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Purpose: Percutaneous abscess drainage (PAD) is the first-line approach for abscess in Crohn’s disease (CD) since it procrastinates or avoids surgery especially in postoperative abscesses [within 30 days post-operative (p.o.)]. We retrospectively evaluated the effectiveness, complications and outcome after PAD in postoperative and spontaneous abscesses and factors influencing the outcomes. Methods: We performed PAD in 91 abscesses, 45 (49,5%) postoperative and 46 (50,5%) spontaneous. We defined the overall success (OS) as clinical (CS) and technical success (TS) when imaging documented the resolution of the abscess with no surgery within 30 days. Conversely, patients without abscess at the time of surgery, were considered as TS but clinical failure (CF). We also analyzed the overall failure (OF) defined as CF with or without technical failure (TF). Overall technical success (OTS) was OS plus TS. Complications were classified as major and minor according to the Interventional Radiology Criteria. Results: In postoperative abscesses we found 91% OS, 9% OF, no TF and 100% OTS. In spontaneous abscesses we found 33% OS, 67% OF, 6.4% TF, 95,6% OTS. A total abscess resolution was achieved in 97,8% of patients. No major complication occurred; only 1 case of minor complication. Factors statistically influencing the outcome were postoperative vs spontaneous collections (OF: 9% vs. 67%, p < 0.0001), multiloculated vs uniloculated collections (OF: 38% vs. 1%, p < 0.0001) and upper abdominal vs lower location (OF: 13% vs. 25%, p

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Our study aimed to investigate whether radiomics on MRI sequences can differentiate responder (R) and non-responder (NR) patients based on the tumour regression grade (TRG) assigned after surgical resection in locally advanced rectal cancer (LARC) treated with neoadjuvant chemoradiotherapy (nCRT). Eighty-five patients undergoing primary staging with MRI were retrospectively evaluated, and 40 patients were finally selected. The ROIs were manually outlined in the tumour site on T2w sequences in the oblique-axial plane. Based on the TRG, patients were grouped as having either a complete or a partial response (TRG = (0,1), n = 15). NR patients had a minimal or poor nCRT response (TRG = (2,3), n = 25). Eighty-four local first-order radiomic features (RFs) were extracted from tumour ROIs. Only single RFs were investigated. Each feature was selected using univariate analysis guided by a one-tailed Wilcoxon rank-sum. ROC curve analysis was performed, using AUC computation and the Youden index (YI) for sensitivity and specificity. The RF measuring the heterogeneity of local skewness of T2w values from tumour ROIs differentiated Rs and NRs with a p-value ≈ 10−5; AUC = 0.90 (95%CI, 0.73–0.96); and YI = 0.68, corresponding to 80% sensitivity and 88% specificity. In conclusion, higher heterogeneity in skewness maps of the baseline tumour correlated with a greater benefit from nCRT.

Background: The number of elderly patients diagnosed with hepatocellular carcinoma (HCC) is progressively increasing. The aim of this study was to determine the safety and efficacy of conventional transarterial chemoembolization (TACE) in elderly HCC patients compared with younger adults. Methods: A consecutive cohort of unresectable HCC patients treated with TACE as a first-line treatment was retrospectively analyzed. Patients were categorized into “elderly” (≥ 70years, 80 patients) and “younger” (< 70years, 145 patients). Liver-related death and progression-free survival after TACE were compared before and after propensity score matching. A competing risk regression analysis was used for univariate/multivariate survival data analysis. Results: cTACE was well tolerated in both groups. The cumulative risk of both liver-related death and progression-free survival after cTACE was comparable between “elderly” and “younger” (death: 73.8% vs 69.4%, P = 0.505; progression-free survival: 48.2% vs 44.8%, P = 0.0668). Propensity model matched 61 patients in each group for gender and Barcelona Clinic Liver Cancer staging. Even after matching, the cumulative risk of liver-related death and of progression-free survival did not differ between the two groups. At multivariate analysis, Child–Pugh class, tumor gross pathology and alpha-fetoprotein were independently associated with the liver-related mortality risk. Conclusions: This study confirms that TACE is well tolerated and effective in patients aged 70years or more with unresectable HCC as it is for their younger counterparts (< 70years). Liver-related mortality was not associated with age ≥ 70years and primarily predicted by tumor multifocality, Child–Pugh class B and an increased alpha-fetoprotein value (> 31ng/ml).

Objectives: Results after trans-arterial radioembolisation (TARE) for intrahepatic cholangiocarcinoma (iCC) depend on the architecture of the tumour. This latter can be quantified through computed tomography (CT) texture analysis. The aims of the present study were to analyse relationships between CT textural features prior to TARE and objective response (OR), progression-free survival (PFS), and overall survival (OS). Methods: Texture analysis was retrospectively applied to 55 pre-TARE CT scans of iCCs, focusing attention on the histogram-based features and the grey-level co-occurrence matrix (GLCM). Texture features were harmonised using the ComBat procedure. Objective response was assessed using the Response Evaluation Criteria In Solid Tumours 1.1. The least absolute shrinkage and selection operator (LASSO) method was applied to select the most useful textural features related to OR. Results: Of the 55 patients, 53 had post-TARE imaging available, showing OR in 56.6% of cases. Texture analysis showed that iCCs showing OR after TARE had a higher uptake of iodine contrast in the arterial phase (higher mean histogram values, p< 0.001) and more homogeneous distribution (lower kurtosis, p= 0.043; GLCM contrast, p= 0.004; GLCM dissimilarity, p= 0.005, and higher GLCM homogeneity, p= 0.005; and GLCM correlation p= 0.030) at the pre-TARE CT scan. A favourable radiomic signature was calculated and observed in 15 of the 55 patients. The median PFS of these 15 patients was 12.1months and that of the remaining 40 patients was 5.1months (p= 0.008). Conclusions: Texture analysis of pre-TARE CT scans can quantify vascularisation and homogeneity of iCC architecture, providing clinical information useful in identifying ideal TARE candidates. Key Points: • Hypervascular tumours with a more homogeneous uptake of iodine contrast in the arterial phase were those most likely to be effectively treated by TARE. • The arterial phase was observed to be the best acquisition phase for providing information regarding the “sensitivity” of the tumour to TARE. • Patients with favourable radiomic signature showed a median progression-free survival of 12.1months versus 5.1months of patients with an unfavourable signature (p = 0.008).

Background: COVID assessment can be performed using the recently developed individual risk score (prediction of severe respiratory failure in hospitalized patients with SARS-COV2 infection, PREDI-CO score) based on High Resolution Computed Tomography. In this study, we evaluated the possibility of automatizing this estimation using semi-supervised AI-based Radiomics, leveraging the possibility of performing non-supervised segmentation of ground-glass areas. Methods: We collected 92 from patients treated in the IRCCS Sant’Orsola-Malpighi Policlinic and public databases, each lung was segmented using a pre-trained AI method, ground-glass opacity was identified using a novel, non-supervised approach, radiomic measurements were collected and used to predict clinically relevant scores, with particular focus on mortality and the PREDI-CO score. We compared the prediction obtained through different machine learning approaches. Results: All the methods obtained a well-balanced accuracy (70%) on the PREDI-CO score but did not obtain satisfying results on other clinical characteristics due to unbalance between the classes. Conclusions: Semi-supervised segmentation, implemented using a combination of non-supervised segmentation and feature extraction, seems to be a viable approach for patient stratification and could be leveraged to train more complex models. This would be useful in a high-demand situation similar to the current pandemic to support gold-standard segmentation for AI training.

Background The percutaneous approach (PA) for management of biliary stones (BS) with or without an underlying biliary stenosis is an option for patients in whom an endoscopic approach (EA) is not possible. The aim of this study was to evaluate the efficacy of a PA in patients with BS unsuitable for an EA. Methods A retrospective review of a database was performed. Inclusion criteria included patients with benign disease, BS who had undergone a PA. The outcomes were technical success rate, short (≤90 days) and long-term (>90 days) efficacy and safety. Results A total of 91 patients enrolled were divided into those with (n = 38) or without (n = 53) a biliary stenosis. A median of 5 (IQR:3–7) treatments/patient were performed. During a median follow-up of 23 months (IQR:3–52), the median time free from recurrence was 21 months (CI:14–29). In the long term, the PA was most efficacious in those patients without a biliary stenosis with long term success in 68% of patients as compared to 36% of patients with a biliary stenosis (p = 0.003). Conclusion A PA is an effective procedure with high initial success rate, however the coexistence of stenosis affects long-term efficacy, especially in patients with chronic biliary disease.

Background: Bile leak (BL) after hepato-pancreato-biliary (HPB) surgery is associated with significant morbidity and mortality. Aim of this study was to evaluate effectiveness and safety of percutaneous transhepatic approach (PTA) to drainage BL after HPB surgery. Methods: Between 2006 and 2018, consecutive patients who were referred to interventional radiology units of three tertiary referral hospitals were retrospectively identified. Technical success and clinical success were analyzed and evaluated according to surgery type, BL-site and grade, catheter size and biochemical variables. Complications of PTA were reported. Results: One-hundred-eighty-five patients underwent PTA for BL. Technical success was 100%. Clinical success was 78% with a median (range) resolution time of 21 (5–221) days. Increased clinical success was associated with patients who underwent hepaticresection (86%,p = 0,168) or cholecystectomy (86%,p = 0,112) while low success rate was associated to liver-transplantation (56%,p < 0,001). BL-site,grade, catheter size and AST/ALT levels were not associated with clinical success. ALT/AST high levels were correlated to short time resolution (17 vs 25 days, p = 0,037 and 16 vs 25 day, p = 0,011, respectively) Complications of PTA were documented in 21 (11%) patients. Conclusion: This study based on a large cohort of patients demonstrated that PTA is a valid and safe approach in BL treatment after HPB surgery.

BCR-ABL1 kinase domain mutation testing in tyrosine kinase inhibitor (TKI)-resistant Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukaemia (ALL) patients is routinely performed by Sanger sequencing (SS). Recently, next-generation sequencing (NGS)-based approaches have been developed that afford greater sensitivity and straightforward discrimination between compound and polyclonal mutations. We performed a study to compare the results of SS and NGS in a consecutive cohort of 171 Ph+ ALL patients. At diagnosis, 0/44 and 3/44 patients were positive for mutations by SS and NGS respectively. Out of 47 patients with haematologic resistance, 45 had mutations according to both methods, but in 25 patients NGS revealed additional mutations undetectable by SS. Out of 80 patients in complete haematologic response but with BCR-ABL1 ≥0·1%, 28 (35%) and 52 (65%) were positive by SS and NGS respectively. Moreover, in 12 patients positive by SS, NGS detected additional mutations. NGS resolved clonal complexity in 34 patients with multiple mutations at the same or different codons and identified 35 compound mutations. Our study demonstrates that, in Ph+ ALL on TKI therapy, NGS enables more accurate assessment of mutation status both in patients who fail therapy and in patients with minimal residual disease above 0·1%.

