The Heterogeneity of Skewness in T2W-Based Radiomics Predicts the Response to Neoadjuvant Chemoradiotherapy in Locally Advanced Rectal Cancer.

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.

Percutaneous management of postoperative Bile leak after hepato-pancreato-biliary surgery: a multi-center experience.

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.

Next-generation sequencing improves BCR-ABL1 mutation detection in Philadelphia chromosome-positive acute lymphoblastic leukaemia.

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%.

Radiomics of cholangiocarcinoma on pretreatment CT can identify patients who would best respond to radioembolisation.

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 12.1 months and that of the remaining 40 patients was 5.1 months (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 12.1 months versus 5.1 months of patients with an unfavourable signature (p = 0.008).

Survival and Tolerability of Transarterial Chemoembolization in Greater Versus less than 70 Years of Age Patients with Unresectable Hepatocellular Carcinoma: A Propensity Score Analysis.

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" (≥ 70 years, 80 patients) and "younger" (< 70 years, 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 70 years or more with unresectable HCC as it is for their younger counterparts (< 70 years). Liver-related mortality was not associated with age ≥ 70 years and primarily predicted by tumor multifocality, Child-Pugh class B and an increased alpha-fetoprotein value (> 31 ng/ml).

Diagnostic Accuracy of North America Expert Consensus Statement on Reporting CT Findings in Patients Suspected of Having COVID-19 Infection: An Italian Single-Center Experience.

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 (RT-PCR), 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" (κ = 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.© RSNA, 2020.

Prospective assessment of NGS-detectable mutations in CML patients with nonoptimal response: the NEXT-in-CML study.

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.

Portal Hypertensive Biliopathy in Adult Patients: Findings and Interventional Radiologic Treatment-A Single-Center Experience.

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.

Outcomes following percutaneous treatment of biliary stones.

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.

Transarterial radioembolization in patients with hepatocellular carcinoma of intermediate B2 substage.

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.

Calreticulin: Challenges Posed by the Intrinsically Disordered Nature of Calreticulin to the Study of Its Function.

Calreticulin is a Ca2+-binding chaperone protein, which resides mainly in the endoplasmic reticulum but also found in other cellular compartments including the plasma membrane. In addition to Ca2+, calreticulin binds and regulates almost all proteins and most of the mRNAs deciding their intracellular fate. The potential functions of calreticulin are so numerous that identification of all of them is becoming a nightmare. Still the recent discovery that patients affected by the Philadelphia-negative myeloproliferative disorders essential thrombocytemia or primary myelofibrosis not harboring JAK2 mutations carry instead calreticulin mutations disrupting its C-terminal domain has highlighted the clinical need to gain a deeper understanding of the biological activity of this protein. However, by contrast with other proteins, such as enzymes or transcription factors, the biological functions of which are strictly defined by a stable spatial structure imprinted by their amino acid sequence, calreticulin contains intrinsically disordered regions, the structure of which represents a highly dynamic conformational ensemble characterized by constant changes between several metastable conformations in response to a variety of environmental cues. This article will illustrate the Theory of calreticulin as an intrinsically disordered protein and discuss the Hypothesis that the dynamic conformational changes to which calreticulin may be subjected by environmental cues, by promoting or restricting the exposure of its active sites, may affect its function under normal and pathological conditions.

Ponatinib as a Valid Alternative Strategy in Patients with Blast Crisis-Chronic Myeloid Leukemia Not Eligible for Allogeneic Stem Cells Transplantation and/or Conventional Chemotherapy: Report of a Case.

Currently, imatinib and dasatinib are the only tyrosine-kinase inhibitors approved in the US and Europe for the treatment of blast crisis of chronic myeloid leukemia (BC-CML) at diagnosis, while ponatinib is the only inhibitor used in patients bearing T315I mutation. Here we report the case of a 61-year-old man diagnosed with B-cell lymphoid BC-CML, initially treated with imatinib 800 mg day and then with dasatinib 140 mg day because of intolerance. A complete cytogenetic response (CCyR) was achieved at three months; however, three months later a relapse was observed, and the T315I mutation was detected. Ponatinib 45 mg once daily was then started together with a short course of chemotherapy. Bone marrow evaluation after six months of therapy showed the regaining of CCyR, together with the achievement of a deep molecular response. However, one year from ponatinib start the patient experienced a new disease relapse; he was effectively treated with ponatinib and chemotherapy once again, but in the meanwhile an ischemic stroke was detected. This case report confirms the high efficacy of ponatinib monotherapy in BC-CML patients, representing a valid option for non-allogeneic stem cells transplantation eligible cases and the only one available for those carrying the T315I mutation.

The clonal evolution of two distinct T315I-positive BCR-ABL1 subclones in a Philadelphia-positive acute lymphoblastic leukemia failing multiple lines of therapy: a case report.

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.

FOXM1 Transcription Factor: A New Component of Chronic Myeloid Leukemia Stem Cell Proliferation Advantage.

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 ß-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 ß-catenin and ß-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 ß-catenin interaction with FOXM1, hence hampering FOXM1 phosphorylation, ß-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.

In chronic myeloid leukemia patients on second-line tyrosine kinase inhibitor therapy, deep sequencing of BCR-ABL1 at the time of warning may allow sensitive detection of emerging drug-resistant mutants.

BACKGROUND: Imatinib-resistant chronic myeloid leukemia (CML) patients receiving second-line tyrosine kinase inhibitor (TKI) therapy with dasatinib or nilotinib have a higher risk of disease relapse and progression and not infrequently BCR-ABL1 kinase domain (KD) mutations are implicated in therapeutic failure. In this setting, earlier detection of emerging BCR-ABL1 KD mutations would offer greater chances of efficacy for subsequent salvage therapy and limit the biological consequences of full BCR-ABL1 kinase reactivation. Taking advantage of an already set up and validated next-generation deep amplicon sequencing (DS) assay, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. METHODS: a total of 125 longitudinal samples from 51 CML patients who had acquired dasatinib- or nilotinib-resistant mutations during second-line therapy were analyzed by DS from the time of failure and mutation detection by conventional sequencing backwards. BCR-ABL1/ABL1%(IS) transcript levels were used to define whether the patient had 'optimal response', 'warning' or 'failure' at the time of first mutation detection by DS. RESULTS: DS was able to backtrack dasatinib- or nilotinib-resistant mutations to the previous sample(s) in 23/51 (45 %) pts. Median mutation burden at the time of first detection by DS was 5.5 % (range, 1.5-17.5 %); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 1-9 months). In 5 cases, the mutations were detectable at baseline. In the remaining cases, response level at the time mutations were first detected by DS could be defined as 'Warning' (according to the 2013 ELN definitions of response to 2nd-line therapy) in 13 cases, as 'Optimal response' in one case, as 'Failure' in 4 cases. No dasatinib- or nilotinib-resistant mutations were detected by DS in 15 randomly selected patients with 'warning' at various timepoints, that later turned into optimal responders with no treatment changes. CONCLUSIONS: DS enables a larger window of detection of emerging BCR-ABL1 KD mutations predicting for an impending relapse. A 'Warning' response may represent a rational trigger, besides 'Failure', for DS-based mutation screening in CML patients undergoing second-line TKI therapy.

Clinical presentation and management practice of systemic mastocytosis. A survey on 460 Italian patients.

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.

Best Practices in Chronic Myeloid Leukemia Monitoring and Management.

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.

Present and future of molecular monitoring in chronic myeloid leukaemia.

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.

Next-generation sequencing for sensitive detection of BCR-ABL1 mutations relevant to tyrosine kinase inhibitor choice in imatinib-resistant patients.

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.