Pyrimidine-Dependent UV-Mediated Cross-Linking Magnifies Minor Genetic or Epigenetic Changes in Clinical Samples.

BACKGROUND: Detection of minor DNA allele alterations is becoming increasingly important for early detection and monitoring of cancer. We describe a new method that uses ultraviolet light to eliminate wild-type DNA alleles and enables improved detection of minor genetic or epigenetic changes. METHODS: Pyrimidine-dependent UV-based minor-allele enrichment (PD-UVME) employed oligonucleotide probes that incorporated a UVA-sensitive 3-cyanovinylcarbazole (CNVK), placed directly opposite interrogated pyrimidines, such as thymine (T) or cytosine (C) in wild-type (WT) DNA. Upon UVA-illumination, CNVK cross-linked with T/C, preventing subsequent amplification. Mutations that removed the T/C escaped cross-linking and were amplified and detected. Similarly, CNVK discriminated between methylated and unmethylated cytosine in CpG dinucleotides, enabling direct enrichment of unmethylated DNA targets. PD-UVME was combined with digital droplet PCR (ddPCR) to detect serine/threonine-protein kinase B-Raf (BRAF) V600E mutations in model systems, thyroid patient cancer tissue samples, and circulating DNA of tumor origin (ctDNA) from melanoma patients. RESULTS: One thyroid cancer sample out of 9, and 6 circulating-DNA samples out of 7 were found to be BRAF V600E-positive via PD-UVME while classified as negative by conventional ddPCR. Positive samples via conventional ddPCR were also found positive via PD-UVME. All 10 circulating cell-free DNA (cfDNA) samples obtained from normal volunteers were negative via both approaches. Furthermore, preferential enrichment of unmethylated alleles in MAGEA1 promoters using PD-UVME was demonstrated. CONCLUSIONS: PD-UVME mutation/methylation enrichment performed prior to ddPCR magnifies low-level mutations or epigenetic changes and increases sensitivity and confidence in the results. It can assist with clinical decisions that hinge on the presence of trace alterations like BRAF V600E.

Development and Validation of a Novel Dual-Drop-off ddPCR Assay for the Simultaneous Detection of Ten Hotspots PIK3CA Mutations.

Breast cancer is the leading cause of cancer-related deaths in women worldwide. Approximately 40% of patients with hormone receptor-positive, human epidermal growth factor receptor-2-negative breast cancer have activating mutations in the PIK3CA gene. We developed a highly sensitive, specific, cost-effective, and reproducible dual-drop-off droplet digital polymerase chain reaction (PCR) assay for the simultaneous detection of ten hotspots of PIK3CA mutations in plasma cell-free (cf) DNA. We first evaluated the analytical specificity, sensitivity, limit of blank, repeatability, and reproducibility of the assay, which simultaneously detects seven mutations in exon9 and three in exon20. We further applied this assay in 11 gDNA and 18 plasma cfDNA samples from healthy donors and 35 plasma cfDNA samples from metastatic breast cancer patients. The assay is highly sensitive, specific, and applicable for clinical samples containing at least 1-5% mutant DNA. We detected PIK3CA mutations in 9/35(26%) plasma cfDNA samples in exon 9 and in 9/35(26%) in exon 20. Direct comparison of the developed assay with amplification refractory mutation system-based PCR (using plasma samples) and with the Food and Drug Administration-approved cobas PIK3CA mutation assay (using formalin fixed paraffin embedded samples) showed high concordance of our developed assay with the cobas PIK3CA assay. The developed assay is cost-effective and can reliably and simultaneously detect ten hotspot PIK3CA mutations in plasma cfDNA. The clinical performance of the assay will be further evaluated in liquid biopsy samples.

Detection and Molecular Characterization of Circulating Tumour Cells: Challenges for the Clinical Setting.

