Circulating microRNA Analysis in a Prospective Co-clinical Trial Identifies MIR652-3p as a Response Biomarker and Driver of Regorafenib Resistance Mechanisms in Colorectal Cancer.

PURPOSE: The multi-kinase inhibitor (mKi) regorafenib has demonstrated efficacy in chemorefractory patients with metastatic colorectal cancer (mCRC). However, lack of predictive biomarkers and concerns over significant toxicities hamper the use of regorafenib in clinical practice. EXPERIMENTAL DESIGN: Serial liquid biopsies were obtained at baseline and monthly until disease progression in chemorefractory patients with mCRC treated with regorafenib in a phase II clinical trial (PROSPECT-R n = 40; NCT03010722) and in a multicentric validation cohort (n = 241). Tissue biopsies collected at baseline, after 2 months and at progression in the PROSPECT-R trial were used to establish patient-derived organoids (PDO) and for molecular analyses. MicroRNA profiling was performed on baseline bloods using the NanoString nCounter platform and results were validated by digital-droplet PCR and/or ISH in paired liquid and tissue biopsies. PDOs co-cultures and PDO-xenotransplants were generated for functional analyses. RESULTS: Large-scale microRNA expression analysis in longitudinal matched liquid and tissue biopsies from the PROSPECT-R trial identified MIR652-3p as a biomarker of clinical benefit to regorafenib. These findings were confirmed in an independent validation cohort and in a "control" group of 100 patients treated with lonsurf. Using ex vivo co-culture assays paired with single-cell RNA-sequencing of PDO established pre- and post-treatment, we modeled regorafenib response observed in vivo and in patients, and showed that MIR652-3p controls resistance to regorafenib by impairing regorafenib-induced lethal autophagy and by orchestrating the switch from neo-angiogenesis to vessel co-option. CONCLUSIONS: Our results identify MIR652-3p as a potential biomarker and as a driver of cell and non-cell-autonomous mechanisms of resistance to regorafenib.

MicroRNAs as biomarkers for trastuzumab-based therapy in HER2-positive advanced oesophago-gastric cancer patients.

Background: This study aimed to identify microRNAs (miRs) as circulating biomarkers of resistance to first-line trastuzumab-based therapy in advanced HER2-positive oesophago-gastric cancer patients. Methods: A high-throughput 1015 Exiqon miRCURY LNA™ microRNA inhibitor library screen was performed in trastuzumab-treated HER2-positive NCI-N87 and HER2-negative FLO-1 oesophago-gastric cancer cell lines. NanoString nCounter® miR analysis was performed in NCI-N87, FLO-1, and MAGIC trial (ISRCTN93793971) formalin-fixed paraffin-embedded (FFPE) oesophago-gastric cancer patient samples. MiR-148a-3p copies in plasma samples were quantified using digital droplet polymerase chain reaction (ddPCR) from HER2-positive oesophago-gastric cancer patients treated with standard-of-care trastuzumab-based therapy within the FOrMAT (NCT02112357) and PLATFORM (NCT02678182) clinical trials. The primary endpoints were overall survival (OS) for plasma miR-148a-3p HIGH (>median) versus LOW (≤median). The secondary endpoints were progression-free survival (PFS) and 3-month progression-free rates (PFRs) miR-148a-3p HIGH versus LOW. PLATFORM sensitivity analysis normalised miR-148a-3p (NmiR-148a-3p). Results: The inhibition of miR-148a-3p reduced NCI-N87 relative cell viability (<0.6) and expression was high (>242) in NCI-N87 and HER2-positive MAGIC trial patients (n=5). Normalised-miR-148a-3p (NmiR-148a-3p) LOW versus HIGH demonstrated a statistically significant difference in 3-month PFRs (n=23; OR, 0.11 [0.02-0.78]; p=0.027; aOR, 0.03 [0.001-0.71], p=0.029) but no difference in OS or PFS. There was no statistically significant relationship between miR-148-3p LOW versus HIGH for OS (PLATFORM, n=62; hazard ratio [HR], 0.98 [0.57-1.66]; p=0.933; FOrMAT, n=8; HR, 0.54 [0.13-2.31]; p=0.322), PFS (n=62; HR, 1.08 [0.65-1.81]; p=0.759; FOrMAT, n=8; HR, 1.26 [0.31-5.07]; p=0.714), or PFRs (PLATFORM, n=31; odds ratio [OR], 0.67 [0.2-2.8]; p=0.577). Conclusion: Normalised miR-148a-3p may be a relevant biomarker for trastuzumab-based therapy in advanced HER2-positive oesophago-gastric cancer patients.

