomas, was performed. Finally, presence of somatic mutations linked to aldosterone-producing adenomas was assessed. RESULTS: The sensitivity and specificity of endoscopic ultrasound to identify multinodularity were 46% and 50%, respectively. We found multinodular hyperplasia in 87% of adrenals with ex vivo magnetic resonance imaging combined with detailed histopathology, and 6 adrenals contained multiple CYP11B2-producing nodules. Every CYP11B2 positive nodule and 61% of CYP11B2 negative nodules showed CXCR4 staining. Finally, in 4 adrenals (27%) we found somatic mutations. In multinodular glands, only 1 nodule harbored this mutation. CONCLUSIONS: Intraoperative endoscopic ultrasound in primary aldosteronism patients has low accuracy to identify multinodularity. Ex vivo magnetic resonance imaging can serve as a tool to direct detailed histopathological examination, which frequently shows CYP11B2 production in multiple nodules. Therefore, partial adrenalectomy is inappropriate in primary aldosteronism as multiple aldosterone-producing nodules easily stay behind.

Polyisocyanide Hydrogels as a Tunable Platform for Mammary Gland Organoid Formation.

In the last decade, organoid technology has developed as a primary research tool in basic biological and clinical research. The reliance on poorly defined animal-derived extracellular matrix, however, severely limits its application in regenerative and translational medicine. Here, a well-defined, synthetic biomimetic matrix based on polyisocyanide (PIC) hydrogels that support efficient and reproducible formation of mammary gland organoids (MGOs) in vitro is presented. Only decorated with the adhesive peptide RGD for cell binding, PIC hydrogels allow MGO formation from mammary fragments or from purified single mammary epithelial cells. The cystic organoids maintain their capacity to branch for over two months, which is a fundamental and complex feature during mammary gland development. It is found that small variations in the 3D matrix give rise to large changes in the MGO: the ratio of the main cell types in the MGO is controlled by the cell-gel interactions via the cell binding peptide density, whereas gel stiffness controls colony formation efficiency, which is indicative of the progenitor density. Simple hydrogel modifications will allow for future introduction and customization of new biophysical and biochemical parameters, making the PIC platform an ideal matrix for in depth studies into organ development and for application in disease models.

Components of the plasminogen activator system and their complexes in renal cell and bladder cancer: comparison between normal and matched cancerous tissues.

OBJECTIVE: To analyse and compare the concentration of plasminogen activator (PA), urokinase-type PA (uPA), tissue-type PA (tPA), PA inhibitor (PAI)-1 and PAI-2, and the complexes uPA-PAI-1 and tPA-PAI-1 and calculated uPA and tPA uncomplexed with PAI-1 ('free') in urothelial cell carcinoma and matched benign urothelium, and in renal cell carcinoma (RCC) and matched benign renal tissue. PATIENTS AND METHODS: Tissue samples were obtained during cystectomy (33 patients) and nephrectomy (55), and specific enzyme-linked immunosorbent assays were used to assess the PA components in extracts of these tissues. RESULTS: Tissue levels of uPA-PAI-1 and tPA-PAI-1, but also PAI-1 itself, were greater in tumorous bladder and kidney tissue than in matched normal tissue (by 1.5-7.8 times). Free tPA was clearly lower in tumour tissue (by 0-0.12-fold). In bladder cancer, but not in RCC, levels of uPA (15.8-fold) and free uPA (16.4-fold) were greater in tumour tissue. Free uPA levels were less in RCC (0.41-fold). For both normal bladder and kidney tissue, there was no clear correlation between uPA-PAI-1 complex and either component. However, the formation of tPA-PAI-1 complexes in normal bladder and kidney tissue was primarily determined by PAI-1. Interestingly, in tumour tissues there was a strong, significant correlation between complex levels and both components. CONCLUSION: RCC and bladder cancer show distinct profiles of components of the PA system. This study provides a basis for further studies into both the (patho)physiological role of the PA system in these tumours, and into a possible relation with tumour progression and prognosis, and as target for therapy.

Intermediate cells in human prostate epithelium are enriched in proliferative inflammatory atrophy.