Over the last decade, liquid biopsy has gained much attention as a powerful tool in personalized medicine since it enables monitoring cancer evolution and follow-up of cancer patients in real time. Through minimally invasive procedures, liquid biopsy provides important information through the analysis of circulating tumour cells (CTCs) and circulating tumour-derived material, such as circulating tumour DNA (ctDNA), circulating miRNAs (cfmiRNAs) and extracellular vehicles (EVs). CTC analysis has already had an important impact on the prognosis, detection of minimal residual disease (MRD), treatment selection and monitoring of cancer patients. Numerous clinical trials nowadays include a liquid biopsy arm. CTC analysis is now an exponentially expanding field in almost all types of solid cancers. Functional studies, mainly based on CTC-derived cell-lines and CTC-derived explants (CDx), provide important insights into the metastatic process. The purpose of this review is to summarize the latest findings on the clinical significance of CTCs for the management of cancer patients, covering the last four years. This review focuses on providing a comprehensive overview of CTC analysis in breast, prostate and non-small-cell lung cancer. The unique potential of CTC single-cell analysis for understanding metastasis biology, and the importance of quality control and standardization of methodologies used in this field, is also discussed.

Highly Sensitive Detection Method of CXCR4 Tumor Hotspot Mutations by Drop-Off Droplet Digital PCR in Patients with IgM Monoclonal Gammopathies.

CXCR4 mutations impact disease presentation and treatment outcomes in Waldenström macroglobulinemia. Current techniques used for CXCR4 mutation detection have a number of limitations. The aim of the present study was to develop and analytically validate a novel droplet digital PCR (ddPCR) assay for the simultaneous detection of five of the most common CXCR4 mutations in bone marrow (BM). In silico novel primers and probes designed for simultaneous detection of five hotspot mutations of CXCR4 were first performed. Experimental conditions were optimized, and the assay was analytically validated. The developed assay was further applied in 95 BM samples from patients with IgM gammopathy, 7 BM samples from patients with non-IgM gammopathy and 12 PBMCs from healthy donors, whereas a direct comparison study of Sanger sequencing and allele-specific PCR was performed by using 95 and 39 identical patient tumor DNA samples, respectively. The drop-off ddPCR assay is a robust, cost-effective, highly sensitive, and highly specific screening tool for CXCR4 mutations. Of 95 patients with IgM gammopathy samples, 27 had at least one CXCR4 mutation in their BM samples. With Sanger sequencing, 12 of the 95 samples tested positive, whereas the direct comparison of the developed assay with allele-specific PCR revealed substantial agreement. The clinical performance of the developed assay will be prospectively evaluated in a large number of patients, and the applicability of this assay will be further evaluated.

Comprehensive liquid biopsy analysis as a tool for the early detection of minimal residual disease in breast cancer.

Liquid biopsy (LB) provides a unique minimally invasive tool to follow-up cancer patients over time, to detect minimal residual disease (MRD), to study metastasis-biology and mechanisms of therapy-resistance. Molecular characterization of CTCs offers additionally the potential to understand resistance to therapy and implement individualized targeted treatments which can be modified during the disease evolution and follow-up period of a patient. In this study, we present a long-term follow-up of operable breast cancer patients based on a comprehensive liquid biopsy analysis. We performed a comprehensive liquid biopsy analysis in peripheral blood of 13 patients with early-stage operable breast cancer at several time points for a period of ten years, consisting of: (a) CTC enumeration using the CellSearch system, (b) phenotypic analysis of CTCs using Immunofluorescence, (c) gene expression analysis, in EpCAM(+) CTCs for CK-19, CD24,CD44, ALDH1, and TWIST1, (d) analysis of PIK3CA and ESR1 mutations in EpCAM(+) CTCs and corresponding plasma ctDNA and (e) DNA methylation of ESR1 in CTCs. 10/13 (77%) patients were found negative for LB markers in PB during the whole follow-up period, and these patients did not relapse during the follow-up. However, 3/13(18%) patients that were positive for at least one LB marker relapsed within the follow-up period. The molecular characteristics of CTCs were highly different even for the same patient at different time points, and always increased before the clinical relapse. Our results indicate that liquid biopsy can reveal the presence of MRD at least 4 years before the appearance of clinically detectable metastatic disease demonstrating that a comprehensive liquid biopsy analysis provides highly important information for the therapeutic management of breast cancer patients.

Pre- and peri-operative characteristics, complications and outcomes of patients with biochemically silent pheochromocytomas; a case series.