Prevalence and type of MMR expression heterogeneity in colorectal adenocarcinoma: therapeutic implications and reporting.

Mismatch repair (MMR) immunohistochemical (IHC) evaluation has entered pathology routine practice as the first-line screening method to identify patients with MMR deficient (MMRd)/microsatellite instability (MSI) colorectal cancer (CRC), and its misdiagnosis may significantly impact the personalization of CRC patient care. To determine the prevalence of MMR protein intratumor heterogeneity in real-world practice, we collected a series of 8282 CRCs tested for MMR proteins in the setting of Lynch syndrome universal screening. Four heterogenous cases were also investigated for tumor infiltrating lymphocytes count, MSI status, and consensus molecular subtypes by Nanostring nCounter® Platform. Overall, 1056 (12.8%) CRCs showed a MMR altered status, with 46 cases showing a heterogeneous MMR profile (0.56% of the total, and 4.36% of all MMRd cases). To conclude, the authors make some critical remarks regarding the approach to MMR heterogeneity in clinical practice and routine diagnostics.

Codon-specific KRAS mutations predict survival benefit of trifluridine/tipiracil in metastatic colorectal cancer.

Genomics has greatly improved how patients with cancer are being treated; however, clinical-grade genomic biomarkers for chemotherapies are currently lacking. Using whole-genome analysis of 37 patients with metastatic colorectal cancer (mCRC) treated with the chemotherapy trifluridine/tipiracil (FTD/TPI), we identified KRAS codon G12 (KRASG12) mutations as a potential biomarker of resistance. Next, we collected real-world data of 960 patients with mCRC receiving FTD/TPI and validated that KRASG12 mutations were significantly associated with poor survival, also in analyses restricted to the RAS/RAF mutant subgroup. We next analyzed the data of the global, double-blind, placebo-controlled, phase 3 RECOURSE trial (n = 800 patients) and found that KRASG12 mutations (n = 279) were predictive biomarkers for reduced overall survival (OS) benefit of FTD/TPI versus placebo (unadjusted interaction P = 0.0031, adjusted interaction P = 0.015). For patients with KRASG12 mutations in the RECOURSE trial, OS was not prolonged with FTD/TPI versus placebo (n = 279; hazard ratio (HR) = 0.97; 95% confidence interval (CI) = 0.73-1.20; P = 0.85). In contrast, patients with KRASG13 mutant tumors showed significantly improved OS with FTD/TPI versus placebo (n = 60; HR = 0.29; 95% CI = 0.15-0.55; P < 0.001). In isogenic cell lines and patient-derived organoids, KRASG12 mutations were associated with increased resistance to FTD-based genotoxicity. In conclusion, these data show that KRASG12 mutations are biomarkers for reduced OS benefit of FTD/TPI treatment, with potential implications for approximately 28% of patients with mCRC under consideration for treatment with FTD/TPI. Furthermore, our data suggest that genomics-based precision medicine may be possible for a subset of chemotherapies.

USP7 inactivation suppresses APC-mutant intestinal hyperproliferation and tumor development.

Adenomatous polyposis coli (APC) mutation is the hallmark of colorectal cancer (CRC), resulting in constitutive WNT activation. Despite decades of research, targeting WNT signaling in cancer remains challenging due to its on-target toxicity. We have previously shown that the deubiquitinating enzyme USP7 is a tumor-specific WNT activator in APC-truncated cells by deubiquitinating and stabilizing ß-catenin, but its role in gut tumorigenesis is unknown. Here, we show in vivo that deletion of Usp7 in Apc-truncated mice inhibits crypt hyperproliferation and intestinal tumor development. Loss of Usp7 prolongs the survival of the sporadic intestinal tumor model. Genetic deletion, but not pharmacological inhibition, of Usp7 in Apc+/- intestine induces colitis and enteritis. USP7 inhibitor treatment suppresses growth of patient-derived cancer organoids carrying APC truncations in vitro and in xenografts. Our findings provide direct evidence that USP7 inhibition may offer a safe and efficacious tumor-specific therapy for both sporadic and germline APC-mutated CRC.

