Over the last decades, the concept of precision medicine has dramatically renewed the field of medical oncology; the introduction of patient-tailored therapies has significantly improved all ...measurable outcomes. Liquid biopsy is a revolutionary technique that is opening previously unexpected perspectives. It consists of the detection and isolation of circulating tumor cells, circulating tumor DNA and exosomes, as a source of genomic and proteomic information in patients with cancer. Many technical hurdles have been resolved thanks to newly developed techniques and next-generation sequencing analyses, allowing a broad application of liquid biopsy in a wide range of settings. Initially correlated to prognosis, liquid biopsy data are now being studied for cancer diagnosis, hopefully including screenings, and most importantly for the prediction of response or resistance to given treatments. In particular, the identification of specific mutations in target genes can aid in therapeutic decisions, both in the appropriateness of treatment and in the advanced identification of secondary resistance, aiming to early diagnose disease progression. Still application is far from reality but ongoing research is leading the way to a new era in oncology. This review summarizes the main techniques and applications of liquid biopsy in cancer.
Abstract
STUDY QUESTION
Are the large cells derived from cultured DEAD box polypeptide 4 (DDX4)-positive oogonial stem cells (OSCs), isolated from the ovarian cortex of non-menopausal and menopausal ...women, oocyte-like cells?
SUMMARY ANSWER
Under appropriate culture conditions, DDX4-positive OSCs from non-menopausal and menopausal women differentiate into large haploid oocyte-like cells expressing the major oocyte markers growth differentiation factor 9 (GDF-9) and synaptonemal complex protein 3 (SYCP3) and then enter meiosis.
WHAT IS KNOWN ALREADY
The recent reports of OSCs in the ovaries of non-menopausal and menopausal women suggest that neo-oogenesis is inducible during ovarian senescence. However, several questions remain regarding the isolation of these cells, their spontaneous maturation in vitro, and the final differentiation state of the resulting putative oocytes.
STUDY DESIGN, SIZE, DURATION
DDX4-positive OSCs were obtained from 19 menopausal and 13 non-menopausal women (who underwent hysterectomy for uterine fibroma, ovarian cyst, or other benign pathologies) and cultured for up to 3 weeks. Large and small cells were individually isolated and typed for early and late differentiation markers.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Ovarian cortex fragments were processed by immuno-magnetic separation using a rabbit anti-human DDX4 antibody and the positive populations were measured by assessing both FRAGILIS and stage-specific embryonic antigen 4 (SSEA-4) expression. After 3 weeks in culture, large oocyte-like cells were individually isolated by DEPArray based on PKH26 red staining and cell size determination. GDF-9 and SYCP3 as final, and developmental pluripotency-associated protein 3 (DPPA3) as primordial, germline markers were measured by droplet digital PCR. The haploid versus diploid chromosomal content of chromosomes X and 5 was investigated using fluorescence in situ hybridization (FISH).
MAIN RESULTS AND THE ROLE OF CHANCE
SSEA-4+ and FRAGILIS+ subsets of DDX4-positive populations were present at lower mean levels in menopausal (SSEA-4+: 46.7%; FRAGILIS+: 47.5%) than in non-menopausal (SSEA-4+: 64.9%; FRAGILIS+: 64.8) women (P < 0.05). A comparison of the women's age with the ratio of DDX4-positive cells/cm3 of ovarian cortex revealed an inverse correlation with OSC number (P < 0.05). Once cultured, cells from both groups differentiated to form large (up to 80 μm) mature oocyte-like cells with typical oocyte morphology. Despite the higher numbers of these cells in cultures from non-menopausal women (37.4 versus 23.7/well; P < 0.001), the intra-culture percentages of large oocyte-like cells did not differ significantly between the two groups. Single large oocyte-like cells isolated from non-menopausal and menopausal women expressed equivalent levels of GDF-9 (e.g. 2.0 and 2.6 copies/μl RNA, respectively) and SYCP3 (e.g. 1.2 and 1.5 copies/μl RNA, respectively) mRNA. The remaining small cells isolated from the cultures expressed large amounts of DPPA3 mRNA (e.g. 5.0 and 5.1 copies/μl RNA, from menopausal and non-menopausal women, respectively), which was undetectable in the large oocyte-like cells. FISH analysis of the large and small cells using probes for chromosomes X and 5 revealed a single signal in the large cells, indicative of chromosome haploidy, whereas in the small cells two distinct signals for each chromosome indicated diploidy.