PURPOSE: Pheochromocytomas are rare tumors and biochemically silent ones with normal catecholamine levels are even rarer. Up to date, biochemically inactive pheochromocytomas are poorly investigated. We aimed to systematically assess the pre- and peri-operative characteristics and the outcomes of patients with these tumors who had been treated and followed-up in 2 tertiary centers. METHODS: Clinical, laboratory and imaging data, treatment outcomes and follow-up of biochemically silent pheochromocytoma patients were recorded. RESULTS: Ten patients (5 men) [median age at diagnosis 52.5 years (24-72)] were included. Adrenal masses were incidentally discovered in all patients except from one who presented with pheochromocytoma-related manifestations. Twenty-four-hour urine metanephrine and normetanephrine levels were in the low-normal, normal and high-normal range in 4, 4 and 2 patients and in 1, 6 and 3 patients, respectively. Tumors were unilateral [median size 46 mm (17-125)] and high density on pre-contrast CT imaging or high signal intensity on T2-weighted MRI scans were found in all cases. Pre-operatively, 5 patients were treated with phenoxybenzamine [median total daily dose 70 mg (20-100)]. Intra-operatively, 4 patients developed hypertension requiring vasodilator administration and 8 developed hypotension; vasoconstrictors were required in 5 cases. One patient, not pre-operatively treated with phenoxybenzamine, developed Takotsubo cardiomyopathy. During a median 24-month (12-88) follow-up period, one patient had disease progression. CONCLUSIONS: The majority (90%) of patients with biochemically silent pheochromocytomas developed hemodynamic instability during adrenal surgery. In patients with biochemically silent adrenal lesions and a high suspicion index for pheochromocytoma based on tumor imaging characteristics, pre-operative alpha-blockade treatment may be advisable.

Detection of ESR1 Mutations in Primary Tumors and Plasma Cell-Free DNA in High-Grade Serous Ovarian Carcinoma Patients.

ESR1 mutations have been recently associated with resistance to endocrine therapy in metastatic breast cancer and their detection has led to the development and current evaluation of novel, highly promising therapeutic strategies. In ovarian cancer there have been just a few reports on the presence of ESR1 mutations. The aim of our study was to evaluate the frequency and the clinical relevance of ESR1 mutations in high-grade serous ovarian cancer (HGSOC). Drop-off droplet digital PCR (ddPCR) was first used to screen for ESR1 mutations in 60 primary tumors (FFPEs) from HGSOC patients and in 80 plasma cell-free DNA (cfDNA) samples from advanced and metastatic ovarian cancer patients. We further used our recently developed ESR1-NAPA assay to identify individual ESR1 mutations in drop-off ddPCR-positive samples. We report for the first time the presence of ESR1 mutations in 15% of FFPEs and in 13.8% of plasma cfDNA samples from advanced and metastatic ovarian cancer patients. To define the clinical significance of this finding, our results should be further validated in a large and well-defined cohort of ovarian cancer patients.

Determination of MYD88L265P mutation fraction in IgM monoclonal gammopathies.

We describe a novel method for the detection of MYD88L265P mutation using a competitive allele-specific polymerase chain reaction (Cast-PCR) assay. This assay has a sensitivity of 1 × 10-3, is applicable in reactions containing very low amounts of DNA (as low as 20 pg), and allowed the detection of MYD88L265P somatic mutation in both tumor-derived DNA (tDNA) and cell-free DNA (cfDNA). In addition, using the Cast-PCR assay, we were able to determine the mutation allele fraction (MAF) in each tested sample. We then analyzed baseline tDNA and cfDNA samples from 163 patients (53 with immunoglobulin M monoclonal gammopathy of undetermined significance and 110 with Waldenström's macroglobulinemia [WM], of whom 54 were asymptomatic and 56 were symptomatic) and also in sequential samples of 37 patients. MAF in both cfDNA and tDNA was higher among patients with symptomatic compared with asymptomatic WM and in those with asymptomatic WM compared with those with immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance. In addition, the evaluation of sequential samples showed that MAF decreased after treatment, whereas it increased in patients who relapsed or progressed to symptomatic WM. Thus, Cast-PCR is a highly sensitive, cost-effective diagnostic tool for MYD88L265P detection, applicable in both tDNA and cfDNA samples, that also provides a quantitative evaluation of the tumor load in patients with IgM monoclonal gammopathies.

Enhanced performance of a modified diagnostic test of primary aldosteronism in patients with adrenal adenomas.

