MicroRNAs expression analysis in high grade gliomas

Several biomarkers had been proposed as useful parameters to better define the prognosis or to delineate new target therapy strategies for glioblastoma (GBM) patients. MicroRNAs could represent interesting molecules, for their role in tumorigenesis and cancer progression and for their specific tissue expression. Although many studies have tried to identify a specific microRNAs signature for glioblastoma, by now an exhaustive GBM microRNAs profile is far to be well defined. In this work we set up a real-time qPCR, based on LNA primers, to investigate the expression of 19 microRNAs in brain tumors, focusing our attention on GBMs. MiRNAs expression in 30 GBM paired FFPE-Fresh/Frozen samples was firstly analyzed. The good correlation obtained comparing miRNAs results confirmed the feasibility of performing miRNAs analysis starting from FFPE tissues. This leads to many advantages, as a good disposal of archival tumor and normal brain specimens and the possibility to verify the percentage of tumor cells in the analyzed sample. In the second part we compared 3 non-neoplastic brain references to use as control in miRNAs analysis. Normal adjacent the tumor, epileptic specimens and a commercial total RNA were analyzed for miRNAs expression and results showed that different non-neoplastic controls could lead to important discrepancies in GBM miRNAs profiles. Analyzing 50 FFPE GBMs using all 3 non-neoplastic references, we defined a putative GBM miRNAs signature: mir-10b, miR-21 and miR-27a resulted upregulated, while miR-7, miR-9, miR-26a, miR-31, miR-101, miR-137, miR-222 and miR-330 were downregulated. Comparing miRNAs expression among GBM group and gliomas of grade I, II and III, we obtained 3 miRNAs (miR-10b, mir-34a and miR-101) showing a different regulation status between high grade and low grade gliomas. Intriguingly, miR-10b was upregulated in high grade and significantly downregulated in low grade gliomas, suggesting that could be a candidate for a GBM target therapy.

Involvement of microRNAs in Androgen Receptor-dependent Breast Cancers

Triple negative breast cancer (TNBC) is a very aggressive tumor subtype characterized by the lack of expression of estrogen receptor 1 (ESR1), due in the most of cases to an increased expression of DNA methyltransferases (DNMTs) and hypermethylation in CpG islands, resulting in gene silencing. Furthermore, in ESR1- negative breast cancers, androgen receptor (AR) is highly expressed and some studies suggest that it can drive tumor progression and might represent a therapeutic target. A correlation between microRNAs, small non-coding RNAs that regulate gene expression, and DNMTs was investigated in a TNBC cell line to restore a normal methylation pattern of ESR1, leading to its re-expression and conferring again sensitivity to selective estrogen receptor modulators (SERMs). miR-148A and miR-29B were found to be involved in the reduction of the expression of DNMT1 and DNMT3A and in a slight increase of ESR1 expression, but not at protein level. Then, we found a down-regulation of AR by miRs-7, -9, -27a, -27b, -29a, -29b, -29c, -127-3p, -127-5p and -376 at 48h post transfection and an up-regulation by miR-15a and miR-16 at every time considered. We concomitantly investigated a possible increase of Tamoxifen, Herceptin and Metformin sensitivity after AR silencing in MDA-MB 453 and T-47D cell lines. Cells seemed more sensitive when silenced for AR only in MDA-MB-453 at 24h post Tamoxifen treatment. Studies on Metformin have basically confirmed an increase of drug sensitivity due to AR silencing in both cell lines. Analysis of Herceptin showed how MDA-MB 453 samples silenced for AR have a slight decrease in the percentage of proliferating cells, demonstrating a possible increase in the response to treatment. These preliminary data provide the basis for further study of the modulation of the expression of AR by microRNAs and it will be interesting to understand the molecular mechanisms underlying these interactions.