Purpose: To evaluate the diagnostic accuracy of the four standardized categories for CT reporting proposed by the Radiological Society of North America (RSNA) to support a faster triage compared with real-time reverse-transcription polymerase chain reaction (RTPCR), which is the reference standard for suspected coronavirus disease 2019 (COVID-19), but has long reporting time (6–48 hours). Materials and Methods: A retrospective analysis of 569 thin-section CT examinations performed for patients suspected of having COVID-19 from February 27 to March 27, 2020 (peak of infection in Italy) was conducted. The imaging pattern was classified according to the statement by the RSNA as “typical,” “indeterminate,” “atypical,” and “negative” and compared with RT-PCR for 460 patients. Interobserver variability in reporting between a senior and a junior radiologist was evaluated. Use of the vascular enlargement sign in indeterminate cases was also assessed. Results: The diagnosis of COVID-19 was made in 45.9% (211/460) of patients. The “typical” pattern (n = 172) showed a sensitivity of 71.6%, a specificity of 91.6%, and a positive predictive value of 87.8% for COVID-19. The “atypical” (n = 67) and “negative” (n =123) pattern demonstrated a positive predictive value of 89.6% and 86.2% for non–COVID-19, respectively. The “indeterminate” (n = 98) pattern was nonspecific, but vascular enlargement was most frequently found in patients with COVID-19 (86.1%; P , .001). Interobserver agreement was good for the “typical” and “negative” pattern and fair for “indeterminate” and “atypical” (k = 0.5; P =.002). Conclusion: In an epidemic setting, the application of the four categories proposed by the RSNA provides a standardized diagnostic hypothesis, strongly linked to the RT-PCR results for the “typical,” “atypical,” and “negative” pattern. In the “indeterminate” pattern, the analysis of the vascular enlargement sign could facilitate the interpretation of imaging features.

In chronic myeloid leukemia (CML) patients, tyrosine kinase inhibitors (TKIs) may select for drug-resistant BCR-ABL1 kinase domain (KD) mutants. Although Sanger sequencing (SS) is considered the gold standard for BCR-ABL1 KD mutation screening, next-generation sequencing (NGS) has recently been assessed in retrospective studies. We conducted a prospective, multicenter study (NEXT-in-CML) to assess the frequency and clinical relevance of low-level mutations and the feasibility, cost, and turnaround times of NGS-based BCR-ABL1 mutation screening in a routine setting. A series of 236 consecutive CML patients with failure (n = 124) or warning (n = 112) response to TKI therapy were analyzed in parallel by SS and NGS in 1 of 4 reference laboratories. Fifty-one patients (22 failure, 29 warning) who were negative for mutations by SS had low-level mutations detectable by NGS. Moreover, 29 (27 failure, 2 warning) of 60 patients who were positive for mutations by SS showed additional low-level mutations. Thus, mutations undetectable by SS were identified in 80 out of 236 patients (34%), of whom 42 (18% of the total) had low-level mutations somehow relevant for clinical decision making. Prospective monitoring of mutation kinetics demonstrated that TKI-resistant low-level mutations are invariably selected if the patients are not switched to another TKI or if they are switched to a inappropriate TKI or TKI dose. The NEXT-in-CML study provides for the first time robust demonstration of the clinical relevance of low-level mutations, supporting the incorporation of NGS-based BCR-ABL1 KD mutation screening results in the clinical decision algorithms.

The aim of this study was to evaluate the morphologic appearance, the clinical scenario, and the outcomes of patients with portal hypertensive biliopathy (PHB), particularly in the symptomatic subgroup treated with interventional radiology (IR) procedures. The outcome of 20 patients with PHB were retrospectively reviewed over a 5-year period. In all cases, the extrahepatic portal vein occlusion (EHPVO) and the compensatory cavernomatosis was the cause of PHB. Eight out of 20 patients had severe symptoms (jaundice and bleeding). Five out of these eight patients were successfully treated with IR procedures. PHB is a rare but serious complication of PH from EHPVO. IR treatments are highly effective in controlling symptoms. Moreover, IR procedures, as drainage and transjugular intrahepatic portosystemic shunt placement, are the first-line treatment in cases of life-threatening bleeding from ruptures of the varices.

Purpose: Patients with hepatocellular carcinoma (HCC) of intermediate stage (BCLC-B according to the Barcelona Clinic Liver Cancer classification) are a heterogeneous group with different degrees of liver function impairment and tumour burden. The recommended treatment is transarterial chemoembolization (TACE). However, patients in this group may be judged as poor candidates for TACE because the risk-benefit ratio is low. Such patients may receive transarterial radioembolization (TARE) only by entering a clinical trial. Experts have proposed that the stage could be further divided into four substages based on available evidence of treatment benefit. We report here, for the first time, the outcome in patients with BCLC-B2 substage HCC treated with TARE. Methods: A retrospective analysis of the survival of 126 patients with BCLC-B2 substage HCC treated with TARE in three European hospitals was performed. Results: Overall median survival in patients with BCLC-B2 substage was not significantly different in relation to tumour characteristics; 19.35 months (95% CI 8.27–30.42 months) in patients with a single large (>7 cm) HCC, and 18.43 months (95% CI 15.08–21.77 months) in patients with multinodular HCC (p = 0.27). However, there was a higher proportion of long-term survivors at 36 months among those with a single large tumour (29%) than among those with multiple tumours (16.8%). Conclusion: Given the poor efficacy of TACE in treating patients with BCLC-B2 substage HCC, TARE treatment could be a better choice, especially in those with a large tumour.

Systemic mastocytosis is a rare heterogeneous myeloproliferative neoplasm characterized by abnormal proliferation and activation of mast cells. We describe a large multicentre series of 460 adult patients with systemic mastocytosis, with a diagnosis based on WHO 2008 criteria, in a "real-life" setting of ten Italian centers with dedicated multidisciplinary programs. We included indolent forms with (n = 255) and without (n = 165) skin lesions, smouldering (n = 20), aggressive (n = 28), associated with other hematological diseases mastocytosis (n = 21) and mast cell leukemia (n = 1). This series was uniquely characterized by a substantial proportion of patients with low burden of neoplastic mast cells; notably, 38% of cases were diagnosed using only minor diagnostic criteria according to WHO 2008 classification, underlying the feasibility of early diagnosis where all diagnostic approaches are made available. This has particular clinical relevance for prevention of anaphylaxis manifestations, that were typically associated with indolent forms. In multivariate analysis, the most important features associated with shortened overall survival were disease subtype and age at diagnosis >60 years. Disease progression was correlated with mastocytosis subtype and thrombocytopenia. As many as 32% of patients with aggressive mastocytosis suffered from early evolution into acute leukemia. Overall, this study provides novel information about diagnostic approaches and current presentation of patients with SM and underlines the importance of networks and specialized centers to facilitate early diagnosis and prevent disease-associated manifestations. Am. J. Hematol. 91:692-699, 2016. © 2016 Wiley Periodicals, Inc.

UNLABELLED : Optimal use of current therapeutic opportunities for chronic myeloid leukemia patients requires integration of clinical and laboratory monitoring. Assessment of molecular response (MR) by real-time quantitative polymerase chain reaction is the most sensitive way to monitor tyrosine kinase inhibitor (TKI) treatment efficacy. Besides major molecular response, which has emerged as a safe haven for survival since the initial studies of first-line imatinib treatment, two additional MR milestones have recently been defined: early molecular response and deep molecular response. The achievement of such MR milestones within defined time points during therapy is thought to draw the ideal trajectory toward optimal long-term outcome and, possibly, successful treatment discontinuation. Sensitive and reproducible MR measurement and proper interpretation of MR results are therefore critical to correctly inform therapeutic decisions. In patients who do not achieve an optimal response to TKI therapy, BCR-ABL1 mutation screening should also be performed, because it may deliver useful information for TKI choice. This review aims to help clinicians apply and translate the latest response definitions and clinical recommendations into practice. We provide a critical update on how these recommendations have incorporated MR levels in the clinical decision algorithms and how detection of BCR-ABL1 mutations should be interpreted. We also include a practical guide for pathologists and molecular biologists to best perform molecular testing and for hematologists and oncologists to best integrate it into routine practice. IMPLICATIONS FOR PRACTICE Ever-more-potent therapeutic strategies have been developed for chronic myeloid leukemia (CML) in parallel with the evolution of therapeutic goals and the refinement of response definitions and monitoring schemes and procedures. Terminology and methodology continue to evolve rapidly, making it difficult for busy hematology/oncology professionals to keep abreast of the newest developments. Optimal CML patient management results from the timely and rational use of molecular testing, the critical assessment of the power and pitfalls of current technology, and the appropriate interpretation and contextualization of results.

Currently, physicians treating chronic myeloid leukaemia (CML) patients can rely on a wide spectrum of therapeutic options: the best use of such options is essential to achieve excellent clinical outcomes and, possibly, treatment-free remission (TFR). To accomplish this, proper integration of expert clinical and laboratory monitoring of CML patients is fundamental. Molecular response (MR) monitoring of patients at defined time points has emerged as an important success factor for optimal disease management and BCR-ABL1 kinase domain mutation screening is useful to guide therapeutic reassessment in patients who do not achieve optimal responses to tyrosine kinase inhibitor therapy. Deeper MRs might be associated with improved long-term survival outcomes. More importantly, they are considered a gateway to TFR. In molecular biology, novel procedures and technologies are continually being developed. More sophisticated molecular tools and automated analytical solutions are emerging as CML treatment endpoints and expectations become more and more ambitious. Here we provide a critical overview of current and novel methodologies, present their strengths and pitfalls and discuss what their present and future role might be.

In chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients who fail imatinib treatment, BCR-ABL1 mutation profiling by Sanger sequencing (SS) is recommended before changing therapy since detection of specific mutations influences second-generation tyrosine kinase inhibitor (2GTKI) choice. We aimed to assess i) in how many patients who relapse on second-line 2GTKI therapy next generation sequencing (NGS) may track resistant mutations back to the sample collected at the time of imatinib resistance, before 2GTKI start (switchover sample) and ii) whether low level mutations identified by NGS always undergo clonal expansion. To this purpose, we used NGS to retrospectively analyze 60 imatinib-resistant patients (CML, n = 45; Ph+ ALL, n = 15) who had failed second-line 2GTKI therapy and had acquired BCR-ABL1 mutations (Group 1) and 25 imatinib-resistant patients (CML, n = 21; Ph+ ALL, n = 4) who had responded to second-line 2GTKI therapy, for comparison (Group 2). NGS uncovered that in 26 (43%) patients in Group 1, the 2GTKI-resistant mutations that triggered relapse were already detectable at low levels in the switchover sample (median mutation burden, 5%; range 1.1%–18.4%). Importantly, none of the low level mutations detected by NGS in switchover samples failed to expand whenever the patient received the 2GTKI to whom they were insensitive. In contrast, no low level mutation that was resistant to the 2GTKI the patients subsequently received was detected in the switchover samples from Group 2. NGS at the time of imatinib failure reliably identifies clinically relevant mutations, thus enabling a more effective therapeutic tailoring.

