Eleonora Rofi

The Detection of Androgen Receptor Splice Variant 7 in Plasma-derived Exosomal RNA Strongly Predicts Resistance to Hormonal Therapy in Metastatic Prostate Cancer Patients.

BACKGROUND The androgen receptor splice variant 7 (AR-V7) is associated with resistance to hormonal therapy in castration-resistant prostate cancer (CRPC). Due to limitations of the methods available for AR-V7 analysis, the identification of a reliable detection method may facilitate the use of this biomarker in clinical practice. OBJECTIVE To confirm AR-V7 as a predictor of resistance to hormonal therapy and develop a new approach to assess AR-V7 by highly sensitive digital droplet polymerase chain reaction (ddPCR) in plasma-derived exosomal RNA. DESIGN, SETTING, AND PARTICIPANTS Plasma samples were collected from 36 CRPC patients before they began second-line hormonal treatment. Exosomes were isolated and RNA extracted for analysis of AR-V7 by ddPCR. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The absolute target gene concentration as copies per milliliter (copies/ml) was determined by ddPCR. Statistical analyses were performed with SPSS software (IBM Corp., Armonk, NY, USA). RESULTS AND LIMITATIONS A total of 26 patients received abiraterone and 10 enzalutamide; 39% of patients were found to be AR-V7 positive (AR-V7+). Median progression-free survival was significantly longer in AR-V7 negative (AR-V7-) versus AR-V7+ patients (20 vs 3 mo; p<0.001). Overall survival was significantly shorter in AR-V7+ participants at baseline compared with AR-V7- participants (8 mo vs not reached; p<0.001). CONCLUSIONS This study demonstrates that plasma-derived exosomal RNA is a reliable source of AR-V7 that can be detected sensitively by ddPCR assay. We also showed that resistance to hormonal therapy may be predicted by AR-V7, making it a clinically relevant biomarker. PATIENT SUMMARY We report a first study on a method for androgen receptor splice variant 7 (AR-V7) detection in RNA extracted from cancer cell vesicles released in blood. Results confirmed the role of AR-V7 as a predictive biomarker of resistance to hormonal therapy. Our assay showed that vesicles are a reliable source of AR-V7 RNA and that the method is fast, highly sensitive, and affordable.

Pharmacogenetics of CYP2D6 and tamoxifen therapy: Light at the end of the tunnel?

The clinical usefulness of assessing the enzymatic activity of CYPD6 in patients taking tamoxifen had been longly debated. In favour of preemptive evaluation of phenotypic profile of patients is the strong pharmacologic rationale, being that the formation of endoxifen, the major and clinically most important metabolite of tamoxifen, is largely dependent on the activity of CYP2D6. This enzyme is highly polymorphic for which the activity is largely depending on genetics, but that can also be inhibited by a number of drugs, i.e. antidepressants, which are frequently used in patients with cancer. Unfortunately, the clinical trials that have been published in the last years are contradicting each other on the association between CYP2D6 and significant clinical endpoints, and for this reason CYP2D6 genotyping is at present not generally recommended. Despite this, the CYP2D6 genotyping test for tamoxifen is available in many laboratories and it may still be an appropriate test to use it in specific cases.

Detection of ALK and KRAS Mutations in Circulating Tumor DNA of Patients With Advanced ALK-Positive NSCLC With Disease Progression During Crizotinib Treatment