RNA Interference and cyclooxygenase-2 (COX-2) regulation in colon cancer cells

Despite new methods and combined strategies, conventional cancer chemotherapy still lacks specificity and induces drug resistance. Gene therapy can offer the potential to obtain the success in the clinical treatment of cancer and this can be achieved by replacing mutated tumour suppressor genes, inhibiting gene transcription, introducing new genes encoding for therapeutic products, or specifically silencing any given target gene. Concerning gene silencing, attention has recently shifted onto the RNA interference (RNAi) phenomenon. Gene silencing mediated by RNAi machinery is based on short RNA molecules, small interfering RNAs (siRNAs) and microRNAs (miRNAs), that are fully o partially homologous to the mRNA of the genes being silenced, respectively. On one hand, synthetic siRNAs appear as an important research tool to understand the function of a gene and the prospect of using siRNAs as potent and specific inhibitors of any target gene provides a new therapeutical approach for many untreatable diseases, particularly cancer. On the other hand, the discovery of the gene regulatory pathways mediated by miRNAs, offered to the research community new important perspectives for the comprehension of the physiological and, above all, the pathological mechanisms underlying the gene regulation. Indeed, changes in miRNAs expression have been identified in several types of neoplasia and it has also been proposed that the overexpression of genes in cancer cells may be due to the disruption of a control network in which relevant miRNA are implicated. For these reasons, I focused my research on a possible link between RNAi and the enzyme cyclooxygenase-2 (COX-2) in the field of colorectal cancer (CRC), since it has been established that the transition adenoma-adenocarcinoma and the progression of CRC depend on aberrant constitutive expression of COX-2 gene. In fact, overexpressed COX-2 is involved in the block of apoptosis, the stimulation of tumor-angiogenesis and promotes cell invasion, tumour growth and metastatization. On the basis of data reported in the literature, the first aim of my research was to develop an innovative and effective tool, based on the RNAi mechanism, able to silence strongly and specifically COX-2 expression in human colorectal cancer cell lines. In this study, I firstly show that an siRNA sequence directed against COX-2 mRNA (siCOX-2), potently downregulated COX-2 gene expression in human umbilical vein endothelial cells (HUVEC) and inhibited PMA-induced angiogenesis in vitro in a specific, non-toxic manner. Moreover, I found that the insertion of a specific cassette carrying anti-COX-2 shRNA sequence (shCOX-2, the precursor of siCOX-2 previously tested) into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT-29) without activating any interferon response. Phenotypically, COX-2 deficient HT-29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, results reported here indicate an easy-to-use, powerful and high selective virus-based method to knockdown COX-2 gene in a stable and long-lasting manner, in colon cancer cells. Furthermore, they open up the possibility of an in vivo application of this anti-COX-2 retroviral vector, as therapeutic agent for human cancers overexpressing COX-2. In order to improve the tumour selectivity, pSUPER.retro vector was modified for the shCOX-2 expression cassette. The aim was to obtain a strong, specific transcription of shCOX-2 followed by COX-2 silencing mediated by siCOX-2 only in cancer cells. For this reason, H1 promoter in basic pSUPER.retro vector [pS(H1)] was substituted with the human Cox-2 promoter [pS(COX2)] and with a promoter containing repeated copies of the TCF binding element (TBE) [pS(TBE)]. These promoters were choosen because they are partculary activated in colon cancer cells. COX-2 was effectively silenced in HT-29 and HCA-7 colon cancer cells by using enhanced pS(COX2) and pS(TBE) vectors. In particular, an higher siCOX-2 production followed by a stronger inhibition of Cox-2 gene were achieved by using pS(TBE) vector, that represents not only the most effective, but also the most specific system to downregulate COX-2 in colon cancer cells. Because of the many limits that a retroviral therapy could have in a possible in vivo treatment of CRC, the next goal was to render the enhanced RNAi-mediate COX-2 silencing more suitable for this kind of application. Xiang and et al. (2006) demonstrated that it is possible to induce RNAi in mammalian cells after infection with engineered E. Coli strains expressing Inv and HlyA genes, which encode for two bacterial factors needed for successful transfer of shRNA in mammalian cells. This system, called “trans-kingdom” RNAi (tkRNAi) could represent an optimal approach for the treatment of colorectal cancer, since E. Coli in normally resident in human intestinal flora and could easily vehicled to the tumor tissue. For this reason, I tested the improved COX-2 silencing mediated by pS(COX2) and pS(TBE) vectors by using tkRNAi system. Results obtained in HT-29 and HCA-7 cell lines were in high agreement with data previously collected after the transfection of pS(COX2) and pS(TBE) vectors in the same cell lines. These findings suggest that tkRNAi system for COX-2 silencing, in particular mediated by pS(TBE) vector, could represent a promising tool for the treatment of colorectal cancer. Flanking the studies addressed to the setting-up of a RNAi-mediated therapeutical strategy, I proposed to get ahead with the comprehension of new molecular basis of human colorectal cancer. In particular, it is known that components of the miRNA/RNAi pathway may be altered during the progressive development of colorectal cancer (CRC), and it has been already demonstrated that some miRNAs work as tumor suppressors or oncomiRs in colon cancer. Thus, my hypothesis was that overexpressed COX-2 protein in colon cancer could be the result of decreased levels of one or more tumor suppressor miRNAs. In this thesis, I clearly show an inverse correlation between COX-2 expression and the human miR- 101(1) levels in colon cancer cell lines, tissues and metastases. I also demonstrate that the in vitro modulating of miR-101(1) expression in colon cancer cell lines leads to significant variations in COX-2 expression, and this phenomenon is based on a direct interaction between miR-101(1) and COX-2 mRNA. Moreover, I started to investigate miR-101(1) regulation in the hypoxic environment since adaptation to hypoxia is critical for tumor cell growth and survival and it is known that COX-2 can be induced directly by hypoxia-inducible factor 1 (HIF-1). Surprisingly, I observed that COX-2 overexpression induced by hypoxia is always coupled to a significant decrease of miR-101(1) levels in colon cancer cell lines, suggesting that miR-101(1) regulation could be involved in the adaption of cancer cells to the hypoxic environment that strongly characterize CRC tissues.