A variety of mechanisms underlie the lack or loss of response to TKIs in patients (pts) with CML, but acquisition of point mutations in the BCR-ABL1 KD is, at present, the only actionable one. Detection of a TKI-resistant mutation by Sanger Sequencing (seq) should trigger a change of therapy and, in some cases, guide TKI selection. Although research studies have shown that NGS may hold added value over Sanger seq for BCR-ABL1 KD mutation screening, routine NGS is technically demanding and expensive and is not yet widely available. Newer methods, like ddPCR, might represent an attractive alternative. Here we set out to 1) assess the actionability of results, hence the potential clinical benefit, of more sensitive NGS-based testing vs Sanger seq in a consecutive series of CML pts who had non-optimal response to TKI therapy according to the 2013 ELN recommendations, and 2) test a novel ddPCR-based multiplex assay for rapid screening for a panel of BCR-ABL1 KD mutations relevant for TKI selection. Between January 2015 and May 2019, samples from 712 CML pts followed at one of 66 GIMEMA CML Working Party hematology centers were referred to our laboratory for BCR-ABL1 KD mutation testing because of a Failure (n=251 pts) or Warning (n=461 pts) response to TKI therapy. In parallel to Sanger seq, NGS of amplicons generated by nested reverse transcription(RT)-PCR was performed on a Roche GS Junior instrument until April 2017, and on an Illumina MiSeq instrument from May 2017 on. Read alignment and variant calling was done using AmpSuite software (SmartSeq). Variants detected in Among Failures, pts positive for BCR-ABL1 KD mutations by Sanger seq were 88/251 (35%). NGS detected low level mutations in 38/251 (15%) additional pts who were negative for mutations by Sanger seq. Moreover, 31/251 (12%) Failure pts were found to have low level mutations additional to those detectable by Sanger seq. Among low level mutations detected by NGS, those actually relevant for TKI selection (2GTKI-resistant) were identified in 22/251 (9%) pts. Compound mutations were found in 10 pts (4%; all progressed to blastic phase). The average turnaround time (TAT) of routine NGS-based analysis was 15 working days (range, 11-32). Thus, in the setting of Failure, NGS-based mutation scanning of the whole KD is of real clinical benefit in a minority of pts while increasing the TAT. Among Warnings, pts positive for BCR-ABL1 KD mutations by Sanger seq were 65/461 (14%). NGS detected low level mutations in 97/461 (21%) additional pts who were negative for mutations by Sanger seq. All Warning pts positive for low level resistant mutations who were not switched to another TKI turned their response into Failure after 3 to 8 months. The multiplex digital PCR assay proved capable to accurately identify and separate the T315I/A, F317L/V/I/C, Y253H, E255K, F359V/I/C, E255V, V299L mutations in five spatially distinct clusters (pan-resistant, Dasatinib-resistant, Nilotinib-resistant, Nilotinib- and Bosutinib-resistant, Dasatinib- and Bosutinib-resistant, respectively) starting from as little as 30ng of total cDNA or 1ng of the 1.7kb 1st PCR product. Very good concordance was observed between ddPCR- and NGS-identified mutations irrespective of mutation frequency or cluster proximity, even for compound mutations. Extensive assay assessment will be presented. TAT of ddPCR was 1 day. In conclusion: - Mutation testing in the Failure setting aims to a timely and rational TKI switch. The incidence of low level mutations relevant to the selection of subsequent-line therapy and the TAT of routine NGS in our cohort suggest that multiplex ddPCR would be an easier and faster alternative. - Mutation testing in the Warning setting may identify pts who need a change in therapy rather than a 'watch and wait' approach. In our cohort, approx 1/5 of the Warning pts negative by Sanger seq had low level mutations resistant to the TKI they were receiving, that ultimately led to Failure. Earlier detection of emerging resistant mutations enabled by NGS (or by ddPCR in pts receiving 2GTKIs) should support proactive TKI switch. Disclosures Soverini: Incyte: Consultancy. Iurlo:Incyte: Other: Speaker Honoraria; Novartis: Other: Speaker Honoraria; Pfizer: Other: Speaker Honoraria. Pregno:Bristol Myers Squibb: Honoraria; Incyte: Consultancy, Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Bonifacio:BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Amgen: Honoraria. Lunghi:Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria. Castagnetti:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Bristol Myers Squiib: Consultancy, Honoraria. Gugliotta:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria. Rosti:BMS: Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Tiribelli:Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Stagno:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; BMS: Honoraria. D'Adda:Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Crugnola:Incyte: Honoraria; Novartis: Honoraria. Corner:BioRad: Employment. Maar:BioRad: Employment. Pane:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: research founding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Saglio:BMS: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Jansen: Consultancy; Celgene: Consultancy. Baccarani:Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Takeda: Consultancy. Martinelli:Pfizer: Consultancy; ARIAD: Consultancy; Roche: Consultancy; BMS: Consultancy; Novartis: Consultancy. Cavo:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria.

PURPOSE: Here, we studied whether amplicon next-generation deep sequencing (NGS) could improve the detection of emerging BCR-ABL1 kinase domain mutations in chronic phase chronic myeloid leukemia (CML) patients under tyrosine kinase inhibitor (TKI) treatment and discussed the clinical relevance of such sensitive mutational detection. METHODS: For NGS data evaluation including extraction of biologically relevant low-level variants from background error noise, we established and applied a robust and versatile bioinformatics approach. RESULTS: Results from a retrospective longitudinal analysis of 135 samples of 15 CML patients showed that NGS could have revealed emerging resistant mutants 2-11 months earlier than conventional sequencing. Interestingly, in cases who later failed first-line imatinib treatment, NGS revealed that TKI-resistant mutations were already detectable at the time of major or deeper molecular response. Identification of emerging mutations by NGS was mirrored by BCR-ABL1 transcript level expressed either fluctuations around 0.1 %(IS) or by slight transcript level increase. NGS also allowed tracing mutations that emerged during second-line TKI therapy back to the time of switchover. Compound mutants could be detected in three cases, but were not found to outcompete single mutants. CONCLUSIONS: This work points out, that next-generation deep sequencing, coupled with a robust bioinformatics approach for mutation calling, may be just in place to ensure reliable detection of emerging BCR-ABL1 mutations, allowing early therapy switch and selection of the most appropriate therapy. Further, prospective assessment of how to best integrate NGS in the molecular monitoring and clinical decision algorithms is warranted.

Introduction Some retrospective studies in tyrosine kinase inhibitor (TKI)-resistant Philadephia-positive (Ph+) leukemia patients (pts) have suggested that deep sequencing (DS) may provide a more accurate picture of BCR-ABL1 kinase domain (KD) mutation status as compared to conventional sequencing (CS). However, the frequency and clinical relevance of low burden mutations remains to be explored prospectively in large series of unselected pts. In addition, the implementation of routine BCR-ABL1 DS in multiple molecular diagnostic laboratories has never been attempted. These open issues led us to design a multi-center, multi-laboratory prospective study ('NEXT-IN-CML') aimed to assess the feasibility, performance and informativity of DS for BCR-ABL1 KD mutation screening. Aims The first phase of the study was aimed to establish a network of 5 reference labs sharing a standardized DS workflow, a joint database for clinical and mutational data storage and a common pipeline of data analysis, interpretation and clinical reporting. The second phase of the study, involving 54 Italian Hematology Units, is aimed to assess the frequency and clinical significance of low burden mutations detectable by DS by prospective collection and analysis of samples from chronic myeloid leukemia (CML) pts who exhibit failure (F) or warning (W) responses and relapsed Ph+ acute lymphoblastic leukemia (ALL) pts. Methods A PCR and an amplicon DS protocol already set up and optimized for the Roche GS Junior in the framework of the IRON II international consortium was adopted. In the first phase, 5 batches of blinded cDNA samples were prepared and shipped to evaluate individual lab performances. The batches included archival samples with known BCR-ABL1 mutation status as assessed by CS and serial dilutions of BaF3 T315I+ cells in BaF3 unmutated cells, simulating mutation loads of 20% down to 1%. In the ongoing second phase prospectively, consecutively collected CML and Ph+ ALL samples are being analyzed in parallel by CS and DS. Clinical history and follow-up data are used for correlations. Results In the first phase of the study, 312/320 amplicons were successfully generated and sequenced. A median of 124,686 (range, 48,181-170,687) high quality reads were obtained across the 5 labs. Median number of forward and reverse reads was 1,757 (range 884-7,838), with no coverage dropouts for any amplicon or index. Comparison of observed vs expected mutations showed that 76/78 evaluable samples were accurately scored. In the remaining two, the analysis software failed to detect the 35bp insertion ('35INS') commonly detectable between exons 8 and 9. Quantitation of point mutation burden was highly reproducible across the entire range of frequencies, from 100% to 1%. The second phase of the study has started in Jan 2016. As of Jul 31st, a total of 106 consecutive pts (CML, n=96; Ph+ ALL, n=10) have been enrolled. The present analysis focuses on the first 75 CML pts (60 F and 15 W), for whom sequencing results are currently available (analysis of the entire population of patients enrolled up to Nov 2016 will be presented at the meeting). Clinically actionable mutations have been detected in 10/75 (14%) pts by CS and in 20/75 pts (27%) by DS. Notably, among the 10 pts positive for clinically actionable mutations by DS but not by CS, 3 had a low burden T315I (2 F [dasatinib, imatinib] and 1 W [dasatinib]). In 5 additional pts negative for mutations by CS (3 F and 2 W), DS identified multiple low burden mutations with unknown IC50, suggesting that the cooperation of individually 'weak' mutants may be a new mechanism underlying reduced TKI efficacy. Longitudinal analysis and follow-up of pts are shaping the clinical significance of different types of low burden mutations and will be presented. Conclusions The 'NEXT-in-CML' study is demonstrating that DS of BCR-ABL1 can successfully be implemented in national lab networks and is an important step forward towards routine use of this technology. We have now adapted the protocol for both the Ion Torrent PGM and the Illumina Miseq platforms. For a minimum of 15 samples per sequencing run, DS costs are estimated to equal those of CS (cost per sample, reagents only: ≈100€ for PGM (314 chip) and Miseq (nano kit v2) vs ≈95€ for CS) with comparable turnaround times for delivery of results. Our study is also contributing useful data for the clinical interpretation of DS findings. Disclosures Soverini: Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Castagnetti:ARIAD Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Ciceri:MolMed SpA: Consultancy. Breccia:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Di Raimondo:Janssen-Cilag: Honoraria. Bassan:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cavo:Millennium: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Rosti:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Baccarani:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Saglio:Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Martinelli:Ariad: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; BMS: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; MSD: Consultancy; Genentech: Consultancy.

