Monitoraggio dinamico delle mutazioni nel DNA tumorale circolante di pazienti affetti da neoplasia polmonare in terapia con target therapy

Tumori caratterizzati dalla stessa istologia possono avere diversa prognosi e diversa risposta al trattamento, a dimostrazione del fatto che sono malattie eterogenee e multifattoriali. Tramite selezione clonale il tumore favorisce la proliferazione delle cellule recanti le alterazioni genetiche utili la sua crescita e sviluppo, inducendo invece apoptosi in coloro che non le hanno. Attualmente, nella pratica clinica la valutazione delle alterazioni molecolari suscettibili di terapia target e le resistenze ai trattamenti farmacologici avviene tramite biopsia. Tuttavia, tale tecnica presenta degli svantaggi, in quanto non sempre è praticabile, è invasiva, può causare l'accidentale migrazione di cellule tumorali dal sito primario in circolo, la conservazione del campione può degradare il DNA da analizzare, ma soprattutto non è in grado di cogliere l’eterogeneità del tumore. A tal riguardo, un metodo alternativo alle ripetute biopsie è rappresentato dalla rilevazione del DNA tumorale circolante (ctDNA) che può essere isolato nel siero o nel plasma dei pazienti affetti da neoplasia ed i cui livelli risultano essere più alti rispetto a quelli rilevati nei soggetti sani. Il ctDNA verrebbe rilasciato in seguito a fenomeno di necrosi, apoptosi e secrezione e viene distinto dalla controparte normale per la presenza di mutazioni e alterazioni genetiche tipiche delle cellule cancerose o pre-cancerose e che non sono evidenziabili nel DNA derivante da cellule sane. Pertanto, l’analisi del ctDNA rappresenta ciò che viene definita “biopsia liquida”, ossia una metodica utile per la caratterizzazione molecolare delle cellule cancerose e per l’identificazione di aberrazioni molecolari specifiche della neoplasia, con il vantaggio, rispetto ad una biopsia classica, di una ridotta invasività. Inoltre, la biopsia liquida può risultare utile nella definizione di meccanismi di resistenza a farmaci biologici per specifici target molecolari, permettendo, durante il trattamento, il monitoraggio dell’ insorgenza di cloni selezionati dalla pressione selettiva del farmaco. Attualmente, il carcinoma polmonare rappresenta nel mondo la prima causa di morte per cancro. Numerosi studi di biologia cellulare e molecolare hanno permesso di stabilire che il processo di cancerogenesi polmonare è multifasico ed evolve in seguito all'accumulo di lesioni genetiche somatiche multiple (10-20 diverse mutazioni) e sequenziali. Dal punto di vista istologico, circa l’85% dei casi di carcinoma polmonare è del tipo non a piccole cellule (Non Small Cell Lung Cancer, NSCLC). La valutazione delle mutazioni “attivanti” che interessano il gene codificante per il recettore del fattore di crescita epiteliale (EGFR) e la traslocazione del gene che codifica per la chinasi del linfoma anaplastico (ALK), ha permesso di stratificare un sottogruppo di pazienti affetti da NSCLC, caratterizzati da un comportamento biologico peculiare della patologia e per i quali sono disponibili farmaci a targeted therapy in grado di bloccare la specifica alterazione individuata. In particolare, tali pazienti sono candidati a ricevere un trattamento con farmaci inibitori tirosin chinasici (TKIs), quali erlotinib, gefitinib e afatinib quando EGFR+ e crizotinib quando ALK+. Tuttavia, è stato osservato che nell’arco di 10-12 mesi, la neoplasia è in grado di sviluppare resistenza al trattamento farmacologico. Tale evento sarebbe da ascrivere all’insorgenza, sotto la pressione selettiva dei TKIs, di cloni caratterizzati da aberrazioni genomiche che conferiscono alle cellule neoplastiche la capacità di proliferare e sopravvivere nonostante la presenza del farmaco. Sulla base di queste evidenze, scopo del presente studio è di analizzare l’andamento nel tempo delle alterazioni molecolari attivanti e della comparsa di mutazioni secondarie responsabili di resistenza in pazienti con neoplasia polmonare in trattamento con targeted therapy, al fine di ottimizzare il trattamento antineoplastico. A tal riguardo, l’analisi del ctDNA isolato dal plasma di pazienti affetti da NSCLC verrà condotta con sistema droplet digital PCR™ QX100™, che permetterà un’analisi estremamente sensibile e precisa degli acidi nucleici, consentendo la rilevazione di alleli mutati che differiscono per un singolo nucleotide e l’analisi di espressione genica