Studio di marcatori immunoistochimici, analisi FISH e ruolo dei microRNA nelle neoplasie gliali di basso e di alto grado

Gliomas are the most common primary brain tumours. Despite advances in surgical techniques, postoperative supportive care, radiation and adjuvant systemic therapy, the life expectancy of patients with high grade glioma has remained essentially poor. Furthermore differential diagnosis among astrocytomas, oligodendrogliomas and oligoastrocytomas is very challenging and subject to inter-observer variability. The purpose of the research was: 1) to investigate a series of high grade and low grade gliomas at gene and protein (immunohistochemistry) levels to disclose possible genetic portraits of malignancy; 2) to verify the utility of Nogo-A, Olig-2 and synaptophysin in providing a correct histological diagnosis of oligodendroglioma and to investigate a possible complementary role in selecting the best areas suitable for detecting 1p/19q codeletion using FISH analysis; 3) to study the role of microRNA in high grade gliomas. In order to obtain these goals large series of brain tumors were studied with DNA microarrays, immunohistochemistry and RT-PCR The results demonstrated that: - Overexpression of IGFBP-2 and CDC20 is highly related to glioblastomas and their immunopositivity can be useful for the identification of glioblastoma in small biopsies. - Nogo-A is the most useful and specific marker in differentiating oigodendrogliomas from other gliomas. Furthermore, using a Nogo-A driven FISH analysis, it is possible to identify a larger number of 1p19q codeletions in gliomas. - microRNAs can be studied in paraffin embedded tissues better than in fresh tissues. A series of six microRNA, significatively deregulated in glioblastomas, may represent a genetic signature with prognostic and predictive value and could constitute candidates for novel anti-cancer therapeutics.

Meccanismi replicativi e di regolazione dell'espressione genica di Parvovirus B19

Il Parvovirus B19, virus patogeno umano della famiglia Parvoviridae, mostra uno specifico tropismo per i precursori eritroidi e una limitata replicazione in alcune linee cellulari megacarioblastoidi. Allo scopo di sviluppare sistemi utili allo studio delle caratteristiche biologiche del virus, diversi laboratori si sono occupati della costruzione di cloni genomici di B19 dotati di competenza funzionale e capaci di generare virus infettante. Parte del presente lavoro ha riguardato l’analisi funzionale di diversi cloni genomici di B19 e ha permesso di caratterizzare le regioni terminali del virus e di identificare requisiti essenziali per la loro funzionalità. Nel contesto intracellulare, esistono differenti livelli di restrizione in relazione alla capacità della cellula di supportare la replicazione virale, non ancora del tutto caratterizzati. Inoltre si sono accumulate evidenze circa la capacità del B19 di instaurare persistenza in numerosi tessuti. Non sono ancora note le caratteristiche funzionali del genoma virale in questo stato, è possibile che il virus persista in forma silente e meccanismi epigenetici possano regolare tale silenziamento. In questo studio è stato analizzato lo stato di metilazione del genoma di B19 e il suo possibile effetto sul ciclo replicativo virale ed è stata investigata la possibile associazione del DNA virale agli istoni cellulari nel corso di infezione in vitro. I risultati ottenuti confermano la presenza di questi meccanismi epigenetici, potendo ipotizzare che giochino un importante ruolo nella regolazione della funzionalità virale e nell’interazione B19-cellula e siano un elemento critico per l’adattamento del virus nell’ambiente in cui si trova. Inoltre l’ipotesi che anche i microRNA possano assumere un importante significato nell’interazione B19-cellula è stata proposta da diversi lavori e nel presente studio è stata valutata la produzione di queste piccole molecole durante l'infezione in vitro, ricercando microRNA (cellulari e/o virali) con omologia di sequenza per il genoma di B19 e quindi specifici per il virus.