OBJECTIVE: Primary aldosteronism (PA) is the commonest cause of endocrine hypertension ranging from 4.6 to 16.6% according to the diagnostic tests employed. The aim of this study was to compare the traditional saline infusion test (SIT) with the modified post-dexamethasone saline infusion test (DSIT) by applying both tests on the same subjects. METHODS: We studied 68 patients (72% hypertensives) with single adrenal adenoma and 55 normotensive controls with normal adrenal imaging. Serum cortisol, aldosterone, and plasma renin concentration (PRC) were measured and the aldosterone-to-renin ratio (ARR) was calculated. Using the mean ± 2 s.d. values from the controls, we defined the upper normal limits for cortisol, aldosterone, and PRC for both the SIT and DSIT. RESULTS: In the controls, the post-DSIT aldosterone levels and the ARR were approximately two-fold and three-fold lower, respectively, than the corresponding post-SIT values (all P = 0.001) leading to lower cut-offs of aldosterone suppression. Applying these cut-offs to patients with adrenal adenomas, the prevalence of PA was 13.2% following the SIT and 29.4% following the DSIT, respectively. In addition, 54.5% of patients with PA had concomitant autonomous cortisol secretion (ACS). Targeted treatment of PA resulted in resolution of hypertension and restoration of normal secretory aldosterone dynamics. CONCLUSIONS: The DSIT improves the diagnostic accuracy of PA, allowing for the detection of milder forms of PA in patients with adrenal adenomas. This is of particular importance as such patients may be at an increased risk of developing cardiovascular and renal morbidity that could be enhanced in the presence of concomitant ACS.

Prevalence of Primary Aldosteronism Across the Stages of Hypertension Based on a New Combined Overnight Test.

Primary aldosteronism (PA) is the most common endocrine cause of arterial hypertension. Despite the increasing incidence of hypertension worldwide, the true prevalence of PA in hypertension was only recently recognized. The objective of the work was to estimate the prevalence of PA in patients at different stages of hypertension based on a newly developed screening-diagnostic overnight test. This is a prospective study with hypertensive patients (n=265) at stage I (n=100), II (n=88), and III (n=77) of hypertension. A group of 103 patients with essential hypertension without PA was used as controls. PA diagnosis was based on a combined screening-diagnostic overnight test, the Dexamethasone-Captopril-Valsartan Test (DCVT) that evaluates aldosterone secretion after pharmaceutical blockade of angiotensin-II and adrenocorticotropic hormone. DCVT was performed in all participants independently of the basal aldosterone to renin ratio (ARR). The calculated upper normal limits for post-DCVT aldosterone levels [3 ng/dl (85 pmol/l)] and post-DCVT ARR [0.32 ng/dl/µU/ml (9 pmol/IU)] from controls, were applied together to establish PA diagnosis. Using these criteria PA was confirmed in 80 of 265 (30%) hypertensives. The prevalence of PA was: 21% (21/100) in stage I, 33% (29/88) in stage II, and 39% (30/77) in stage III. Serum K+ levels were negatively correlated and urinary K+ was positively correlated in PA patients with post-DCVT ARR (r=-0.349, p <0.01, and r=0.27, p <0.05 respectively). In conclusion, DCVT revealed that PA is a highly prevalent cause of hypertension. DCVT could be employed as a diagnostic tool in all subjects with arterial hypertension of unknown cause.

The Effect of Genomic DNA Contamination on the Detection of Circulating Long Non-Coding RNAs: The Paradigm of MALAT1.

The presence of contaminating gDNA in RNA preparations is a frequent cause of false positives in RT-PCR-based analysis. However, in some cases, this cannot be avoided, especially when there are no exons-intron junctions in the lncRNA sequences. Due to the lack of exons in few of long noncoding RNAs (lncRNAs) and the lack of DNAse treatment step in most studies reported so far, serious questions are raised about the specificity of lncRNA detection and the potential of reporting false-positive results. We hypothesized that minute amounts of gDNA usually co-extracted with RNA could give false-positive signals since primers would specifically bind to gDNA due to the lack of junction. In the current study, we evaluated the effect of gDNA and other forms of DNA like extrachromosomal circular DNAs (eccDNAs) contamination and the importance of including a DNAse treatment step on lncRNAsexpression.As a model, we have chosen as one of the most widely studied lncRNAs in cancer namely MALAT1, which lacks exons. When we tested this hypothesis in plasma and primary tissue samples from NSCLC patients, our findings clearly indicated that results on MALAT1 expression are highly affected by the presence of DNA contamination and that the DNAse treatment step is absolutely necessary to avoid false positive results.