Implications of KRAS mutations in acquired resistance to treatment in NSCLC

Rationale: KRAS is the most common and, simultaneously, the most ambiguous oncogene implicated in human cancer. Despite KRAS mutations were identified in Non Small Cell Lung Cancers (NSCLCs) more than 20 years ago, selective and specific inhibitors aimed at directly abrogating KRAS activity are not yet available. Nevertheless, many therapeutic approaches have been developed potentially useful to treat NSCLC patients mutated for KRAS and refractory to both standard chemotherapy and targeted therapies. The focus of this review will be to provide an overview of the network related to the intricate molecular KRAS pathways, stressing on preclinical and clinical studies that investigate the predictive value of KRAS mutations in NSCLC patients. Materials and Methods: A bibliographic search of the Medline database was conducted for articles published in English, with the keywords KRAS, KRAS mutations in non-small cell lung cancer, KRAS and tumorigenesis, KRAS and TKIs, KRAS and chemotherapy, KRAS and monoclonal antibody, KRAS and immunotherapy, KRAS and drugs, KRAS and drug resistance

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

Liquid biopsy to monitor genetic alterations in plasma as non-invasive pharmacogenetic-based approach to evaluate predictive biomarkers during treatment in cancer patients.

At present, in oncology the therapeutic strategies are defined according to the molecular landscape assessed with tissue biopsies and, thus, using DNA and/or RNA obtained from a fragment of the primary tumour or a single metastatic lesion. Anyway, mutational analysis in a single-site biopsy is encumbered by several limitations. A huge step forward in oncology to characterize tumor heterogeneity and plasticity at diagnosis and during the course of treatment was done with the use of a promising minimally invasive tool., i.e. via a blood draw. All cells can release in human blood their materials, including cell-free DNA (cfDNA), RNA, proteins and vesicles (such as exosomes). This approach is the expression of the overall spatial heterogeneity from the overall sites of disease and it is able to identify emerging sub-clones responsible for treatment resistance. This thesis was aimed at evaluating if a pharmacogenetic approach based on circulating predictive biomarkers (i.e. circulating cell-free tumor DNA and RNA extracted from exosomes) may be a good and useful tool to monitor treatment outcome in solid tumors. Patients affected by lung, breast, prostate, pancreatic cancer and melanoma have been enrolled and monitored during different treatment, i.e. targeted therapy, hormonal therapy, chemotherapy and immunotherapy by circulating tumor nucleic acids. Nucleic acids were analysed by digital droplet PCR for targetable drivers, for acquired mutations and gene expression selected during therapy. Circulating tumor nucleic acids were find to correlate with tumor burden, disease status and response to treatment, beeing a good candidate to monitor tumor response and support routine clinical practice

Should CYP2D6 be genotyped when treating with tamoxifen?

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Should CYP2D6 be genotyped when treating with tamoxifen? [Letter in reply]

Letter in regards to: Del Re M, Rofi E, Citi V, Fidilio L, Danesi R. Should CYP2D6 be genotyped when treating with tamoxifen? Pharmacogenomics 17(18), 1967-1969 (2016). In response to: Damkier P. Don't think twice it's all right: tamoxifen and CYP2D6 genotyping in the treatment of breast cancer patients. Pharmacogenomics 18(8), XXX (2017)

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

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