Caratterizzazione del soppressore tumorale miR-101 nel colon carcinoma

I microRNA sono una classe di piccole molecole di RNA non codificante che controllano la stabilità di numerosi RNA messaggeri, perciò sono considerati come “master regulator” dell’espressione genica. Ogni tumore è caratterizzato da un profilo di espressione alterato dei microRNA. Il miR-101 è un oncosoppressore represso nei tessuti tumorali ed è candidato come biomarcatore del cancro colon-rettale. È regolato da numerosi eventi fisiologici e patologici, come angiogenesi e carcinogenesi. Gli eventi molecolari coinvolti nella regolazione dell’espressione del miR-101 sono scarsamente conosciuti, poiché è trascritto da due loci genici non caratterizzati. L’obiettivo di questo lavoro è di caratterizzare i geni del miR-101 ed individuarne i regolatori molecolari coinvolti nella cancerogenesi colon-rettale.

Role of Non-coding RNAs in chemotherapeutic treatments

The transcribed ultraconserved regions (T-UCRs) are a group of long non-coding RNAs involved in human carcinogenesis. The factors regulating the expression of T-UCRs and their mechanism of action in human cancers are unknown. In this work it was shown that high expression of uc.339 associates with lower survival in 204 non-small cell lung cancer (NSCLC) patients. Moreover, it was shown that uc.339 found up-regulated in archival NSCLC samples, acts as a decoy RNA for miR-339-3p, -663-3p and -95-5p. So, Cyclin E2, a direct target of three microRNAs is up-regulated, inducing cancer growth and migration. Evidence of this mechanism was provided from cell lines and primary samples confirming that TP53 directly regulates uc.339. These results support a key role for uc.339 in lung cancer.

Tumor heterogeneity in Esophageal Adenocarcinoma: a genomic approach

INTRODUCTION: Esophageal adenocarcinoma (EAC) is a severe malignancy in terms of prognosis and mortality rate. Because its great genetic heterogeneity, disputes regarding classification, prevention and treatments are still unsolved. AIM: We investigated intra- and inter-EAC heterogeneity by defining EAC’s somatic mutational profile and the role of candidate microRNAs, to correlate the molecular profile of tumors to clinical outcomes and to identify biomarkers for classification. METHODS: 38 EAC cases were analyzed via high-throughput cell sorting technology combined with targeted sequencing and whole genome low-pass sequencing. Targeted sequencing of further 169 cases was performed to widen the study. miR221 and miR483-3p expression was profiled via qPCR in 112 EACs and correlation with clinical outcomes was investigated. RESULTS: 35/38 EACs carried at least one somatic mutation absent in stromal cells. TP53 was found mutated in 73.7% of cases. Selective sorting revealed tumor subclones with different mutational loads and copy number alterations, confirming the high intra-tumor heterogeneity of EAC. Mutations were in most cases at homozygous state, and we identified alterations that were missed with the whole-tumor analysis. Mutations in HNF1A gene, not previously associated with EAC, were identified in both cohorts. Higher expression of miR483-3p and miR221 was associated with poorer cancer specific survival (P=0.0293 and P=0.0059), and recurrence in the Lauren intestinal subtype (P=0.0459 and P=0.0002). Median expression levels of miRNAs were higher in patients with advanced tumor stages. The loss of SMAD4 immunoreactivity was significantly associated with poorer cancer specific survival and recurrence (P=0.0452; P=0.022 respectively). CONCLUSION: Combining selective sorting technology and next generation sequencing allowed to better define EAC inter- and intra-tumor heterogeneity. We identified HNF1A as a new mutated gene associated to EAC that could be involved in tumor progression and promising biomarkers such as SMAD4, miR221 and miR483-3p to identify patients at higher risk for more aggressive tumors.