LARGE SCALE DATA
Not applicable.
LIMITATIONS, REASONS FOR CAUTION
Our study demonstrated the final differentiation of OSCs, collected from the ovarian cortex of adult women, to oocyte-like cells. However, because the rate of differentiation was low, a major role of the stem cell niche housing these OSCs cannot be ruled out.
WIDER IMPLICATIONS OF THE FINDINGS
Since the ability of OSCs to generate mature oocytes in vitro is highly variable, the viability of these cells in the ovarian cortex of non-menopausal and menopausal women may well be determined by the stem cell niche and the woman's concurrent reproductive state. Our study showed that the oocyte-like cells obtained by OSC differentiation in vitro, including those from the OSCs of menopausal women, express markers of meiosis. This model of ovarian neo-oogenesis will contribute to the development of approaches to treat female infertility.
STUDY FUNDING/COMPETING INTEREST(S)
The study was funded by Italian Association for Cancer Research (IG grant 17536), and from the Apulia Region ('Oncogenomic Project' and 'Jonico-Salentino Project'). All Authors declare no competing interests.
Breast cancer (BC) is the most common malignancy in women with a significant increasing incidence during the reproductive life. However, based on the newest anti-cancer molecular targeting drugs, ...successful treatments lead to the disease healing particularly in young patients, thus refreshing their motherhood programs. However, as effect of the BC treatment, a premature depletion of the ovarian follicle reserve occurs in more than one-third of patients resulting in permanent infertility. To prevent the cancer treatment-related infertility (CTRI), several options are today utilized. Besides the ovary suppression by gonadotropin-releasing hormone agonist (GnRHa), other procedures include either oocytes or embryos cryopreservation as well as ovarian cortex cryopreservation that are currently adopted before anti-cancer therapies. These modern techniques appear variably successful in terms of pregnancy rate though their safety concerning the hormonal stimulation to promote the folliculogenesis is still debated in relation to the potential oncogenic risk in patients bearing hormone-sensitive tumors as BC, while the ovarian cortex re-implantation often results in a low number of regenerated follicles including oocytes of unknown quality. Recent studies on ovarian stem cells (OSCs) suggest their use for future application in CTRI. In fact, OSCs from ovarian cortex have been shown to differentiate in vitro into oocyte-like cells (OLCs) and express molecular markers of mature oocytes. Once the OSC technology will be optimized and translated to clinical use, oocytes derived from these cells will be molecularly assessed before fertilization to assure their best embryo quality resulting in a safe procedure to treat CTRI in patients as young women with BC.
The existence of ovarian stem cells (OSCs) in women as well as their physiological role in post-menopausal age are disputed. However, accumulating evidence demonstrated that, besides the animal ...models including primarily mice, even in adult women putative OSCs obtained from ovarian cortex are capable to differentiate in vitro into oocyte-like cells (OLCs) expressing molecular markers typical of terminal stage of oogonial cell lineage. Recent studies describe that, similarly to mature oocytes, the OSC-derived OLCs also contain haploid karyotype. As proof of concept of their stem commitment, OSCs from mice differentiated to oocytes in vitro are suitable to be fertilized and implanted in sterilized animals resulting in embryo development. Despite enthusiasm for these data, which definitely require extended confirmation before considering potential application in humans for treatment of ovarian insufficiency, OSCs appear suitable for other clinical uses, restoring the endocrine derangements in premature ovarian failure or for fertility preservation in oncologic patients after anti-cancer treatments. In this context, the selection of viable oocytes generated from OSCs before chemotherapy protocols would overcome the potential adjunct oncogenic risk in women bearing hormone-dependent tumors who are repeatedly stimulated with high dose estrogens to induce oocyte maturation for their egg recruitment and cryopreservation.