Generation of Non-Small Cell Lung Cancer Patient-Derived Xenografts to Study Intratumor Heterogeneity.

Recent advances in sequencing technologies have allowed the in-depth molecular study of tumors, even at the single cell level. Sequencing efforts have uncovered a previously unappreciated heterogeneity among tumor cells, which has been postulated to be the driving force of tumor evolution and to facilitate recurrence, metastasis, and drug resistance. In the current study, focused on early-stage operable non-small cell lung cancer, we used tumor growth in patient-derived xenograft (PDX) models in mice as a fast-forward tumor evolution process to investigate the molecular characteristics of tumor cells that grow in mice, as well as the parameters that affect the grafting efficiency. We found that squamous cell carcinomas grafted significantly more efficiently compared with adenocarcinomas. Advanced stage, patient age and primary tumor size were positively correlated with grafting. Additionally, we isolated and characterized circulating tumor cells (CTC) from patients' peripheral blood and found that the presence of CTCs expressing epithelial-to-mesenchymal (EMT) markers correlated with the grafting potential. Interestingly, exome sequencing of the PDX tumor identified genetic alterations in DNA repair and genome integrity genes that were under-represented in the human primary counterpart. In conclusion, through the generation of a PDX biobank of NSCLC, we identified the clinical and molecular properties of tumors that affected growth in mice.

Evaluation of Monocarboxylate Transporter 4 (MCT4) Expression and Its Prognostic Significance in Circulating Tumor Cells From Patients With Early Stage Non-Small-Cell Lung Cancer.

Purpose: Monocarboxylate transporter 4 (MCT4) can influence the amount of lactate in the tumor microenvironment and further control cancer cell proliferation, migration, and angiogenesis. We investigated for the first time the expression of MCT4 in circulating tumor cells (CTCs) derived from early stage Non-Small Cell Lung Cancer patients (NSCLC) and whether this is associated with clinical outcome. Experimental Design: A highly sensitive RT-qPCR assay for quantification of MCT4 transcripts was developed and validated and applied to study MCT4 expression in CTC isolated through the Parsortix size-dependent microfluidic device from 53 and 9 peripheral blood (PB) samples of NSCLC patients at baseline (pre-surgery) and at relapse, respectively, as well as the "background noise" was evaluated using peripheral blood samples from 10 healthy donors (HD) in exactly the same way as patients. Results: MCT4 was differentially expressed between HD and NSCLC patients. Overexpression of MCT4 was detected in 14/53 (26.4%) and 3/9 (33.3%) patients at baseline and at progression disease (PD), respectively. The expression levels of MCT4 was found to increase in CTCs at the time of relapse. Kaplan-Meier analysis showed that the overexpression of MCT4 was significantly (P = 0.045) associated with progression-free survival (median: 12.5 months, range 5-31 months). Conclusion: MCT4 overexpression was observed at a high frequency in CTCs from early NSCLC patients supporting its role in metastatic process. MCT4 investigated as clinically relevant tumor biomarker characterizing tumor aggressiveness and its potential value as target for cancer therapy. We are totally convinced that MCT4 overexpression in CTCs merits further evaluation as a non-invasive circulating tumor biomarker in a large and well-defined cohort of patients with NSCLC.

Unravelling the Genetic Basis of Primary Aldosteronism.

Primary aldosteronism (PA), a condition characterized by autonomous aldosterone hypersecretion, constitutes the most common cause of secondary hypertension. Over the last decade, major breakthroughs have been made in the field of genetics underpinning PA. The advent and wide application of Next Generation Sequencing (NGS) technology led to the identification of several somatic and germline mutations associated with sporadic and familial forms of PA. Somatic mutations in ion-channel genes that participate in aldosterone biosynthesis, including KCNJ5, CACNA1D, ATP1A1, and ATP2B3, have been implicated in the development of aldosterone-producing adenomas (APAs). On the other hand, germline variants in CLCN2, KCNJ5, CACNA1H, and CACNA1D genes have been implicated in the pathogenesis of the familial forms of PA, FH-II, FH-III, and F-IV, as well as PA associated with seizures and neurological abnormalities. However, recent studies have shown that the prevalence of PA is higher than previously thought, indicating the need for an improvement of our diagnostic tools. Further research is required to recognize mild forms of PA and to investigate the underlying molecular mechanisms.

Surgical treatment outcome of primary aldosteronism assessed using new modified diagnostic tests.