Identification of a molecular signature as potential biomarker in blood of symptomatic and asymptomatic patients with carotid atherosclerosis

Background and Objectives: Carotid revascularization to prevent future vascular events is reasonable in patients with high-grade carotid stenosis. Currently, several biomarkers to predict carotid plaque development and progression have been investigated, among which microRNAs (miRs) are promising tools for the diagnosis of atherosclerosis. Methods and Results: A total of 49 participants were included in the study, divided into two main populations: Population 1 comprising symptomatic and asymptomatic inpatients, and Population 2 comprising asymptomatic outpatients. The study consisted of two main phases: a preliminary discovery phase and a validation phase, applying different techniques. MiR-profiles were performed on plasma and plaque tissue samples obtained from 4 symptomatic and 4 asymptomatic inpatients. MiRs emerging from profiling comparisons, i.e. miR-126-5p, miR-134-5p, miR-145-5p, miR-151a-5p, miR-34b, miR-451a, miR-720 and miR-1271-5p, were subjected to validation through RT-qPCR analysis in the total cohort of donors. Comparing asymptomatic and symptomatic inpatients, significant differences were reported in the expression levels of c-miRs for miR-126-5p and miR-1271-5p in blood, being more expressed in symptomatic subjects. In contrast, simultaneous evaluation of the selected miRs in plaque tissue samples did not confirm data obtained by the miR profiling, and no significant differences were observed. Using Receiver-Operating Characteristic (ROC) analysis, a circulating molecular signature (mir-126-5p, miR-1271-5p, albumin, C-reactive protein, and monocytes) was identified, allowing the distinction of the two groups in Population 1 (AUC = 0.795). Conclusions: Data emerging from this thesis suggest that c-miRs (i.e. miR-126-5p, miR-1271-5p) combined with selected haemato-biochemical parameters (albumin, C-reactive protein, and monocytes) produced a good molecular 'signature' to distinguish asymptomatic and symptomatic inpatients. C-miRs in blood do not necessarily reflect the expression levels of the same miRs in carotid plaque tissues since different mechanism can influence their expression.

Role of non-coding RNA alterations in melanoma

Cutaneous melanoma (CM) is a potentially lethal form of skin cancer and its most important histopathologic factor for staging is Breslow thickness (BT). Its correct determination is fundamental for pathologists. A deeper understanding of the molecular processes guiding CM pathogenesis could improve diagnosis, treatment and prognosis. MicroRNAs (miRNAs) play a key role in CM biology. The firs aim was to investigate miRNA expression in reference to BT assessment. We found that the combined miRNA expression of miR-21-5p and miR-146a-5p above or below 1.5 was significantly associated with overall survival and successfully identified all superficially spreading melanoma (SSM) patients with relapsing suggesting that the combined assessment of these miRNAs expression could aid in SSM staging. Secondly, we focus on multiple primary melanoma (MPM) patients, which develop multiple primary melanomas in their lifetime, and represent a model of high-risk CM occurrence. We explored the miRNome of single CM and MPM: CM and MPM present several dysregulated miRNAs, including key miRNAs involved in epithelial-mesenchymal transition. A different miRNA profile was observed between 1st and 2nd melanoma from the same patient. MiRNA target analysis revealed a more differentiated and less invasive status of MPMs compared to CMs. This characterization of the miRNA regulatory network of MPMs highlights molecular features differentiating this subtype from CM. Recently, NGS experiments revealed the existence of miRNA variants (isomiRs) with different length and sequence. We identified a shorter 3’isoform as tenfold over-represented compared to the canonical form of miR-125a-5p. Target analysis revealed that miRNA shortening could change the pattern of target gene regulation. Finally, we study miRNA and isomiR dysregulation in benign nevi (BN) and CM and in CM and melanoma metastasis. The reported non-random dysregulation of specific isomiRs contributes to the understanding of the complex melanoma pathogenesis and serves as the basis for further functional studies.