Background Innovative therapies have improved the overall survival in melanoma, although a high number of patients still experience disease progression or recurrence. Ex-vivo culture of circulating ...tumour cells (CTCs) represents a valuable laboratory resource for in-depth characterization of rare cell populations responsible for disease progression. Methods CTCs from patients with metastatic melanoma were in-vitro established. Their stemness was demonstrated by both phenotypic and genotypic assays, as well as by functional studies. Xenograft experiments in NOD.CB17 mice injected with CTCs from a single patient were completed. Data were analysed by Student's test and results expressed as mean + or - SEM. Results CTCs share the mutational profile with primary cells, an intermediate epithelial-mesenchymal transition (EMT) phenotype and high expression of the immunosuppressive factors. A subclonal CTC population exhibited stem cell properties as high aldehyde dehydrogenase 1 activity, melanosphere-forming ability, and expression of major stemness transcription factors. Xenograft experiments confirmed the CTC ability to generate melanoma in-vivo and revealed enhanced metastatic propensity. Conclusions CTCs play a relevant role in melanoma and may actively contribute to drive the disease progression and metastasis. Thus, they are a unique potential tool for pharmacogenomic studies to guide treatment strategies in advanced disease. Keywords: Melanoma, Circulating tumor cells, Phenotypic plasticity, Stemness, Metastases; NOD.CB17 mice
Although statins are lipid-lowering drugs that block cholesterol biosynthesis, they exert immunomodulatory, anti-inflammatory, anti-angiogenic and anti-proliferative functions by reducing the ...isoprenylation of proteins involved in cell signal transduction such as Ras and RhoA. In this study, we provide evidence that several natural (lovastatin, simvastatin and pravastatin) and synthetic (cerivastatin and atorvastatin) statins exert a cytotoxic effect on human T, B and myeloma tumor cells by promoting their apoptosis. Dissimilar susceptibility to apoptosis has been detected in these lines, presumably in relation to the altered expression of proteins involved in the regulation of cellular signals. Cerivastatin promptly activated the cell death even in doxorubicin resistant cell lines such as MCC-2, whereas pravastatin, a hydrophilic compound, failed to induce any effect on either proliferation or apoptosis. The statin-induced apoptotic pathway in these cell lines was presumably regulated by altered prenylation of either Ras or RhoA, as measured by the defective membrane localization of these small GTPases. In addition the cell proliferation was rescued by both farnesylpyrophosphate (FPP) and geranyl-geranylpyrophosphate (GGPP), whereas no effect was obtained with squalene, a direct precursor of cholesterol. Statins primed apoptosis through its intrinsic pathway involving the mitochondria. In fact, we observed the reduction of mitochondrial membrane potential and the cytosolic release of the second mitochondria-derived activator of caspases (Smac/DIABLO). The apoptotic pathway was caspase-dependent since caspases 9, 3 and 8 were efficiently activated. These results support the potential use of statins in association with conventional treatment as apoptosis-triggering agents in these tumors.
The usefulness of this review is to highlight how a fertility preservation (FP) approach is currently feasible for patients diagnosed with uterine cervical cancer. To this regard, a fertility sparing ...surgery has just overcome its traditional limits, gained acceptance within the major gynecologic oncology societies thanks to the ability to identify the "ideal" candidates to this conservative treatment. On the other hand, the use of other FPs for oocyte and ovarian cortex cryopreservation is still extremely debated. In fact, the existing risk of tumor spreading during oocyte retrieval necessary for oocyte cryostorage for patients' candidates for neo-adjuvant therapy, as well as the potential hazard of cancer cell dissemination after ovarian tissue replacement in cases of non-squamous type cervical carcinomas should not be underestimated. Therefore, in consideration of the encountered limitations and the need to ensure adequate reproductive health for young uterine cervical cancer survivors, translational research regarding the FP has progressively collected innovative insights into the employment of stemness technology. In this context, the property of ovarian stem cells obtained from the ovarian cortex to generate functional oocytes in women could represent a promising therapeutic alternative to the current procedures for a novel and safer FP approach in cancer survivors.