PURPOSE: Primary aldosteronism (PA) is the most frequent type of endocrine hypertension. In our previous studies, we introduced two modified diagnostic tests for PA, the post-dexamethasone saline infusion test (DSIT) and the overnight dexamethasone, captopril, and valsartan test (DCVT). In this study, we aimed to validate both tests in respect to the biochemical and clinical response of a cohort of hypertensive patients in pre- and post-surgical setting. METHODS: We retrospectively studied 41 hypertensive patients (16 males), with a median (IQR, range) age of 50 (16, 35-74) years and positive histology for adrenal adenoma. Preoperatively, all patients had a single adenoma on CT and a diagnosis of PA with either DSIT or DCVT. The defined daily dose (DDD) of hypertensive drugs was assessed pre- and postoperatively. DSIT or DCVT and basal ARR were reassessed postoperatively. RESULTS: Two of the 41 patients failed to suppress aldosterone post-surgery, leading to a post-adrenalectomy biochemical cure rate of 95%, while blood pressure was improved in 36 patients, leading to a clinical cure rate of 88% as assessed by the DDD methodology. CONCLUSIONS: The present study was a proof-of-concept process to validate two modified diagnostic tests for PA in clinical practice. These tests, used to diagnose a group of patients with PA, successfully assessed their biochemical cure post-adrenalectomy at rates similar to those reported in the literature.

ESR1 NAPA Assay: Development and Analytical Validation of a Highly Sensitive and Specific Blood-Based Assay for the Detection of ESR1 Mutations in Liquid Biopsies.

A considerable number of estrogen receptor-positive breast cancer (ER+ BrCa) patients develop resistance to endocrine treatment. One of the most important resistance mechanisms is the presence of ESR1 mutations. We developed and analytically validated a highly sensitive and specific NaME-PrO-assisted ARMS (NAPA) assay for the detection of four ESR1 mutations (Y537S, Y537C, Y537N and D538G) in circulating tumour cells (CTCs) and paired plasma circulating tumour DNA (ctDNA) in patients with ER+ BrCa. The analytical specificity, analytical sensitivity and reproducibility of the assay were validated using synthetic oligos standards. We further applied the developed ESR1 NAPA assay in 13 ER+ BrCa primary tumour tissues, 13 non-cancerous breast tissues (mammoplasties) and 64 liquid biopsy samples: 32 EpCAM-positive cell fractions and 32 paired plasma ctDNA samples obtained at different time points from 8 ER+ metastatic breast cancer patients, during a 5-year follow-up period. Peripheral blood from 11 healthy donors (HD) was used as a control. The developed assay is highly sensitive (a detection of mutation-allelic-frequency (MAF) of 0.5% for D538G and 0.1% for Y537S, Y537C, Y537N), and highly specific (0/13 mammoplasties and 0/11 HD for all mutations). In the plasma ctDNA, ESR1 mutations were not identified at the baseline, whereas the D538G mutation was detected in five sequential ctDNA samples during the follow-up period in the same patient. In the EpCAM-isolated cell fractions, only the Y537C mutation was detected in one patient sample at the baseline. A direct comparison of the ESR1 NAPA assay with the drop-off ddPCR using 32 identical plasma ctDNA samples gave a concordance of 90.6%. We present a low cost, highly specific, sensitive and robust assay for blood-based ESR1 profiling. The clinical performance of the ESR1 NAPA assay will be prospectively evaluated in a large number of well-characterized patient cohorts.

Circulating miRNAs as Tumor Biomarkers: A Preface to the Special Issue.

Nowadays, therapeutic strategies in cancer are subsequently defined according to the molecular profile of the tissue [...].

Effectiveness of intramuscular ergocalciferol treatment in a patient with osteomalacia and insufficiency fractures due to severe vitamin D deficiency after bariatric surgery.