Papillary Thyroid Carcinoma: Molecular Distinction by MicroRNA Profiling.

Papillary thyroid carcinoma (PTC) is a miscellaneous disease with a variety of histological variants, each with its own mutational profile, and clinical and prognostic characteristics. Identification of microRNA (miRNA) expression profiles represents an important benchmark for understanding the molecular mechanisms underlying the biological behavior of these unique PTC subtypes in order that they be better characterized. We considered a series of 35 PTC samples with a histological diagnosis of either hobnail (17 cases) or classical variant (nine cases) and with a specific BRAF p.K601E mutation (nine cases). We determined the overall miRNA expression profile with NanoString technology, and both quantitative reverse transcription-PCR and in situ hybridization were used to confirm selected miRNAs. The miRNA signature was found to consistently differentiate specific histotypes and mutational profiles. In contrast to the BRAF p.K601E mutation and classic PTCs, three miRNAs (miR-21-5p, miR-146b-5p, and miR-205-5p) were substantially overexpressed in the hobnail variant. The current study found that different miRNA signature profiles were linked to unique histological variants and BRAF mutations in PTC. Further studies focusing on the downstream pathogenetic functions of mRNAs in thyroid neoplasms are warranted.

DCE-MRI is more sensitive than IVIM-DWI for assessing anti-angiogenic treatment-induced changes in colorectal liver metastases.

BACKGROUND: Diffusion weighted imaging (DWI) with intravoxel incoherent motion (IVIM) modelling can inform on tissue perfusion without exogenous contrast administration. Dynamic-contrast-enhanced (DCE) MRI can also characterise tissue perfusion, but requires a bolus injection of a Gadolinium-based contrast agent. This study compares the use of DCE-MRI and IVIM-DWI methods in assessing response to anti-angiogenic treatment in patients with colorectal liver metastases in a cohort with confirmed treatment response. METHODS: This prospective imaging study enrolled 25 participants with colorectal liver metastases to receive Regorafenib treatment. A target metastasis > 2 cm in each patient was imaged before and at 15 days after treatment on a 1.5T MR scanner using slice-matched IVIM-DWI and DCE-MRI protocols. MRI data were motion-corrected and tumour volumes of interest drawn on b=900 s/mm2 diffusion-weighted images were transferred to DCE-MRI data for further analysis. The median value of four IVIM-DWI parameters [diffusion coefficient D (10-3 mm2/s), perfusion fraction f (ml/ml), pseudodiffusion coefficient D* (10-3 mm2/s), and their product fD* (mm2/s)] and three DCE-MRI parameters [volume transfer constant Ktrans (min-1), enhancement fraction EF (%), and their product KEF (min-1)] were recorded at each visit, before and after treatment. Changes in pre- and post-treatment measurements of all MR parameters were assessed using Wilcoxon signed-rank tests (P<0.05 was considered significant). DCE-MRI and IVIM-DWI parameter correlations were evaluated with Spearman rank tests. Functional MR parameters were also compared against Response Evaluation Criteria In Solid Tumours v.1.1 (RECIST) evaluations. RESULTS: Significant treatment-induced reductions of DCE-MRI parameters across the cohort were observed for EF (91.2 to 50.8%, P<0.001), KEF (0.095 to 0.045 min-1, P<0.001) and Ktrans (0.109 to 0.078 min-1, P=0.002). For IVIM-DWI, only D (a non-perfusion parameter) increased significantly post treatment (0.83 to 0.97 × 10-3 mm2/s, P<0.001), while perfusion-related parameters showed no change. No strong correlations were found between DCE-MRI and IVIM-DWI parameters. A moderate correlation was found, after treatment, between Ktrans and D* (r=0.60; P=0.002) and fD* (r=0.67; P<0.001). When compared to RECIST v.1.1 evaluations, KEF and D correctly identified most clinical responders, whilst non-responders were incorrectly identified. CONCLUSION: IVIM-DWI perfusion-related parameters showed limited sensitivity to the anti-angiogenic effects of Regorafenib treatment in colorectal liver metastases and showed low correlation with DCE-MRI parameters, despite profound and significant post-treatment reductions in DCE-MRI measurements. TRIAL REGISTRATION: NCT03010722 clinicaltrials.gov; registration date 6th January 2015.