Summary
Interleukin 17A (IL17A), a cytokine involved in allergy, inflammation and osteoclastogenesis, was investigated in multiple myeloma (MM) to assess its role in the osteoclast (OC)‐like activity ...of marrow immature dendritic cells (iDCs). Comparing nine MM patients with control subjects affected by monoclonal gammopathy of undetermined significance, we found high IL17A expression in the marrow plasma of MM patients in parallel with its deposits within the stromal matrix. Increased expression of the IL17A receptor (IL17RA) was also found in primary myeloma iDCs, which underwent OC‐like transdifferentiation after IL17A stimulation. To assess the role of IL17A, we measured the activity of the IL17/IL17RA pathway in IL17A‐transdifferentiated iDCs and the expression of functional OC genes by Western blotting and real‐time polymerase chain reaction. These cells showed increased RNA transcription of genes enrolled in the maturation of OCs, while NFATC1 and FOS were induced by IL17A, independently of NFKB1 phosphorylation. Moreover, the concurrent phosphorylation of the Lip isoform of CEBPB and the down‐regulation of MAFB supported the activation of IL17RA pathway in OC‐like transdifferentiated iDCs that was apparently unrelated to TNFRSF11A signalling. These data emphasize the involvement of iDCs in MM hyperactive osteoclastogenesis and suggest that their bone resorption activity is also regulated, at least in vitro, by IL17RA.
The clinical management of metastatic urothelial carcinoma (mUC) is undergoing a major paradigm shift; the integration of immune checkpoint inhibitors (ICIs) and antibody-drug conjugates (ADCs) into ...the mUC therapeutic strategy has succeeded in improving platinum-based chemotherapy outcomes. Given the expanding therapeutic armamentarium, it is crucial to identify efficacy-predictive biomarkers that can guide an individual patient's therapeutic strategy. We reviewed the literature data on mUC genomic alterations of clinical interest, discussing their prognostic and predictive role. In particular, we explored the role of the fibroblast growth factor receptor (FGFR) family, epidermal growth factor receptor 2 (HER2), mechanistic target of rapamycin (mTOR) axis, DNA repair genes, and microsatellite instability. Currently, based on the available clinical data, FGFR inhibitors and HER2-directed ADCs are effective therapeutic options for later lines of biomarker-driven mUC. However, emerging genomic data highlight the opportunity for earlier use and/or combination with other drugs of both FGFR inhibitors and HER2-directed ADCs and also reveal additional potential drug targets that could change mUC management.
Background:
Circulating tumor cells (CTCs) have recently emerged as a new dynamic soluble marker for several malignancies including cutaneous melanoma (CM) and are suitable for prognostic evaluations ...and treatment monitoring. However, to date many limitations still hamper the wide-scale application of CTCs in CM setting, including the lack of standardized methods as well as both low levels and heterogeneity of these cells.
Methods:
We developed a protocol for CTC detection in CM based on immune-magnetic sorting to deplete CD45-, CD31- or CD34-positive cells, followed by dielectrophoretic DEPArray separation according to cell morphology and immunophenotype. To this end, we explored the expression of melanoma stem cell antigens (CD271, ABCB5, and RANK) and the epithelial-to-mesenchymal transition markers (N-Cad, -CD44, and -MCAM/CD146) on CTCs from 17 stage IV CM patients, and investigated their BRAF mutational status by droplet digital PCR.
Results:
The number of CTCs isolated from CM patients ranged from 2 to 91 cells (38 ± 6.4) with respect to healthy donors (p < 0.0002). To confirm the melanoma origin of isolated cells, we observed an 80% agreement between their BRAFV600 mutational status and matched primary tumors. The characterization of the immune phenotype of isolated cells revealed high interindividual and intraindividual heterogeneity that was found to correlate with the clinical outcome.
Conclusions:
The dual-step protocol of immune-magnetic sorting and subsequent dielectrophoretic DEPArray separation, turned out to be a suitable method to isolate viable CTCs from stage IV melanoma patients and enabled quantitative and qualitative analyses on these cells, which may deserve prospective evaluation for potential use in the clinical practice.
PDF