Background The treatment of Philadelphia chromosome-positive Acute Lymphoblastic Leukemia (Ph+ ALL) patients who harbor the T315I BCR-ABL1 mutation or who have two or more mutations in the same BCR-ABL1 molecule is particularly challenging since first and second-generation Tyrosine Kinase Inhibitors (TKIs) are ineffective. Ponatinib, blinatumomab, chemotherapy and transplant are the currently available options in these cases. Case presentation We here report the case of a young Ph+ ALL patient who relapsed on front-line dasatinib therapy because of two independent T315I-positive subclones, resulting from different nucleotide substitutions -one of whom never reported previously- and where additional mutant clones outgrew and persisted despite ponatinib, transplant, blinatumomab and conventional chemotherapy. Deep Sequencing (DS) was used to dissect the complexity of BCR-ABL1 kinase domain (KD) mutation status and follow the kinetics of different mutant clones across the sequential therapeutic approaches. Conclusions This case presents several peculiar and remarkable aspects: i) distinct clones may acquire the same amino acid substitution via different nucleotide changes; ii) the T315I mutation may arise also from an ‘act’ to ‘atc’ codon change; iii) the strategy of temporarily replacing TKI therapy with chemo or immunotherapy, in order to remove the selective pressure and deselect aggressive mutant clones, cannot always be expected to be effective; iv) BCR-ABL1-mutated sub-clones may persist at very low levels (undetectable even by Deep Sequencing) for long time and then outcompete BCR-ABL1-unmutated ones becoming dominant even in the absence of any TKI selective pressure. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3511-2) contains supplementary material, which is available to authorized users.

FOXM1 transcription factor is a central component of tumor initiation, growth, and progression due to its multiple effects on cell cycle, DNA repair, angiogenesis and invasion, chromatin, protein anabolism, and cell adhesion. Moreover, FOXM1 interacts with β-catenin promoting its nuclear import and transcriptional activation. Here, we show that FOXM1 is involved in the advantage of chronic myeloid leukemia hematopoiesis over the normal counterpart. FOXM1 hyper-activation associated with BCR-ABL1 results from phosphorylation by the fusion protein kinase-dependent activation of Polo-like kinase 1. FOXM1 phosphorylation lets its binding with β-catenin and β-catenin transcriptional activation, a key event for persistence of the leukemic stem cell compartment under tyrosine kinase inhibitor therapy. Polo-like kinase 1 inhibitor BI6727, already advanced for clinical use, breaks β-catenin interaction with FOXM1, hence hampering FOXM1 phosphorylation, β-catenin binding, nuclear import, and downstream signaling. In conclusion, our results support Polo-like kinase 1/FOXM1 axis as a complementary target to eradicate leukemic early progenitor/stem cell compartment in chronic myeloid leukemia. J. Cell. Biochem. 118: 3968-3975, 2017. © 2017 Wiley Periodicals, Inc.

Adult B-ALL patients still have a dismal prognosis, due to a high incidence of relapse even after allogenic SCT. Safety and efficacy of Blinatumomab, an anti CD3-CD19 Bite antibody, has been demonstrated both in MRD positive patients and in relapsed/refractory (R/R) setting. To evaluate safety profile and efficacy of Blinatumomab, obtained through a compassionate use, in a cohort of 18 adult patients affected by MRD+ or R/R B-ALL treated at Bologna University. From March 2015 to December 2017, 18 patients received Blinatumomab at the standard dosage (9 mcg/d x 7 days, 28 mcg/d x 21 days) in 28-days courses. All the patients were hospitalized to receive the first course of therapy. The following courses, based on the good safety profile of the compound, were administered in outpatient setting. 18 patients (M/F = 10/8; median age 43, range 18-73) have been treated. Philadelphia (Ph) chromosome was detected in 8/18 patients. 10 patients were MRD+ (5 Ph pos and 5 Ph neg); E2A-PBX1 and MLL-AF4 rearrangements were found in two patients. 8 patients had a R/R disease (3 Ph pos and 5 Ph neg). Median WBC count before starting therapy with Blinatumomab was 5400/mmc (range 500-76500). All the patients had previously received many lines of therapy (median 4, range 1-7). In 4 cases an alloTMO was already performed, and two patients had received two transplants. 12/18 patients were referred to us by other Italian Institutions. All the patients received at least one course of Blinatumomab. In one case three courses were administered; an elderly patient is actually receiving the fifth course. Globally, 32 courses of therapy have been administered (median 2, range 1-5). Bone marrow evaluations, including cytogenetics, molecular biology and immunophenotyping analysis were performed at the beginning of every course of therapy in order to assess patients' disease status. MRD evaluation was assessed through BCR-ABL fusion transcript quantitative analysis in Ph pos ALL patients and Ig rearrangment in Ph negative patients. Monitoring of adverse events was periodically performed. 16/18 patients are evaluable for response, at least to one cycle (one patient died during the first course, one patient is still receiving the first course). 9/16 (56%) patients obtained a CR (7/9 MRD+ and 2/7 R/R). In 7/9 (78%) responders patients a molecular CR was reached, (in 6 patients after the first course, in one case after the second one). 5 responders proceeded to alloBMT and are actually alive in CR (median follow-up after transplant 240 days). In terms of toxicity, one patient developed a grade IV neurological event (mental confusion, tremor), which completely resolved after a transient drug withdrawal. Our results confirm the high rate of response and to Blinatumomab in a poor patients' population, and the good management profile of the compound. Note: This abstract was not presented at the meeting. Citation Format: Giovanni Martinelli, Stefania Paolini, Claudio Cerchione, Alessandra Santoro, Valentina Robustelli, Simona Soverini, Caterina De Benedittis, Enrica Imbrogno, Andrea Ghelli Luserna Di Rora, Sarah Parisi, Chiara Sartor, Giovanni Marconi, Silvia Lo Monaco, Maria Chiara Abbenante, Maria Chiara Fontana, Antonella Padella, Anna Ferrari, Giorgia Simonetti, Elena Tenti, Federica Frabetti, Francesca Volpato, Samantha Bruno, Fabiana Mammoli, Maria Teresa Bocchicchio, Carmen Baldazzi, Cristina Papayannidis. Blinatumomab is safe and effective in relapsed and MRD positive B-ALL CD19+ patients: The bologna compassionate program experience [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3100.

Next-Generation Sequencing (NGS)-based BCR-ABL1 kinase domain (KD) mutation screening has been shown to enable greater accuracy and sensitivity and straightforward identification of compound mutants (CM) as compared to Sanger sequencing (seq). However, the prevalence of CMs has never been assessed in prospective studies, and although in vitro data suggest that many of them may be challenging for all tyrosine kinase inhibitors (TKIs) including ponatinib, attempts to correlate such data with in vivo responses have never been made. To address these issues, we have reviewed the results of routine NGS-based BCR-ABL1 KD mutation screening performed over the past 3 years. Between 2015 and 2018, we have prospectively used NGS to analyze a consecutive series of 751 Ph+ leukemia patients (pts) on TKI therapy who were eligible for BCR-ABL1 KD mutation screening according to ELN/NCCN/ESMO recommendations. The study population included 664 chronic myeloid leukemia (CML) pts with failure or warning response (chronic phase [CP], n=593; accelerated or blastic phase [AP/BP], n=71) and 87 Ph+ acute lymphoblastic leukemia (ALL) pts with relapsed/refractory disease. NGS of ≈400bp amplicons generated by nested RT-PCR was performed on a Roche GS Junior (until April 2017) or on an Illumina MiSeq (from May 2017 on) using custom protocols whose accuracy, sensitivity and reproducibility was checked by national and international (EUTOS) control rounds. Read alignment and variant calling was done using the AmpSuite software (SmartSeq srl), with a lower detection limit set to 3%. Cis or trans configuration of mutation pairs, indicating CMs or polyclonality, respectively, was determined correcting for the likelihood of PCR recombination. The 35INS insertion/truncation mutant was excluded from the analysis. NGS identified mutations in the BCR-ABL1 KD in a total of 313/664 (47%) CML pts (255/593 [43%] CP-CML and 58/71 [82%] AP/BP-CML) and 69/87 (79%) Ph+ ALL pts. Ninety-one percent of the mutations could be recognized as conferring resistance to at least one TKI on the basis of publicly available IC50 data or published reports. In 42/593 (7%) CP-CML, 6/71 (8.5%) AP/BP-CML and 12/87 (14%) Ph+ ALL pts, low burden mutations (i.e., mutations carried by a proportion of transcripts 15% - hence detectable by Sanger seq). Fifty-five (9.2%) CP-CML, 51 (72%) AP/BP-CML and 56 (49%) Ph+ ALL pts had ≥2 mutations (CP-CML: 1-5 mutations; AP/BP-CML: 1-6 mutations; Ph+ ALL: 1-13 mutations). Identification of CMs in pts with ≥2 mutations was fully possible (i.e., all the candidate pairs mapped within a distance of 400bp) in 71% of cases and partially possible (i.e., some, but not all the candidate pairs mapped within a distance of 400bp) in another 12% of cases. A total of 86 CMs (85 double and 1 triple) in 73 pts (21 [3.5%] CP-CML, 23 [32%] AP/BP-CML and 29 [37%] Ph+ ALL pts) could be catalogued (Figure 1A). All but two (T315I+D276G, M244V+E255K) were detected in pts who had received ≥2 TKIs and all included at least a 2nd-generation TKI-resistant mutation. The most frequent CMs were T315I+E255K, T315I+E255V, T315I+F359V, F317L+Y253H (Figure 1A). The triple CM, detected in a ponatinib-resistant pt, was F317I+Y253F+Q252H. Correlation of IC50 data with in vivo responses (the TKIs pts were clinically resistant to) confirmed only partially in vitro predictions (Figure 1B). In particular, although ponatinib was shown in vitro to be poorly effective against several CMs, only the T315I+E255V was consistently found to be associated with ponatinib failure. In conclusion, our results in a large unselected series of TKI-resistant pts analyzed by NGS show that:CMs are relatively infrequent in CP-CML, but may be a relevant issue in AP/BP-CML and Ph+ ALL;among pts with multiple mutations, those who have failed 1 line of therapy have most often polyclonality, whereas those who have failed ≥2 lines of therapy may have CMs or polyclonality;in vitro predictions of sensitivity and insensitivity based on IC50 data should be regarded with caution. In particular, the only compound mutant that we consistently found to be associated with ponatinib failure was the T315I+E255V. Supported by EUTOS 2016. Disclosures Soverini: Novartis: Consultancy; Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy. Pagano:Pfizer: Speakers Bureau; Gilead: Speakers Bureau; Basilea: Speakers Bureau; Merck: Speakers Bureau; Janssen: Speakers Bureau. Gugliotta:Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Castagnetti:Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Meyers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Angelucci:Roche Italy: Other: Local (national) advisory board; Novartis: Honoraria, Other: Chair Steering Comiittee TELESTO Protocol; Celgene: Honoraria, Other: Chair DMC; Jazz Pharmaceuticals Italy: Other: Local ( national) advisory board; Vertex Pharmaceuticals Incorporated (MA) and CRISPR CAS9 Therapeutics AG (CH): Other: Chair DMC. Martinelli:Abbvie: Consultancy; Ariad/Incyte: Consultancy; Janssen: Consultancy; Novartis: Speakers Bureau; Jazz Pharmaceuticals: Consultancy; Roche: Consultancy; Pfizer: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy. Cavo:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees.