Modulation of pancreatic cancer cell sensitivity to FOLFIRINOX through microRNA-mediated regulation of DNA damage.

FOLFIRINOX, a combination of chemotherapy drugs (Fluorouracil, Oxaliplatin, Irinotecan -FOI), provides the best clinical benefit in pancreatic ductal adenocarcinoma (PDAC) patients. In this study we explore the role of miRNAs (MIR) as modulators of chemosensitivity to identify potential biomarkers of response. We find that 41 and 84 microRNA inhibitors enhance the sensitivity of Capan1 and MiaPaCa2 PDAC cells respectively. These include a MIR1307-inhibitor that we validate in further PDAC cell lines. Chemotherapy-induced apoptosis and DNA damage accumulation are higher in MIR1307 knock-out (MIR1307KO) versus control PDAC cells, while re-expression of MIR1307 in MIR1307KO cells rescues these effects. We identify binding of MIR1307 to CLIC5 mRNA through covalent ligation of endogenous Argonaute-bound RNAs cross-linking immunoprecipitation assay. We validate these findings in an in vivo model with MIR1307 disruption. In a pilot cohort of PDAC patients undergoing FOLFIRONX chemotherapy, circulating MIR1307 correlates with clinical outcome.

A phospho-proteomic study of cetuximab resistance in KRAS/NRAS/BRAFV600 wild-type colorectal cancer.

PURPOSE: We hypothesised that plasticity in signal transduction may be a mechanism of drug resistance and tested this hypothesis in the setting of cetuximab resistance in patients with KRAS/NRAS/BRAFV600 wild-type colorectal cancer (CRC). METHODS: A multiplex antibody-based platform was used to study simultaneous changes in signal transduction of 55 phospho-proteins in 12 KRAS/NRAS/BRAFV600 wild-type CRC cell lines (6 cetuximab sensitive versus 6 cetuximab resistant) following 1 and 4 h in vitro cetuximab exposure. We validated our results in CRC patient samples (n = 4) using ex vivo exposure to cetuximab in KRAS/NRAS/BRAFV600 cells that were immunomagnetically separated from the serous effusions of patients with known cetuximab resistance. RESULTS: Differences in levels of phospho-proteins in cetuximab sensitive and resistant cell lines included reductions in phospho-RPS6 and phospho-PRAS40 in cetuximab sensitive, but not cetuximab resistant cell lines at 1 and 4 h, respectively. In addition, phospho-AKT levels were found to be elevated in 3/4 patient samples following ex vivo incubation with cetuximab for 1 h. We further explored these findings by studying the effects of combinations of cetuximab and two PI3K pathway inhibitors in 3 cetuximab resistant cell lines. The addition of PI3K pathway inhibitors to cetuximab led to a significantly higher reduction in colony formation capacity compared to cetuximab alone. CONCLUSION: Our findings suggest activation of the PI3K pathway as a mechanism of cetuximab resistance in KRAS/NRAS/BRAFV600 wild-type CRC.

MIR21-induced loss of junctional adhesion molecule A promotes activation of oncogenic pathways, progression and metastasis in colorectal cancer.

Junctional adhesion molecules (JAMs) play a critical role in cell permeability, polarity and migration. JAM-A, a key protein of the JAM family, is altered in a number of conditions including cancer; however, consequences of JAM-A dysregulation on carcinogenesis appear to be tissue dependent and organ dependent with significant implications for the use of JAM-A as a biomarker or therapeutic target. Here, we test the expression and prognostic role of JAM-A downregulation in primary and metastatic colorectal cancer (CRC) (n = 947). We show that JAM-A downregulation is observed in ~60% of CRC and correlates with poor outcome in four cohorts of stages II and III CRC (n = 1098). Using JAM-A knockdown, re-expression and rescue experiments in cell line monolayers, 3D spheroids, patient-derived organoids and xenotransplants, we demonstrate that JAM-A silencing promotes proliferation and migration in 2D and 3D cell models and increases tumour volume and metastases in vivo. Using gene-expression and proteomic analyses, we show that JAM-A downregulation results in the activation of ERK, AKT and ROCK pathways and leads to decreased bone morphogenetic protein 7 expression. We identify MIR21 upregulation as the cause of JAM-A downregulation and show that JAM-A rescue mitigates the effects of MIR21 overexpression on cancer phenotype. Our results identify a novel molecular loop involving MIR21 dysregulation, JAM-A silencing and activation of multiple oncogenic pathways in promoting invasiveness and metastasis in CRC.