Within the human prostate epithelium four cell populations can be discriminated based on their expression of keratins (K). Basal cells express high levels of K5 and K14, as well as p63, whereas they have very low levels of androgen receptor, prostate-specific antigen (PSA), K8, and K18. Luminal secretory cells lack p63, K5, and K14 but express high levels of K8, K18, androgen receptor, and PSA. Additionally, cells have been identified with a keratin phenotype intermediate between basal and luminal cells that co-express high levels of K5 and K18 (K5/18) as well as hepatocyte growth factor receptor c-MET. Although intermediate cells have been proposed as precursor cells of prostate cancer, their biology is ill defined. Epithelial cells in proliferative inflammatory atrophy (PIA) appear to be cycling rapidly as indicated by expression of Ki-67, and morphological transitions have been identified between PIA and high-grade prostate intraepithelial neoplasia. Many of the atrophic epithelial luminal cells in PIA are candidates for intermediate cells based in part on weak expression of PSA and androgen receptor, high levels of K8/18, and lack of p63. The objective of this study was to further clarify the phenotype of the proposed intermediate cells in PIA and to quantitatively determine the level in which these intermediate cells preferentially occur in PIA lesions. Intermediate cells were immunohistochemically demonstrated using antibodies to K5, K14, K18, and c-MET. Using radical prostatectomy specimens (n = 15) the area fraction of intermediate cells in normally differentiated prostate epithelium and PIA were quantified by a grid point counting method. Atrophic luminal cells of PIA lesions expressed K5 in 39.2 +/- 7.4% of cells compared to 2.4 +/- 2.3% in normal epithelium (P < 0.00001). By contrast, K14 was only expressed in 3.0 +/- 3.2% of the luminal cells. Previous studies have shown that virtually 100% of these atrophic luminal cells are strongly positive for K8/18. c-MET was present in 44.1 +/- 14.1% of luminal cells in PIA but only in 2.1 +/- 2.8% of luminal cells in normal epithelium (P < 0.00001). To unambiguously determine whether intermediate luminal cells in PIA show increased proliferative activity and decreased p27(kip1) expression, double-staining immunofluorescence of Ki-67 and K5, as well as p27(Kip1) and K5 was performed. Luminal cells in PIA often co-expressed K5 and Ki-67. Although p27(Kip1) was strongly expressed in K5-negative differentiated cells in normal epithelium, p27(Kip1) staining was absent in many of the K5-positive cells in the luminal compartment of PIA. We conclude that cells phenotypically intermediate between basal and secretory cells are enriched in PIA lesions. The finding of a large number of highly proliferating intermediate cells in PIA provides further support that these cells may serve as preferred target cells in prostate carcinogenesis.

DD3(PCA3), a very sensitive and specific marker to detect prostate tumors.

We identified DD3(PCA3) as one of the most prostate cancer-specific genes at present (M. J. Bussemakers et al. Cancer Res., 59: 5975-5979, 1999). Consequently, DD3(PCA3) is an interesting candidate for use as a diagnostic and/or prognostic marker. In this study we developed a method for the accurate quantification of DD3(PCA3) mRNA, using real-time quantitative reverse transcription-PCR. DD3(PCA3) was expressed at low levels in normal prostate but not in 21 selected other normal tissues, blood, or 39 tumor samples other than prostate. The diagnostic and prognostic value of DD3(PCA3) in normal, hyperplastic, and malignant prostate tissues was determined and compared with another promising tumor marker for prostate cancer, telomerase reverse transcriptase (hTERT gene), the expression of which is related to telomerase activity. Sensitivity and specificity estimates for both genes were calculated as the area under the receiver-operating characteristics curve (AUC-ROC). DD3(PCA3) (AUC-ROC, 0.98) demonstrated better diagnostic efficacy than hTERT (AUC-ROC, 0.88). Moreover, the median increase in mRNA expression in tumor tissues compared with nonmalignant prostate tissues was much higher for DD3(PCA3) (34-fold) than for hTERT (6-fold). In tumor tissues, the median expression of DD3(PCA3) was much higher than hTERT (5849 versus 10 normalized mRNA copies). A significant relationship was observed only between tumor stage and hTERT gene expression. We conclude that expression of the DD3(PCA3) gene is a very sensitive and specific marker for the detection of prostate tumor cells in a high background of normal (prostate) cells. Consequently, DD3 measurements may be used for clinical application in prostate needle biopsies or bodily fluids such as blood, ejaculate, urine, or prostate massage fluid.