In Philadelphia-positive (Ph+) Acute Lymphoblastic Leukemia (ALL) patients (pts), resistance to tyrosine kinase inhibitors (TKIs) is frequently associated with the selection of one or more mutations in the BCR-ABL1 kinase domain (KD). The swift emergence of mutant clones as early as during induction therapy supports the hypothesis that, at least in some cases, mutations may already be present at diagnosis. Next Generaton Sequencing (NGS) has been proposed as an alternative to Sanger sequencing (seq) for BCR-ABL1 KD mutation screening because of its greater sensitivity and accuracy, but no studies have so far evaluated its prospective use in Ph+ ALL. Between 2015 and 2018, we have used NGS in parallel to Sanger seq to analyze a consecutive series of 126 Ph+ ALL pts who were newly diagnosed (n=39) or who had relapsed/refractory disease (n=87) on TKI therapy. In 22 cases, both bone marrow and peripheral blood were analyzed and compared. NGS of ≈400bp amplicons generated by nested RT-PCR was performed on a Roche GS Junior (until April 2017) or on an Illumina MiSeq (from May 2017 on). Read alignment and variant calling (with a lower limit set to 3%) were done with the AmpSuite software (SmartSeq srl). When multiple mutations mapped within the same sequence reads, assessment of cis vs trans configuration was done correcting for the probability of PCR recombination. Three out of 39 (7.7%) de novo Ph+ ALL pts had low burden point mutations detectable by NGS: one had a V289A (variant frequency, 3.4%); one had a D276G (4.0%) and a F359V (3.5%); one had an E255K mutation (3.3%). The first pt was enrolled in the GIMEMA LAL1811 study of frontline ponatinib; the second and the third pts were enrolled in the GIMEMA D-ALBA study of frontline sequential treatment with dasatinib and blinatumomab. All pts achieved molecular remission, consistently with the mutations being sensitive to the TKIs received. The 35INS insertion/truncation mutant was detected in 27 (69%) pts, who all have so far achieved molecular remission. This is in line with the report by O'Hare et al (Blood 2011) suggesting that the 35INS variant is kinase-inactive and does not contribute to TKI resistance. For this reason, the 35INS was excluded from subsequent analyses. Relapsed/refractory pts positive for mutations by Sanger seq were 57 (65%); those positive for mutations by NGS were 69 (79%). Fifty-six out of 87 (49%) pts had >1 mutation (up to 13) detected by NGS. NGS identified low burden mutations (i.e., mutations present in a proportion of transcripts between 3 and 20%) in 12 pts who were negative for mutations by Sanger seq. Most importantly, NGS provided a more accurate picture of BCR-ABL1 mutations status in 40 (46%) pts who turned out to have one or more low burden mutations in addition to the dominant mutation(s) detectable by Sanger seq. In all cases, each low burden mutation detected by NGS could be recognized as poorly sensitive either to the TKI the pt was receiving at the time of testing, or to the previous TKI. The clonal nature of NGS-based analysis further proved its utility i) in 4 pts where Sanger seq had shown 2 base substitutions in the same codon so that the actual amino-acid change(s) were impossible to infer (a ponatinib-resistant pt with a T315M mutation, 2 dasatinib-resistant pts with various combinations of F317I, F317C and/or F1317L, a dasatinib-resistant pt with 2 different nucleotide substitutions both leading to the V299L), and ii) in 48/56 pts who had ≥2 mutations whose clonal configuration could not be resolved. Twenty-eight out of these 48 pts were found to carry one or more (up to 3) compound mutants. Compound mutants were more common in pts who had failed ≥2 lines of therapy, whereas polyclonality was more common in pts who had failed first line therapy. The most frequent compound mutants were T315I+E255K and T315I+E255V. Interestingly, the latter was associated with poor or no response to ponatinib. Our results in a relatively large series of Ph+ ALL pts suggest that an NGS-based approach provides a more accurate characterization of the complexity of BCR-ABL1 KD mutation status, including compound mutants some of whom may be poorly sensitive even to ponatinib. Mutations may already be detected at the time of diagnosis. It remains to be assessed whether more sensitive techniques like digital PCR may identify a greater number of pts with pre-therapy mutations and whether the detection of pre-therapy mutations may be used to guide 1st-line treatment selection. Disclosures Soverini: Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Pagano:Gilead: Speakers Bureau; Basilea: Speakers Bureau; Merck: Speakers Bureau; Janssen: Speakers Bureau; Pfizer: Speakers Bureau. Abruzzese:Ariad: Consultancy; BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Martinelli:Roche: Consultancy; Celgene: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Abbvie: Consultancy; Janssen: Consultancy; Ariad/Incyte: Consultancy; Amgen: Consultancy. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Objectives and background: According to the World Health Organization (WHO) classification, the diagnosis of Systemic Mastocytosis (SM) relies on bone marrow (BM) examination and is based on a major and four minor criteria. The somatic ‘autoactivating’ point mutation D816V in the KIT receptor gene is one of the minor criteria, founded in the great majority of patients (90%) and it plays a central role in the pathogenesis of the disease. Indolent Systemic Mastocytosis (ISM) is the most common variant of SM, characterized by a very low MC burden and associated with very different clinical pictures. A highly sensitive diagnostic methods for D816V detection are required to assure an appropriate diagnosis and to reduce false-negative results. The recent development of “ultra-deep amplicon sequencing” (UDS) technologies has opened the way to a more accurate characterization of molecular aberrations with higher sensitivity of screening for known and unknown mutations. Our aims were: i) to set-up and optimize a UDS-based mutation screening strategy of the KIT gene on the Roche GS Junior Instrument; ii) to test the sensitivity of our UDS assay to detect the D816V mutation; iii) to investigate the presence of additional KIT mutations in SM. Methods: We decided to take advantage of a next generation sequencing approach to perform an UDS KIT gene mutation analysis on 20 bone marrow (BM) samples from patients whit ISM that were negative for the D816V mutation by Sanger Sequencing which has a sensitivity of 20%. Fusion primers were designed to generate ten partially overlapping amplicon covering the whole KIT transcript (exons 1-21) by RT-PCR. To determine the lower detection limit of our UDS-assay, serial dilutions of the HMC-1 cell line (harboring the D816V mutation) into an unmutated K562 cell line in ratios such as to simulate the following mutation loads were sequenced: 50%, 37.5%, 25%, 12.5%, 5%; 2.5%, 1.25%, 0.5%, 0.25%. Results and significance: UDS of cell line dilutions showed a high accuracy of D816V mutation detection and linearity of mutation calling over the entire range down to 0.25%. The UDS technology allowed to detected the D816V mutation, below the lower detection limit of Sanger Sequencing, with an abundance from 0.5% to 11%, in 12/20 ISM patients. Two additional sequence variations were detected in a large proportion of patients. These two variations included a 3bp in-frame deletion in exon 15 (GenBank X06182.1: c.2164_2166delAGC; p.S715del) found in 11/20 patients and a 12bp in frame-deletion in exon 9 in all patients, whit an abundance ranging from 83% to 97% (GenBank X06182.1: c.1550_1561delGTAACAACAAAG; p.G510_K513del). Previously published studies indicate that the KIT Gly-Asn-Asn-Lys510-513+/- alternatively spliced located immediately downstream to the extracellular KIT domain and KIT Ser715+/-, an interkinase KIT domain, are expressed in normal human hematopoietic cell, leukemic cell lines, acute myeloid leukemia blast and GISTs and represent rather a splice variant of KIT transcript. Interestingly our results showed the presence of the transmembrane domain M541L (GenBank X06182.1: c.1642A>C; p.Met541Leu) KIT-activating mutation in exon 10, with an abundance of 50%, in addition to D816V, in 2/20 ISM. This mutation is known to retain sensitivity to imatinib mesylate. Conclusions: Our preliminary results suggest that our-UDS based KIT gene mutation screening assay might be a reliable and sensitive alternative to conventional sequencing methods for the detection of the D816V. We are now planning to investigate whether the greater sensitivity of UDS allows to detect the D816V mutation in peripheral blood mononuclear cells from patients with a suspected clonal mast cell disorder. These results could represent a starting point to plan other extensive studies to better understand the exact role of KIT receptor alterations in SM. Supported by ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Cavo: Celgene: Consultancy, Honoraria, Speakers Bureau. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Background According to the 2013 European LeukemiaNet recommendations, BCR-ABL kinase domain (KD) mutation screening by conventional Sanger sequencing (SS) is recommended in all patients (pts) with progression, failure or warning, since detection of second-generation (2G) TKI-resistant mutations (T315I, F317L/V/I/C, V299L, Y253F/H, E255K/V, F359V/I/C) helps in the selection of the most suitable alternative therapy. However, SS has limited sensitivity. A recent study (Parker et al, J Clin Oncol 2011) has shown that low level mutations (i.e., undetectable by SS) may be identified by mass spectrometry and this may aid in more appropriate selection of the second-line treatment strategy for imatinib-resistant pts. Aims We sought to determine i) whether an UDS-based approach for BCR-ABL KD mutation screening might allow more sensitive detection of 2GTKI-resistant mutations at the time of switchover to second or third-line therapy and ii) whether low level mutations identified by UDS undergo clonal expansion, thus predicting for subsequent failure, if the 2GTKI to which they are insensitive to happens to be chosen. Methods To this purpose, we used UDS to scan the BCR-ABL KD in a cohort of 75 chronic myeloid leukemia (n=50) or Philadelphia chromosome-positive acute lymphoblastic leukemia (n=25) pts who were treated with a 2GTKI (dasatinib or nilotinib) after having failed first-line (n=62) or second-line (n=13) TKI therapy. A total of 235 samples collected at the time of switchover and at subsequent timepoints during second-or third-line treatment were analyzed. All the samples had already been evaluated by SS. UDS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study. Runs were designed to achieve high sequencing depth. However, raw data analysis with Amplicon Variant Analyzer software (Roche Applied Science) was performed filtering out variants Results Forty-three imatinib-resistant pts failed subsequent second- (n=37) or third-line (n=6) treatment with 2GTKIs (dasatinib, n=28; nilotinib, n=15). By routine SS analysis, 32 BCR-ABL KD mutations had been identified in 29/43 (67%) pts at the time of switchover - informing subsequent-line treatment selection in 12 pts who were found to harbor 2GTKI-resistant mutations. At the time of treatment failure (after a median of 9 months; range, 2-30), SS had detected newly acquired 2GTKI-resistant mutations in all 43 pts. We thus wondered how many of these mutations could have been detected at the time of switchover using a more sensitive approach. By UDS re-analysis, 73 mutations were identified in 35/43 (81%) pts at switchover – UDS detected all the 32 mutations previously identified by SS plus 41 low level mutations (i.e., with an abundance between 1 and 15%). Twenty-four of the 41 low level mutations were resistant to the 2GTKI that happened to be selected (T315I=10; E255K=2; E255V=1; Y253H/F=4; F359V=1; F317L=4; V299L=1) and all invariably expanded becoming detectable by SS at relapse, alone or in combination with pre-existing mutations. Thus, mutations that would have influenced therapeutic decision after first or second TKI failure could have been detected in 17 more pts by UDS (p Thirty pts who achieved a stable response to second- (n=26) or third-line (n=4) treatment with 2GTKIs after having failed first- or second-line treatment, respectively, were also analyzed for comparison. By UDS, a total of 13 mutations were detected – including 6 mutations (6 pts) that had already been identified by SS plus 7 low level mutations (7 pts). No low level mutation resistant to the 2GTKI the pts actually received was detected. Conclusions In comparison to SS, UDS could identify 2GTKI-resistant mutations at the time of switchover in a significantly greater proportion of pts. Low level mutations (down to 1% abundance) detected by UDS were consistently found to expand when the 2GTKI they are insensitive to happened to be chosen. UDS-based screening of the BCR-ABL KD at switchover might thus have enabled more effective therapeutic tailoring. Further evaluation of whether this technology may be implemented in a diagnostic setting is highly warranted. Supported by PRIN 2009 (prot.2009JSMKY), Fondazione CARISBO, AIL, AIRC, FP7 ‘NGS-PTL’, IGA NT 11555 and 13899. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Machova Polakova:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Castagnetti:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Rosti:Novartis: Consultancy, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau. Baccarani:Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees Other; Bristol-Myers Squibb: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees, Speaker fees Other; Ariad: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees, Speaker fees Other; Pfizer: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees, Speaker fees Other; Teva: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees Other. Martinelli:Novartis: Consultancy, Speaker fees Other; Bristol-Myers Squibb: Consultancy, Speaker fees, Speaker fees Other; Pfizer: Consultancy, Speaker fees, Speaker fees Other; Ariad: Consultancy, Speaker fees, Speaker fees Other.