Background In patients with anaplastic lymphoma kinase (ALK)‐positive non–small‐cell lung cancer (NSCLC), disease progression occurs after a median of 9 to 10 months of crizotinib treatment. Several mechanisms of resistance have been identified and include ALK mutations and amplification or the activation of bypassing signaling pathways. Rebiopsy in NSCLC patients represents a critical issue and the analysis of circulating cell‐free DNA (cfDNA) has a promising role for the identification of resistance mechanisms. Patients and Methods Twenty patients with advanced ALK‐positive NSCLC were enrolled after disease progression during crizotinib treatment; cfDNA was analyzed using digital droplet polymerase chain reaction (BioRad, Hercules, CA) for ALK (p.L1196M, p.G1269A, and p.F1174L) and Kirsten rat sarcoma (KRAS) (codons 12 and 13) mutations. Results ALK secondary mutations (p.L1196M, p.G1269A, and p.F1174L) were identified in 5 patients; 1 patient had 2 ALK mutations (p.L1196M and p.G1269A). Overall, 10 patients presented KRAS mutations (7 p.G12D, 2 p.G12V, and 1 p.G12C mutations, respectively). In 3 patients KRAS mutations were associated with ALK mutations. cfDNA was monitored during the treatment with second‐generation ALK inhibitors and the amount of ALK as well as KRAS mutations decreased along with tumor regression. Conclusion ALK and KRAS mutations are associated with acquired resistance to crizotinib in ALK‐positive NSCLC. In particular, ALK acquired mutations can be detected in plasma and could represent a promising tumor marker for response monitoring. Micro‐Abstract We investigated mechanisms of acquired resistance in a group of anaplastic lymphoma kinase (ALK) translocated lung cancer patients whose disease progressed during crizotinib treatment using plasmatic circulating DNA extracted from blood samples. We identified 5 patients who presented ALK plasmatic mutations and 10 patients with Kirsten rat sarcoma (KRAS) gene mutations, both associated with crizotinib resistance. We showed that plasmatic decrease of mutation levels was associated with radiological response confirming that resistance ALK and KRAS mutations are detectable and their monitoring could serve as response parameters.

Pharmacogenetics and Metabolism from Science to Implementation in Clinical Practice: The Example of Dihydropyrimidine Dehydrogenase

BACKGROUND Fluoropyrimidines are widely used in the treatment of solid tumors and remain the backbone of many combination chemotherapy regimens. Despite their clinical benefit, they are associated with frequent gastrointestinal and hematological toxicities, which often lead to treatment discontinuation. Fluoropyrimidines undergo complex anabolic and catabolic biotransformation. Enzymes involved in this pathway include dihydropyrimidine dehydrogenase (DPD), which breaks down 5-FU and its prodrugs. Candidate gene approaches have demonstrated associations between 5-FU treatment outcomes and germline polymorphisms in DPD. The aim of this review is to report and discuss the latest results on fluoropyrimidine pharmacogenetics. METHODS Literature from PubMed databases and bibliography from retrieved publications have been analyzed according to terms such DPD, DPYD, fluoropyrimdines, polymorphisms, toxicity, pharmacogenetics. RESULTS To date, many sequence variations have been identified within DPYD gene, although the majority of these have no functional consequences on enzymatic activity. Nowadays, there is a general agreement on the clinical significance of the importance of DPD deficiency in patients who suffer from severe, life-threatening drug toxicity although preemptive testing is not applied to all patients. CONCLUSION Considering the published literature, clinicians are strongly encouraged to consider testing for DPD poor metabolizer variants as a rational pre-treatment screening for patients candidate to a fluoropyrimidine-based regimens, in order to prevent toxicities and personalise treatments.

Primary resistance to osimertinib due to SCLC transformation: Issue of T790M determination on liquid re-biopsy