Challenges and perspectives for immunotherapy in oesophageal cancer: A look to the future (Review).

Oesophageal cancer is one of the most aggressive malignancies with limited treatment options, thus resulting in a high morbidity and mortality. With 5­year survival rates of only 5­10%, oesophageal cancer holds a dismal prognosis for patients. In order to improve overall survival, the early diagnosis and tools for patient stratification for personalized treatment are urgent needs. A minority of oesophageal cancers belong to the spectrum of Lynch syndrome­associated cancers and are characterized by microsatellite instability (MSI). Microsatellite instability is a consequence of defective mismatch repair protein functions and it has been well characterized in other gastrointestinal tumours, such as colorectal and gastric cancer. In the latter, high levels of MSI are associated with a better prognosis and with an increased benefit to immune­based therapies. Therefore, similar therapeutic approaches could offer an opportunity of treatment for oesophageal cancer patients with MSI. Apart from immune checkpoint inhibitors, other immunotherapies such as adoptive T­cell transfer, peptide vaccine and oncolytic viruses are under investigation in oesophageal cancer patients. In the present review, the rationale and current knowledge about immunotherapies in oesophageal cancer are summarised.

Back from the Brink: EGFR Inhibition in Gastroesophageal Cancer.

Gastroesophageal adenocarcinomas (GEA) remain difficult to treat with limited targeted therapeutics. Negative results from randomized trials of EGFR inhibitors (EGFRi) in patients with molecularly unselected GEA have hampered the development of EGFRi in the gastroesophageal cancer space. A recent study reopens the game.See related article by Corso et al., p. 3126.

Serine synthesis pathway inhibition cooperates with dietary serine and glycine limitation for cancer therapy.

Many tumour cells show dependence on exogenous serine and dietary serine and glycine starvation can inhibit the growth of these cancers and extend survival in mice. However, numerous mechanisms promote resistance to this therapeutic approach, including enhanced expression of the de novo serine synthesis pathway (SSP) enzymes or activation of oncogenes that drive enhanced serine synthesis. Here we show that inhibition of PHGDH, the first step in the SSP, cooperates with serine and glycine depletion to inhibit one-carbon metabolism and cancer growth. In vitro, inhibition of PHGDH combined with serine starvation leads to a defect in global protein synthesis, which blocks the activation of an ATF-4 response and more broadly impacts the protective stress response to amino acid depletion. In vivo, the combination of diet and inhibitor shows therapeutic efficacy against tumours that are resistant to diet or drug alone, with evidence of reduced one-carbon availability. However, the defect in ATF4-response seen in vitro following complete depletion of available serine is not seen in mice, where dietary serine and glycine depletion and treatment with the PHGDH inhibitor lower but do not eliminate serine. Our results indicate that inhibition of PHGDH will augment the therapeutic efficacy of a serine depleted diet.

EGFR amplification and outcome in a randomised phase III trial of chemotherapy alone or chemotherapy plus panitumumab for advanced gastro-oesophageal cancers.

OBJECTIVE: Epidermal growth factor receptor (EGFR) inhibition may be effective in biomarker-selected populations of advanced gastro-oesophageal adenocarcinoma (aGEA) patients. Here, we tested the association between outcome and EGFR copy number (CN) in pretreatment tissue and plasma cell-free DNA (cfDNA) of patients enrolled in a randomised first-line phase III clinical trial of chemotherapy or chemotherapy plus the anti-EGFR monoclonal antibody panitumumab in aGEA (NCT00824785). DESIGN: EGFR CN by either fluorescence in situ hybridisation (n=114) or digital-droplet PCR in tissues (n=250) and plasma cfDNAs (n=354) was available for 474 (86%) patients in the intention-to-treat (ITT) population. Tissue and plasma low-pass whole-genome sequencing was used to screen for coamplifications in receptor tyrosine kinases. Interaction between chemotherapy and EGFR inhibitors was modelled in patient-derived organoids (PDOs) from aGEA patients. RESULTS: EGFR amplification in cfDNA correlated with poor survival in the ITT population and similar trends were observed when the analysis was conducted in tissue and plasma by treatment arm. EGFR inhibition in combination with chemotherapy did not correlate with improved survival, even in patients with significant EGFR CN gains. Addition of anti-EGFR inhibitors to the chemotherapy agent epirubicin in PDOs, resulted in a paradoxical increase in viability and accelerated progression through the cell cycle, associated with p21 and cyclin B1 downregulation and cyclin E1 upregulation, selectively in organoids from EGFR-amplified aGEA. CONCLUSION: EGFR CN can be accurately measured in tissue and liquid biopsies and may be used for the selection of aGEA patients. EGFR inhibitors may antagonise the antitumour effect of anthracyclines with important implications for the design of future combinatorial trials.