Patients with Systemic Mastocytosis (SM) need a highly sensitive diagnostic test for D816V detection of the KIT receptor gene.Along with histology/cytology and flow cytometry evaluation, bone marrow (BM) from 110 consecutive adult patients referred with a suspicion of SM to Multidisciplinary Outpatient Clinic for Mastocytosis in Verona were tested both by Amplification Refractory Mutation System Reverse Transcriptase quantitative real time Polymerase Chain Reaction (ARMS-RT-qPCR) and RT-PCR. +. Restriction Fragment Length Polymorphism (RFLP) followed by Denaturing-High Performance Liquid Chromatography (D-HPLC) and Sanger sequencing.ARMS-RT-qPCR identified D816V mutation in 77 patients, corresponding to 100% of cases showing CD25+ mast cells (MCs) whereas RT-PCR+RFLP/D-HPLC+sequencing revealed D816V mutations in 47 patients.According to the 2008 WHO criteria 75 SM, 1 Cutaneous Mastocytosis (CM), 1 monoclonal MC activation syndrome (MMAS), and 1 SM Associated with Haematologic Non-Mast Cell Disorder (SM-AHNMD) were diagnosed. Seventeen out 75 SM patients (23%) would have not satisfied sufficient WHO criteria on the basis of the sole RT-PCR+RFLP: these patients had significantly lower serum tryptase levels and amount of CD25+ MCs.Therefore, ARMS-RT-qPCR might result particularly useful, in patients that do not fulfil major BM histological criterion, for the recognition of indolent SM with a very low MC burden.

Chronic myeloid leukemia (CML) has been the first human malignancy to be associated, more than 50 years ago, with a consistent chromosomal abnormality--the t(9;22)(q34;q11) chromosomal translocation. The resulting BCR-ABL1 fusion gene, encoding a tyrosine kinase with deregulated activity, has a central role in the pathogenesis of CML. Ancestral or additional genetic events necessary for CML to develop have long been hypothesized but never really demonstrated. CML can successfully be treated with tyrosine kinase inhibitors (TKIs). Mutations in the BCR-ABL1 kinase domain might arise, however, that confer resistance to 1 or more of the currently available TKIs. Hence, the critical role of BCR-ABL1 mutation screening for optimal therapeutic management, with the current gold standard technique, conventional sequencing, likely to be replaced soon by ultra-deep sequencing. Mutations in genes other than BCR-ABL1 include ASXL1, TET2, RUNX1, DNMT3A, EZH2, and TP53 in chronic phase patients and RUNX1, ASXL1, IKZF1, WT1, TET2, NPM1, IDH1, IDH2, NRAS, KRAS, CBL, TP53, CDKN2A, RB1, and GATA-2 mutations in advanced phase patients. The latter also display additional cytogenetic abnormalities, including submicroscopic regions of gain or loss that only single nucleotide polymorphism arrays or array comparative genomic hybridization can detect. Whether whole genome/exome sequencing studies will uncover novel mutations relevant for pathogenesis, progression, and risk-adapted therapy is still unclear.

Abstract 2601 Background: Tyrosine Kinase Inhibitors (TKI) have been shown to be very effective for the treatment of Acute Lymphoblastic Leukemia (ALL), with a Complete Hematologic Remission (CHR) rate close to 100%, and a high rate of Complete Cytogenetic and Molecular responses (CCgR and CMR). However, when they are used alone, as single agents, most patients relapse, so that they are currently used in combination with chemotherapy and as a preparation to allogeneic stem cell transplantation (SCT). Since Ph+ ALL is more frequent in the elderly, many patients cannot tolerate intensive chemotherapy and are not eligible for SCT. We have explored if the administration of two TKIs, Nilotinib (NIL) and Imatinib (IM) can improve the results without increasing the toxicity. Aims: To evaluate the response and the outcome of Ph+ ALL patients treated with the sequential administration of NIL and IM, to investigate the type and number of BCR-ABL kinase domain mutations developing during and after the study. Methods: We have designed a study (ClinicalTrials.gov. NCT01025505) in which patients more than 60 years old or unfit for intensive chemotherapy and SCT where treated with two TKIs, NIL 400 mg twice daily, and IM 300 mg twice daily, alternating for 6 weeks for a minimum of 24 weeks (study core) and indefinitely in case of response. The 6-weeks rotation schedule was respected, irrespectively of temporary discontinuations. The primary end-point was the rate of Disease Free Survival (DFS) at 24 weeks (4 courses of treatment); the secondary end points included the evaluation of CHR, CCgR and CMR rates. Mutation analysis was performed by nested RT-PCR amplification of the ABL kinase domain of the BCR-ABL transcript (codons 206 through 421). Amplified products were screened by denaturing-high performance liquid chromatography (D-HPLC). Samples scored positive for the presence of sequence variations were then subjected to direct automatic sequencing to characterize the mutation. Results: 39 patients have been enrolled in 15 Italian hematologic Centers (median age 66 years, range 28–84). Among these, 8 patients were unfit for standard chemotherapy or SCT (median age 50 years, range 28–59). 27 patients were p190, 5 were p210 and 7 were p190/p210. After 6 weeks of treatment, 36 patients were evaluable for response: 34 were in CHR (94%) and 2 in PHR (6%). 23 patients have already completed the study core (24 weeks), 87% were in CHR and 17 are currently continuing therapy in the protocol extension phase. Thus, the OS at 1 year is 79%, and 64% at 2 years. Overall, 1 patient was primarily resistant and 13 patients have relapsed, with a median time to relapse of 7.6 months (range 0.8–16.1 months), for a DFS of 51.3% at 12 months (Figure 1). Mutations detected were T315I in 2 cases, Y253H in 3 cases, T315I and Y253H in 1 case, E255K in 1 case, T315I and E255K in 1 case, E255V and Y253H in 1 case. Two patients were WT. A detailed kinetics of Molecular responses is shown in Table 1. Data on mutational analysis are reported in Table 2. Further details about Cytogenetic and Molecular responses, and about Adverse Events will be provided on site. Conclusions: In this small cohort of Ph+ ALL elderly/unfit patients, the rates of relapse and progression were not likely to be different from the rates observed with Imatinib alone (Vignetti et al, Blood 2007, May 1;109(9):3676-8) and Dasatinib alone (Foà, Blood 2011, Dec 15;118(25):6521-8). It's important to notice that the mutations that occurred at the time of relapse were sensitive to other TKIs (Dasatinib and Ponatinib). Acknowledgments: COFIN, Bologna University, BolognAIL, PRIN, Fondazione del Monte di Bologna e Ravenna, INPDAP. Disclosures: Pizzolo: Hoffmann-La Roche: Consultancy, Honoraria. Luppi:CELGENE CORPORATION: Research Funding. Vallisa:CELGENE CORPORATION: Research Funding. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau.