OBJECTIVES EGFR T790M mutation is the most common mechanism of resistance to first-/second-generation EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) and could be overcome by third-generation EGFR-TKIs, such as osimertinib. Liquid biopsy, a non-invasive technique used to test the presence of the resistant mutation, may help avoiding tissue re-biopsy. However, analysing only circulating-free DNA, information about other less frequent and coexisting resistance mechanisms may remain unrevealed. MATERIALS AND METHODS All patients reported in this series participated in the ASTRIS trial, a real world treatment study testing the efficacy of osimertinib (80mg os die) in advanced T790M-positive NSCLC progressed to prior EGFR-TKI. Patients were considered eligible to osimertinib if T790M positive on tissue or plasma samples. In our patients, EGFR molecular testing on blood sample was conducted with digital droplet PCR (ddPCR). RESULTS We report our experience of five patients treated with osimertinib after T790M detection on liquid biopsy that presented a disease progression at first tumor assessment mediated by SCLC transformation, as evidenced at tissue re-biopsies. All patients showed low ratio T790M/activating mutation in the blood before osimertinib (lower than 0.03). For three patients, EGFR mutational analysis was T790M-negative when re-assessed by using a less sensitive method (therascreen®) on the same liquid biopsy sample analysed by ddPCR before osimertinib therapy. CONCLUSION Although liquid biopsy is a relevant tool to diagnose T790M presence in NSCLC patients resistant to EGFR-TKI, in case of a low ratio T790M/activating mutation, tissue biopsy should be considered to exclude the presence of SCLC transformation and/or other concomitant resistance mechanisms.

Early changes in plasma DNA levels of mutant KRAS as a sensitive marker of response to chemotherapy in pancreatic cancer

Pancreatic cancer (PDAC) is still lacking of reliable markers to monitor tumor response. CA 19-9 is the only biomarker approved, despite it has several limitations in sensitivity and specificity. Since mutations of KRAS occur in more than 90% of tumors, its detection in circulating free tumor DNA (cftDNA) could represent a biomarker to monitor chemotherapy response. Twenty-seven advanced PDAC patients given first-line 5-fluorouracil, irinotecan and oxaliplatin or gemcitabine and nab-paclitaxel were enrolled. Three ml of plasma were collected: 1) before starting chemotherapy (baseline); 2) at day 15 of treatment; and 3) at each clinical follow-up. cftDNA was extracted and analysed for KRAS mutations (mutKRAS) by digital droplet PCR. Nineteen patients displayed a mutKRAS in baseline plasma samples. There was a statistically significant difference in progression-free survival (PFS) and overall survival (OS) in patients with increase vs. stability/reduction of cftDNA in the sample collected at day 15 (median PFS 2.5 vs 7.5 months, p = 0.03; median OS 6.5 vs 11.5 months, p = 0.009). The results of this study demonstrate that cftDNA mutKRAS changes are associated with tumor response to chemotherapy and support the evidence that mutKRAS in plasma may be used as a new marker for monitoring treatment outcome and disease progression in PDAC.

Concise Review: Chronic Myeloid Leukemia: Stem Cell Niche and Response to Pharmacologic Treatment

Nowadays, more than 90% of patients affected by chronic myeloid leukemia (CML) survive with a good quality of life, thanks to the clinical efficacy of tyrosine kinase inhibitors (TKIs). Nevertheless, point mutations of the ABL1 pocket occurring during treatment may reduce binding of TKIs, being responsible of about 20% of cases of resistance among CML patients. In addition, the presence of leukemic stem cells (LSCs) represents the most important event in leukemia progression related to TKI resistance. LSCs express stem cell markers, including active efflux pumps and genetic and epigenetic alterations together with deregulated cell signaling pathways involved in self‐renewal, such as Wnt/β‐catenin, Notch, and Hedgehog. Moreover, the interaction with the bone marrow microenvironment, also known as hematopoietic niche, may influence the phenotype of surrounding cells, which evade mechanisms controlling cell proliferation and are less sensitive or frankly resistant to TKIs. This Review focuses on the role of LSCs and stem cell niche in relation to response to pharmacological treatments. A literature search from PubMed database was performed until April 30, 2017, and it has been analyzed according to keywords such as chronic myeloid leukemia, stem cell, leukemic stem cells, hematopoietic niche, tyrosine kinase inhibitors, and drug resistance. Stem Cells Translational Medicine 2018;7:305–314

EGFR-TKIs in non-small-cell lung cancer: focus on clinical pharmacology and mechanisms of resistance.