MNK Inhibition Sensitizes KRAS-Mutant Colorectal Cancer to mTORC1 Inhibition by Reducing eIF4E Phosphorylation and c-MYC Expression.

KRAS-mutant colorectal cancers are resistant to therapeutics, presenting a significant problem for ∼40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant colorectal cancer. Using Kras-mutant mouse models and mouse- and patient-derived organoids, we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small-molecule targeting of this pathway, we acutely sensitize KRASG12D models to rapamycin via suppression of c-MYC. We show that 45% of colorectal cancers have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC-dependent cotargeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population that may benefit from its clinical application. SIGNIFICANCE: KRAS mutation and elevated c-MYC are widespread in many tumors but remain predominantly untargetable. We find that mutant KRAS modulates translation, culminating in increased expression of c-MYC. We describe an effective strategy targeting mTORC1 and MNK in KRAS-mutant mouse and human models, pathways that are also commonly co-upregulated in colorectal cancer.This article is highlighted in the In This Issue feature, p. 995.

Vault RNAs: hidden gems in RNA and protein regulation.

Non-coding RNAs are important regulators of differentiation during embryogenesis as well as key players in the fine-tuning of transcription and furthermore, they control the post-transcriptional regulation of mRNAs under physiological conditions. Deregulated expression of non-coding RNAs is often identified as one major contribution in a number of pathological conditions. Non-coding RNAs are a heterogenous group of RNAs and they represent the majority of nuclear transcripts in eukaryotes. An evolutionary highly conserved sub-group of non-coding RNAs is represented by vault RNAs, named since firstly discovered as component of the largest known ribonucleoprotein complexes called "vault". Although they have been initially described 30 years ago, vault RNAs are largely unknown and their molecular role is still under investigation. In this review we will summarize the known functions of vault RNAs and their involvement in cellular mechanisms.

Diagnostic Accuracy and Safety of Coaxial System in Oncology Patients Treated in a Specialist Cancer Center With Prospective Validation Within Clinical Trial Data.

BACKGROUND: Image-guided tissue biopsies are critically important in the diagnosis and management of cancer patients. High-yield samples are also vital for biomarker and resistance mechanism discovery through molecular/genomic analyses. PATIENTS AND METHODS: All consecutive patients who underwent plugged image-guided biopsy at Royal Marsden from June 2013 until September 2016 were included in the analysis. In the next step, a second cohort of patients prospectively treated within two clinical trials (PROSPECT-C and PROSPECT-R) were assessed for the DNA yield from biopsies assessed for complex genomic analysis. RESULTS: A total of 522 plugged core biopsies were performed in 457 patients [men, 52%; median age, 63 years (range, 17-93)]. Histological diagnosis was achieved in 501 of 522 (96%) performed biopsies. Age, gender, modality, metastatic site, and seniority of the interventionist were not found to be significant factors associated with odds of failure on a logistic regression. Seventeen (3.3%) were admitted due to biopsy-related complications; nine, three, two, one, one, and one were admitted for grade I/II pain control, sepsis, vasovagal syncope, thrombosis, hematuria, and deranged liver functions, respectively; two patients with right upper quadrant pain after liver biopsy were found to have radiologically confirmed subcapsular hematoma requiring conservative treatment. One patient (0.2%) developed grade III hemorrhage following biopsy of a gastric gastrointestinal stromal tumor (GIST). Overall molecular analysis was successful in 89% (197/222 biopsies). Prospective validation in 62 biopsies gave success rates of 92.06 and 79.03% for DNA extraction of >1 µm and tmour content of >20%, respectively. CONCLUSION: The probability of diagnostic success for complex molecular analysis is increased with plugged large coaxial needle biopsy technique, which also minimizes complications and reduces hospital stay. High-yield DNA acquisition allows genomic molecular characterization for personalized medicine.