Abstract 1749 Introduction. Mastocytosis is a myeloid neoplasm characterized by abnormal accumulation and frequent activation of mast cells (MCs) in various organs, mostly bone marrow, skin, liver and gastrointestinal tract. In most adult patients, the systemic form of mastocytosis (SM) is diagnosed, which includes an indolent, an aggressive and a leukemic subvariant. The c-kit mutation D816V is detectable in most adult patients with SM. Treatment of SM usually focuses on symptom relief by histamine receptor antagonists and other supportive therapy. However, in aggressive and leukemic variants, cytoreductive and targeted drugs must be applied. Methods. From 2008, 11 patients (male/female=3/8) affected by Mastocytosis, have been referred to our Institution. The median age was 53 years. All the patients underwent a bone marrow biopsy, with flow citometry and molecular biology analysis, in order to identify the presence of D816V mutation of c-Kit gene. Serum tryptase level was tested, resulting elevated in all cases. According to 2008 WHO diagnostic criteria, 9 patients presented with SM, whereas in the other cases a skin isolated involvement was detected. Systemic symptoms were characterized by nausea, diarrhoea, asthenia, weight loss, pruritus and serotine fever, identifying an aggressive form of the disease in 5/11 patients, due to skeletal involvement in three cases, ascitis and liver function impairment in another one and bone marrow disfunction in the fifth one. Therefore, since a first line therapy with supportive care and histamine receptor antagonists wasn't followed by a significant benefit, a personalized use of PKC412 was asked and obtained for all these five patients. Results. From March 2011 three out of the five patients with aggressive SM have received a prolonged period of treatment with PKC412, which was administered orally, at the dosage of 100 mg twice daily, without rest periods. The drug was well tolerated. No serious adverse events were observed. All the patients obtained a quick and prolonged improvement of clinical symptoms, in terms of weight gain, bowel function and skeletal pain. At the bone marrow evaluation, the persistence of the D816V c-kit mutation was observed, despite a significant decrease of mast cell marrow involvement. In one case we observed, after a first good response, a disease progression, characterized by the sudden reoccurrence of the same symptoms detected at diagnosis, confirmed by a relevant expansion of a pathologic mast cell population in the bone marrow. After a rest period, the drug was readministered, and a second remission was obtained. Conclusions. PKC412 is safe and effective in patients with SM, being able to significantly improve not only the gastrointestinal and systemic symptoms, but also the haematological profile. The persistence of the D816V c-kit mutation, despite a morphologic remission, suggests that many other oncogenic factors may be responsible for the pathogenesis of the disease. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN, Fondazione del Monte di Bologna e Ravenna, University of Bologna. Disclosures: Baccarani: Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy, Honoraria, Speakers Bureau; ARIAD: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Abstract 284 Background and Aims: Selection of drug-resistant mutations in the Bcr-Abl kinase domain (KD) is a critical problem undermining the long-term efficacy of tyrosine kinase inhibitor (TKI)-based therapies in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients. Bcr-Abl KD mutation screening is routinely performed by Sanger sequencing (SS). Before the advent of ultra-deep sequencing (UDS) technologies, no method was available that could conjugate the possibility to scan the KD for the so many mutations known to be associated with TKI resistance with a sensitivity higher than that of SS. UDS technologies also allow high throughputness and accurate quantitation of mutated clones and their application in a diagnostic setting is not far to come. We used an UDS strategy for Bcr-Abl KD mutation screening in order to study the dynamics of expansion of mutated clones in Ph+ ALL patients receiving TKI-based therapies and to test the ability of UDS to highlight emerging clones harboring critical mutations. Methods: 72 samples from 25 Ph+ ALL patients who had developed resistance to one or multiple lines of TKI (imatinib, dasatinib, nilotinib, bosutinib, ponatinib) therapy were selected for this retrospective analysis. All the patients had previously been analyzed by Sanger sequencing (SS) and were known to have developed one or more TKI-resistant Bcr-Abl KD mutations on treatment. In order to reconstruct the dynamics of mutation emergence, longitudinal re-analysis of monthly collected samples was perfomed with UDS on a Roche GS Junior. UDS allowed to achieve a lower detection limit of at least 0.1% (by generating a minimum of 5,000 sequence reads/patient), as compared to 20% of SS. Results: 39 samples were known to harbor one (n=27 samples) or more (n=12 samples) TKI-resistant mutations with >20% abundance, as assessed by SS. UDS could successfully detect all the 54 mutations previously identified by SS. In addition, UDS detected one or multiple lower-level (20% abundance. The type of lower-level mutations detected by UDS could easily be accounted for by TKI exposure history, since the majority were known to be poorly sensitive either to the TKI being administered or to the previous TKI received. Overall, 44 samples turned out to carry multiple (two to five) mutations at any level, distributed in the same and/or in different subpopulations with a complex clonal architecture that UDS allowed to reconstruct. Of note, in 14/25 (56%) patients with molecularly detectable disease but not yet evidence of cytogenetic or hematologic relapse, UDS could identify emerging TKI-resistant mutations 1 to 2 months before they became detectable by SS. These outgrowing mutations were detected at 1–19% abundance in 12 patients and at 0.1–1% abundance in 2 patients. In the remaining 11 patients, dynamics of outgrow of the TKI-resistant mutations (five T315I, two Y253H, two E255K, one E255V and one F317L) was so rapid that not even strict monthly monitoring could allow to pick them up before they became dominant. Conclusions: Now that multiple options are available, Bcr-Abl KD mutation monitoring has become a precious tool for rational decision-making in order to maximize the efficacy of TKI-based regimens as induction or salvage therapy for Ph+ ALL patients. UDS proved as reliable as SS for the detection of mutations with >20% abundance and to have comparable costs. As a key advantage, UDS added precious quantitative and qualitative information on the full repertoire of mutated populations, that SS failed to appreciate in more than half of the samples analyzed. TKI-resistant mutations leading to patient relapse were not necessarily preexisting at low levels at diagnosis or at the time of switchover to another TKI, underlining the importance of regular monitoring of patients. Although TKI-resistant populations may arise and take over very rapidly, in approximately half of the patients monthly monitoring with UDS would have allowed to identify them earlier than SS and well in advance of clinical relapse, thus allowing a more timely therapeutic intervention. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Luppi:CELGENE CORPORATION: Research Funding. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Background and Aims: The term Systemic Mastocytosis (SM) identifies a poorly understood group of rare and clinically heterogenous myeloproliferative neoplasms characterized by abnormal growth and activation of mast cells (MCs) and their precursors in the bone marrow and in various tissues and organs. Based on phenotype and extent of organ infiltration/dysfunction, a spectrum of disease variants can be recognized ranging from indolent SM (ISM) to aggressive SM (ASM) and mast cell leukemia (MCL). The fact that in all cases, including ISM who have a (near) normal life expectancy, neoplastic MCs display the same D816V KIT gene mutation points to additional mechanisms and molecular defects as responsible for ASM and MCL. So far, however, this issue has mainly been addressed with targeted resequencing studies of candidate gene panels. We thus decided to undertake an integrated molecular characterization study of ASM and MCL to identify novel, functionally relevant molecular lesions and/or clinically actionable signaling pathways. Methods: A discovery panel including 6 patients with ASM and 6 patients with MCL was studied using whole exome sequencing (WES) and copy number variation (CNV) analysis. WES (80x) was performed on a Hiseq 2500 (Illumina). CNV was done using Cytoscan HD Arrays (Affymetrix). Paired normal/MC DNA was analyzed in all but 2 archival MCL cases for whom germline DNA was not available. A validation panel of 30 ISM, 5 smoldering SM and 20 additional ASM was also included in this study. Results: In the discovery panel, WES identified a total of 1554 point mutations, small insertions and deletions. Seven hundred and eighty-five were non-silent mutations in 698 genes, with an average of 51 (range, 30-186) non-silent mutations per patient. Non-silent mutations included 354 missense mutations, 188 nonsense mutations, 145 frameshift insertions/deletions, 98 non-frameshift insertions/deletions. C to T transitions were by far the most frequent. Orthogonal validation estimated the accuracy of mutation calls at >95%. Interrogation of the COSMIC and OMIM databases revealed 42 known cancer genes. Among the missense mutations, 87 were predicted to have a high probability of being deleterious by Condel. MCL cases were found not to harbour a higher mutation load as compared to ASM cases. High resolution CN analysis showed that focal amplifications/deletions/loss-of-heterozygosity (LOH) were prevalent over arm-level alterations (found in 3 patients only). Genes were selected for further assessment when recurrently mutated in ≥2 patients or concurrently identified in WES and CNV analyses or previously associated with leukemogenesis or cancer pathogenesis. Among these, genes already reported to be affected by mutations in SM included TET2, NRAS, ASXL1, CBL, IDH1, SRSF2, SF3B1, RUNX1. We also identified genetic alterations in genes not previously implicated in SM pathogenesis including TP53BP1, RUNX3, NCOR2, CDC27, CCND3, EI24, MLL3, ARID1B, ARID3B, ARID4A, SETD1A, SETD1B, KDM1B, PRDM1, ATM, WRN. A long tail of infrequently mutated genes dominated, resulting in significant intertumoural heterogeneity. However, when genes were assigned to functional pathways to discern patterns of mutations across different patients, we found that PI3K/Akt and MAPK pathways, calcium pathway, chromatin modification, DNA methylation, and DNA damage repair were consistently affected (Figure 1). Further assessment of the mutation frequency of selected genes within each pathway and functional validation at the protein level are currently ongoing in the validation panel. Preliminary findings on a tumor suppressor selected among those identified by WES show transcript and/or protein downmodulation due to inactivating mutations, transcriptional silencing or enhanced degradation in 17/20 ASM. Detailed results will be presented at the meeting. Conclusions: WES and CNV analyses of ASM and MCL revealed a complex landscape, not unexpected when considering the clinical heterogeneity of these patients. Nonetheless, key pathways were found to be recurrently altered. Further investigation of selected candidate genes and pathways is warranted and will cast light on the cooperative genetic (and epigenetic?) events underlying the more aggressive forms of SM - paving the way to a better prognostic stratification and more effective treatment. <>This study was supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; Pfizer: Consultancy; Ariad: Consultancy; AMGEN: Consultancy; MSD: Consultancy.

Background: Adult B-ALL patients still have a dismal prognosis, due to a high incidence of relapse even after allogenic SCT. Safety and efficacy of Blinatumomab, an anti CD3-CD19 Bite antibody, has been demonstrated both in MRD positive patients and in relapsed/refractory (R/R) setting. Aim: To evaluate safety profile and efficacy of Blinatumomab, obtained through a compassionate use, in a cohort of 18 adult patients affected by MRD+ or R/R B-ALL treated at Bologna University. Design: From March 2015 to July 2016, 18 patients received Blinatumomab at the standard dosage (9 mcg/d x 7 days, 28 mcg/d x 21 days) in 28-days courses. All the patients were hospitalized to receive the first course of therapy. The following courses, based on the good safety profile of the compound, were administered in outpatient setting. Patients: 18 patients (M/F = 10/8; median age 43, range 18-73) have been treated. Philadelphia (Ph) chromosome was detected in 8/18 patients. 10 patients were MRD+ (5 Ph pos and 5 Ph neg); E2A-PBX1 and MLL-AF4 rearrangements were found in two patients. 8 patients had a R/R disease (3 Ph pos and 5 Ph neg). Median WBC count before starting therapy with Blinatumomab was 5400/mmc (range 500-76500). All the patients had previously received many lines of therapy (median 4, range 1-7). In 4 cases an alloTMO was already performed, and two patients had received two transplants. 12/18 patients were referred to us by other Italian Institutions. All the patients received at least one course of Blinatumomab. In one case three courses were administered; an elderly patient is actually receiving the fifth course. Globally, 32 courses of therapy have been administered (median 2, range 1-5). Bone marrow evaluations, including cytogenetics, molecular biology and immunophenotyping analysis were performed at the beginning of every course of therapy in order to assess patients' disease status. MRD evaluation was assessed through BCR-ABL fusion transcript quantitative analysis in Ph pos ALL patients and Ig rearrangment in Ph negative patients. Monitoring of adverse events was periodically performed. Results: 16/18 patients are evaluable for response, at least to one cycle (one patient died during the first course, one patient is still receiving the first course). 9/16 (56%) patients obtained a CR (7/9 MRD+ and 2/7 R/R). In 7/9 (78%) responders patients a molecular CR was reached, (in 6 patients after the first course, in one case after the second one). 5 responders proceeded to alloBMT and are actually alive in CR (median follow-up after transplant 240 days). In terms of toxicity, one patient developed a grade IV neurological event (mental confusion, tremor), which completely resolved after a transient drug withdrawal. Conclusions: Our results confirm the high rate of response and to Blinatumomab in a poor patients' population, and the good management profile of the compound. Acknowledgments: Work supported by ALN, AIL, AIRC, PRIN, Progetto Regione-Universita 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy.