The clinical introduction of EGFR-TKIs within the oncologic armamentarium has changed the therapeutic landscape of non-small-cell lung cancer (NSCLC) creating widespread expectations both in patients and clinicians. However, several gaps in current understanding leave open important questions regarding the use of these drugs in clinical practice. For instance, there is uncertainty in regard to which EGFR-TKI should be given first in naive patients with EGFR-driven malignancies since different generations of drugs are available with different pharmacological profiles. Furthermore, acquired drug resistance may limit the therapeutic potential of EGFR-TKIs and the choice of the best treatment strategy after first-line treatment failure is still debated. This review article is aimed at describing the pharmacological properties of EGFR-TKIs and the current treatment options for NSCLC patients who develop acquired resistance. This information might be useful to design new rational and more effective pharmacological strategies in patients with EGFR-mutant NSCLC.

Clinical pharmacology of intravitreal anti-VEGF drugs

Clinical efficacy of intravitreal anti-VEGF drugs has been widely demonstrated in several angiogenesis-driven eye diseases including diabetic macular edema and the neovascular form of age-related macular degeneration. Pegaptanib, ranibizumab, and aflibercept have been approved for use in the eye, whereas bevacizumab is widely used by ophthalmologists to treat patients “off-label”. These drugs are active in the nanomolar to picomolar range; however, caution is required when establishing the rank order of affinity and potency due to in vitro inter-experimental variation. Despite the small doses used for eye diseases and the intravitreal route of administration may limit systemic side effects, these drugs can penetrate into blood circulation and alter systemic VEGF with unknown clinical consequences, particularly in vulnerable groups of patients. Clinical pharmacokinetics of ocular anti-VEGF agents should therefore be taken into account when choosing the right drug for the individual patient. The gaps in current understanding that leave open important questions are as follows: (i) uncertainty about which drug should be given first, (ii) how long these drugs can be used safely, and (iii) the choice of the best pharmacological strategy after first-line treatment failure. The current review article, based on the information published in peer-reviewed published papers relevant to anti-VEGF treatments and available on the PubMed database, describes in detail the clinical pharmacology of this class of drugs to provide a sound pharmacological basis for their proper use in ophthalmology clinical practice.

Patients with NSCLC may display a low ratio of p.T790M vs. activating EGFR mutations in plasma at disease progression: implications for personalised treatment

Introduction NSCLC harboring activating mutations of EGFR is highly sensitive to first-line EGFR-tyrosine kinase inhibitors (TKIs), but drug resistance depending on the EGFR mutation p.T790M will occur in about 50-60% of patients. Detailed information on the amount of p.T790M plasmatic level associated with resistance to EGFR-TKIs and guidance to treatment with p.T790M-effective TKI depending on these levels, is lacking. Methods This study enrolled p.T790M-positive patients (n=49) affected by EGFR-mutated NSCLC at progression to first-line EGFR-TKIs and, in selected cases (n=5), after second-line treatment with osimertinib. Cell-free circulating tumor DNA (cftDNA) was extracted from plasma and the quantitative analysis of EGFR ex19del, p.L858R and p.T790M was performed by digital droplet PCR. Results The mean amount of mutated alleles at progression to first-line EGFR-TKIs was 108,492 copies/ml for ex19del, 97,336 copies/ml for p.L858R, but only 8,754 copies/ml for p.T790M. There was no significant correlation between progression-free survival and the ratio of p.T790M over EGFR activating mutations. The analysis of cftDNA in 5 patients treated with osimertinib revealed a marked decrease of all EGFR mutant alleles. Conclusions The amount of p.T790M in plasma can be much lower than activating EGFR mutations. Despite this finding, osimertinib is effective in p.T790M-positive patients. These results indicate that clones driving resistance to EGFR-TKIs represent a minority among cells bearing activating EGFR-mutations. In addition, the identification of a threshold level of p.T790M is not a strict requirement for the selection of patients to be treated with osimertinib, since treatment showed a decrease in all EGFR mutated cells.