Vitamin D (vitD) deficiency and bone loss may occur after bariatric surgery and hence, supplementation with high oral doses of vitD may be required. Alternatively, intramuscular depot ergocalciferol, which slowly releases vitD and bypasses the gastrointestinal tract, could be administrated. We present a case of severe vitD deficiency-osteomalacia after gastric bypass operation for morbid obesity, treated with ergocalciferol intramuscularly. A 45-year-old woman was presented with hip pain and muscle weakness, which led ultimately to immobilization in a wheelchair. Fifteen years ago, she underwent roux-en-Y gastric by-pass for morbid obesity. Occasionally, she was treated with multivitamin supplements. On admission, iron deficiency anaemia, vitD deficiency (25OHD: 3.7 ng/ml) and secondary hyperparathyroidism were revealed. Bone turnover markers (BTM) were elevated. Radiological evaluation demonstrated insufficiency fractures on the pubic and left femur and reduced BMD. Osteomalacia due to vitD deficiency and calcium malabsorption were diagnosed. Calcium citrate 500 mg qid and intramuscular ergocalciferol 600,000 IU every 20 days were initiated. One month later, musculoskeletal pain and weakness were resolved and the patient was mobilized. Few months later, vitD, BTM and BMD showed substantial improvement. Intramuscular ergocalciferol administration can improve the clinical and biochemical status and thus, is suggested to prevent and/or treat osteomalacia in such patients.

PIM-1 Is Overexpressed at a High Frequency in Circulating Tumor Cells from Metastatic Castration-Resistant Prostate Cancer Patients.

PIM-1 is an oncogene involved in cell cycle progression, cell growth, cell survival and therapy resistance, activated in many types of cancer, and is now considered as a very promising target for cancer therapy. We report for the first time that PIM-1 is overexpressed in circulating tumor cells (CTCs) from metastatic castration-resistant prostate cancer patients (mCRPC). We first developed and validated a highly sensitive RT-qPCR assay for quantification of PIM-1 transcripts. We further applied this assay to study PIM-1 expression in EpCAM(+) CTC fraction isolated from 64 peripheral blood samples of 50 mCRPC patients. CTC enumeration in all samples was performed using the FDA-cleared CellSearch® system. PIM-1 overexpression was detected in 24/64 (37.5%) cases, while in 20/24 (83.3%) cases that were positive for PIM-1 expression, at least one CTC/7.5 mL PB was detected in the CellSearch®. Our data indicate that PIM-1 overexpression is observed at high frequency in CTCs from mCRPC patients and this finding, in combination with androgen receptor splice variant 7 (AR-V7) expression in CTCs, suggest its potential role as a very promising target for cancer therapy. We strongly believe that PIM-1 overexpression in EpCAM(+) CTC fraction merits to be further evaluated and validated as a non-invasive circulating tumor biomarker in a large and well-defined patient cohort with mCRPC.

Direct comparison study between droplet digital PCR and a combination of allele-specific PCR, asymmetric rapid PCR and melting curve analysis for the detection of BRAF V600E mutation in plasma from melanoma patients.

Background In metastatic melanoma, 40%-50% of patients harbor a BRAF V600E mutation and are thereby eligible to receive a combined BRAF/MEK inhibitor therapy. Compared to standard-of-care tissue-based genetic testing, analysis of circulating tumor DNA (ctDNA) from blood enables a comprehensive assessment of tumor mutational status in real-time and can be used for monitoring response to therapy. The aim of our study was to directly compare the performance of two highly sensitive methodologies, droplet digital PCR (ddPCR) and a combination of ARMS/asymmetric-rapid PCR/melting curve analysis, for the detection of BRAF V600E in plasma from melanoma patients. Methods Cell-free DNA (cfDNA) was isolated from 120 plasma samples of stage I to IV melanoma patients. Identical plasma-cfDNA samples were subjected to BRAF V600E mutational analysis using in parallel, ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis. Results BRAF V600E mutation was detected in 9/117 (7.7%) ctDNA samples by ddPCR and in 22/117 (18.8%) ctDNA samples by the combination of ARMS/asymmetric- rapid PCR/melting curve analysis. The concordance between these two methodologies was 85.5% (100/117). The comparison of plasma-ctDNA analysis using ddPCR and tissue testing revealed an overall agreement of 79.4% (27/34), while the corresponding agreement using the combination of ARMS/asymmetric-rapid PCR/melting curve analysis was 73.5% (25/34). Moreover, comparing the detection of BRAF-mutant ctDNA with the clinics, overall agreement of 87.2% (48/55) for ddPCR and 79.2% (42/53) was demonstrated. Remarkably, the duration of sample storage was negatively correlated with correctness of genotyping results highlighting the importance of pre-analytical factors. Conclusions Our direct comparison study has shown a high level of concordance between ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis for the detection of BRAF V600E mutations in plasma.