The course of chronic myeloid leukemia (CML) and the response to treatment with tyrosine kinase inhibitors (TKIs) are best monitored and assessed using two molecular tests: the first is real-time quantitative reverse transcription-polymerase chain reaction (RQ-PCR), which measures the size of residual disease that is expressed as BCR-ABL1% (the ratio between BCR-ABL1 and a control gene) and the other is mutational analysis by Sanger sequencing, which checks for the presence of BCR-ABL1 kinase domain point mutations. Both tests are technically demanding and require a high level of specialization and standardization. RQ-PCR, when performed on a regular basis, allows for the defining of molecular response (MR) levels as log reduction from a standardized baseline: major molecular response (MMR or MR3) that is the best predictor of survival; and the deeper molecular response (MR4, MR4.5, and MR5) that is necessary to enroll a patient in a trial aiming at treatment-free remission (TFR). Mutational analysis, to be performed in case of failure or warning by Sanger sequencing, allows for screening of the BCR-ABL1 kinase domain for mutations conferring resistance to TKIs. Since different mutations have different degrees of sensitivity to each of the currently available TKI, the knowledge of BCR-ABL1 kinase domain–mutation status is necessary for subsequent treatment choice. Optimal patient management requires that MR and mutational information be rationally interpreted at both the technical and at the biologic level, and put into context—therapeutic decisions also take into account other factors, such as age, comorbidities, side effects, compliance, and treatment-related complications.

BACKGROUND Patients with Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL) frequently relapse on imatinib with acquisition of BCR-ABL kinase domain (KD) mutations. To analyze the changes that second-generation tyrosine kinase inhibitors (TKIs) have brought in mutation frequency and type, a database review was undertaken of the results of all the BCR-ABL KD mutation analyses performed in the authors' laboratory from January 2004 to January 2013. METHODS Interrogation of the database retrieved 450 mutation analyses in 272 patients with Ph+ ALL. Prescreening of samples was performed with denaturing high-performance liquid chromatography (D-HPLC), followed by direct sequencing of D-HPLC–positive cases. RESULTS BCR-ABL KD mutations were detected in 70% of imatinib-resistant patients, with T315I, E255K, and Y253H mutations accounting for 75% of cases. Seventy-eight percent of the patients reported to be resistant to second-generation TKIs after imatinib failure were positive for mutations, and 58% of them had multiple mutations. Analysis of patients relapsing on dasatinib revealed a newly acquired T315I mutation in almost two-thirds of the cases. Direct sequencing detected no mutations at diagnosis, even in patients who relapsed after a few months. CONCLUSIONS Second-generation TKIs ensure a more rapid debulking of the leukemic clone and have much fewer insensitive mutations, but long-term disease control remains a problem, and the T315I mutation is revealed to be an even more frequent enemy. BCR-ABL KD mutation screening of patients with Ph+ ALL who are receiving imatinib or second-generation TKIs would be a precious ally for timely treatment optimization. In contrast, the clinical usefulness of conventional direct sequencing at diagnosis seems to be very low. Cancer 2014;120:1002–1009. © 2013 American Cancer Society.

Systemic mastocytosis (SM) includes a heterogeneous group of disorders ranging from indolent SM to mast cell leukemia (MCL). Somatic mutations in the Kit receptor tyrosine kinase (most frequently, D816V) can be detected in >90% of patients with SM and are thought to play a key pathogenetic role. Nevertheless, morphological and clinical diversity, as well as the fact that some patients are negative for KIT mutations, suggest that the underlying molecular picture is far from being fully elucidated. To shed further light on this issue, we undertook an integrated molecular genetic study of a KIT gene mutation-negative MCL patient who came to our attention in 2012 – a 63 year-old woman diagnosed with MCL, aleukemic variant (50-60% atypical mast-cells in the bone marrow [BM] smear; CD117+/CD2+/CD13+-/CD33+/CD59+ immunophenotype; serum tryptase, 2500 µg/L; no C-findings). The patient had received imatinib for 6 months, with no clinical benefit. The disease, however, had had an overall chronic clinical course for 6 more months until severe anemia occurred. The patient rapidly progressed and died after 21 months from diagnosis. After having obtained written informed consent, we extracted genomic DNA and total RNA from purified MCs isolated from BM at diagnosis and at progression, as well as DNA from saliva, and performed whole exome sequencing (WES) and RNA sequencing on an HiSeq1000 (Illumina, San Diego CA). Cytoscan HD arrays (Affymetrix, Santa Clara CA) were also used to scan for chromosomal gains and losses as well as for loss of heterozigosity (LOH). Among the mutated genes detected by WES, SETD2 stood out among others because two distinct putatively inactivating heterozygous mutations were identified, a frameshift insertion of a C in exon 20 (NM_014159:c.7595_7596insC: p.Gly2515ArgfsTer5) and a nonsense mutation in exon 15 (NM_014159:c.G6753T:p.Glu2234Ter). The two mutations were found to hit distinct alleles, pointing to a loss-of-function event. Western Blotting (WB), however, showed that only the 2234 a.a. Setd2 truncated isoform resulting from the nonsense mutation, losing the highly conserved WW and SRI functional domains, was detectable in the sample. The SETD2 gene encodes a histone methyltransferase nonredundantly responsible for trimethylation of lysine 36 of histone H3, a key hystone mark associated not only with active chromatin but also with transcriptional elongation, alternative splicing, DNA replication and repair. SETD2 gene mutations have been described in a variety of cancers and, more recently, have been found to be cooperating events in acute leukemia initiation and progression. In yeast, deletion of the SRI domain abolishes Set2-RNA polymerase II (PolII) interaction causing transcription elongation defects and abolishes K36 methylation. The truncated SETD2 isoform was actually found to lose the ability to bind RNAPolII, as shown by co-immunoprecipitation. Accordingly, RNA-sequencing showed evidence of spurious transcripts initiated from cryptic promoter-like sequences within genes rather than from canonical promoters. More importantly, WB confirmed that H3K36Me3 was completely abrogated. In line with the recently reported role of SETD2-dependent H3K36Me3 in homologous recombination (HR) repair and genome stability, Cytoscan HD arrays showed that LOH and several gains and losses at many chromosomal loci, undetectable at diagnosis, had been acquired at the time of progression. Haploinsufficiency of PSIP1 (recruiting HR machinery at double strand breaks) at 9p24.3 might have represented a cooperating event. Downmodulation of the Setd2 protein (in the presence of LOH but in the apparent absence of sequence variations other than polymorphisms) and reduced H3K36Me3 levels were detected in two more MCL cases, in which putative cooperative lesions were also identified. Results of WES and high resolution karyotyping of additional SM cases will be presented. Our findings point to epigenetic regulation and/or DNA repair as two candidate pathways deserving further investigation in an attempt to identify novel actors or mechanisms contributing to the pathogenesis and progression of SM, or novel modulators of disease phenotype. They also extend the recent observation that the molecular landscape of SM is much more complex than the initial finding of KIT mutations allowed to imagine. Supported by FP7 NGS-PTL project and Progetto Regione-Università 2010-12 (L. Bolondi) Disclosures Soverini: Novartis: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Speakers Bureau.

Effective management of tyrosine kinase inhibitor (TKI) therapy for patients with CML requires frequent patient testing to monitor patient disease status. The gold standard for this testing is a PCR measurement of the BCR-ABL/ABL transcript ratio. Hence, standardized, accurate and reproducible molecular analyses are fundamental for clinicians in order to correctly address therapeutic decision and to achieve a better patient management. Based on these clinical needs, it is widely recognized that inter-laboratory reproducibility is a crucial issue that requires standardization and strict alignment of BCR-ABL values on the international scale (IS), as established by the International Randomized Study of Interferon and STI571 (IRIS) laboratories. In addition to this, another important aspect in terms of patient management is the availability of a rapid response, improving patient quality of life by reducing anxiety linked to delay in results delivery. Xpert® BCR-ABL Monitor, developed and manufactured by Cepheid (Sunnyvale, CA), is a cartridge-based automated assay for quantification of BCR-ABL1 mRNA, reporting results in International Scale (IS). Alignment to the IS, based upon the quality control standards derived from the WHO BCR-ABL Standards, is performed lot to lot automatically by the software of the Cepheid GeneXpert® DX Instrument. Xpert® BCR-ABL Monitor automates and integrates sample processing, nucleic acids extraction and amplification and target sequence detection in peripheral blood specimens collected either in EDTA or PAXgene tubes using real-time RT-PCR. The cartridge includes reagents to detect BCR-ABL fusion genes resulting from two major breakpoints, translocation e13a2 (b2a2) and e14a2 (b3a2) and the ABL transcript as an endogenous control. In the following study, Xpert® BCR-ABL Monitor was independently evaluated in the three Italian reference centers for BCR-ABL mRNA monitoring of the LabNet project. A total of 150 peripheral blood samples from CML patients, equally divided between centers, were analyzed both with the standard laboratory method and with Xpert® BCR-ABL Monitor Assay, in order to compare the results expressed in International Scale. BCR-ABL mRNA of the specimens covered a broad IS range, allowing to compare data also at low levels of disease (MR 4.5, 0.00316% BCR-ABL/ABL). Overall, linear regression demonstrated that there was a good correlation between Xpert® BCR-ABL Monitor and reference centers data (R2= 0.92). Correlation slightly increased when the data produced with Xpert® BCR-ABL Monitor were refined using a correction tool not automatically adopted by the current version of the assay software (R2= 0.93). Moreover, using assay comparison criteria proposed by Müller et al. (Leukemia 2009), Xpert® BCR-ABL Monitor was considered comparable to the standard laboratory methods of the reference centers, considering that all the three acceptance criteria were satisfied (58% of the samples between 0.5 and 2-fold variation, 78% of the samples between 0.33 and 3-fold variation, 91% of the samples between 0.2 and 5-fold variation). In conclusion, Xpert® BCR-ABL Monitor demonstrated to be a rapid, reliable and accurate test for monitoring of BCR-ABL mRNA transcript levels. Results were available within approximately 2 hours, potentially allowing clinicians to report results to patient the same day of the visit. From the technical point of view, cartridge-based automated system significantly reduced operator hands-on time from several hours to approximately 15 minutes. Concerning the assay comparative performance, Xpert® BCR-ABL showed to have a good inter-laboratory reproducibility, with no significant differences between the three reference centers standard methods. Acknowledgments: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Saglio: NOVARTIS: Consultancy. Pane:NOVARTIS: Consultancy, Speakers Bureau. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.