Exploratory analysis of the role of circulating microRNA in metastatic melanoma patients

MicroRNAs act as oncogene or tumor suppressor gene regulators and are actively released from tumor cells in the circulation. Specific microRNAs can be isolated and quantified in the blood, usually in serum or plasma fractions, where they are uncommonly stable. Cell-free microRNAs serve many, and possibly yet unexplored, functional roles and microRNA levels reflect underlying conditions and have been associated with skin cancer presence, stage and evolution. However, the clinical potential of circulating miRNAs in metastatic melanoma remains largely undefined. From May 2020 to September 2022, we conducted a spontaneous, monocentric, exploratory study on human tissues in vitro, which aimed to evaluate the prognostic and predictive role of circulating miRNAs in metastatic melanoma patients. At the Medical Oncology Unit of Policlinico Sant’Orsola-Malpighi of Bologna, peripheral venous blood samples from patients with metastatic melanoma treated with checkpoint inhibitors (CPI) were collected before the start of CPI (baseline, T0) and longitudinally, approximately every 3 months (T1, T2, etc). Circulating miRNA quantification was performed by droplet digital PCR (Biorad) using an EvaGreen and LNA primer-based assays. QuantaSoft Program (Biorad) calculated the absolute quantifications of each miRNA, indicated as copies/µL. After analysis of the literature, we chose to analyze miR-155-5p, miR-320a and miR-424-5p level. All miRNAs except miR-424-5p show a significantly higher level in plasma of patients who are alive after 1 year of follow-up. High/low levels of baseline miR-155-5p, miR-320a and miR-424-5p are significantly associated with overall survival and progression-free survival. Furthermore, a preliminary analysis on the group of patients who received first-line with anti-PD-1 (N=7), baseline miR-155-5p shows higher levels in responder vs. non responder patients (p 0.06). These data, though promising, are preliminary and need to be further investigated in a larger cohort of patients.

Identification of potential miRNAs related to classification and treatment response of actinic keratosis

The Workflow activity was the following: Preliminary phase: Identification of 18 Formalin-fixed paraffin embedded (FFPE) samples (9 patients) («matched» 9 AK lesions and 9 SCC lesions). Working on biopsies samples we perform an extraction and RNA analysis with droplet Digital PCR (ddPCR) and we perform the data analysis. Second and final step phase: Evaluation of additional 39 subjects (36 men and 3 women). Results: We perform an evaluation and comparison of the following miRNA: miR-320 (a miRNA involved in apoptosis and cell proliferation control; miR-204, a miRNA involved in cell proliferation in and miRNA-16-5p, a miRNA involved in apoptosis).Conclusion: Our data suggest that there is no significant variation in the expression of the three tested microRNAs between adjacent AK lesions and squamous-cell carcinoma. However, a relevant trend has been observed Furthermore, by evaluating the miRNA expression trend between keratosis and carcinoma of the same patient, it is observed that there is no "uniform trend": for some samples the expression rises for the transition from AK to SCC and viceversa.

Cancers of unknown primary site (CUPs) as a model of metastatic process.

Cancers of unknown primary site (CUPs) are a rare group of metastatic tumours, with a frequency of 3-5%, with an overall survival of 6-10 month. The identification of tumour primary site is usually reached by a combination of diagnostic investigations and immunohistochemical testing of the tumour tissue. In CUP patients, these investigations are inconclusive. Since international guidelines for treatment are based on primary site indication, CUP treatment requires a blind approach. As a consequence, CUPs are usually empiric treated with poorly effective. In this study, we applied a set of microRNAs using EvaGreen-based Droplet Digital PCR in a retrospective and prospective collection of formalin-fixed paraffin-embedded tissue samples. We assessed miRNA expression of 155 samples including primary tumours (N=94), metastases of known origin (N=10) and metastases of unknown origin (N=50). Then, we applied the shrunken centroids predictive algorithm to obtain the CUP’s site(s)-of-origin. The molecular test was successfully applied to all CUP samples and provided a site-of-origin identification for all samples, potentially within a one-week time frame from sample inclusion. In the second part of the study we derived two CUP cell lines, and corresponding patient-derived xenografts (PDXs). CUP cell lines and PDXs underwent histological, molecular, and genomic characterization confirming the features of the original tumour. Tissues-of-origin prediction was obtained from the tumour microRNA expression profile and confirmed by single cell RNA sequencing. Genomic testing analysis identified FGFR2 amplification in both models. Drug-screening assays were performed to test the activity of FGFR2-targeting drug and the combination treatment with the MEK inhibitor trametinib, which proved to be synergic and exceptionally active, both in vitro and in vivo. In conclusion, our study demonstrated that miRNA expression profiling could be employed as diagnostic test. Then we successfully derived two CUP models from patients, used for therapy tests, bringing personalized therapy closer to CUP patients.