BL-MOL-AR Project, Preliminary Results about Liquid Biopsy: Molecular Approach Experience and Research Activity in Oncological Settings.

Background Liquid biopsy is mainly used to identify tumor cells in pulmonary neoplasms. It is more often used in research than in clinical practice. The BL-MOL-AR study aims to investigate the efficacy of next-generation sequencing (NGS) and clinical interpretation of the circulating free DNA (cfDNA) levels. This study reports the preliminary results from the first samples analyzed from patients affected by various neoplasms: lung, intestinal, mammary, gastric, biliary, and cutaneous. Methods The Biopsia Liquida-Molecolare-Arezzo study aims to enroll cancer patients affected by various malignancies, including pulmonary, intestinal, advanced urothelial, biliary, breast, cutaneous, and gastric malignancies. Thirty-nine patients were included in this preliminary report. At time zero, a liquid biopsy is executed, and two types of NGS panels are performed, comprising 17 genes in panel 1, which is already used in the routine tissue setting, and 52 genes in panel 2. From the 7th month after enrollment, 10 sequential liquid biopsies are performed up to the 17th month. The variant allele frequency (%) and cfDNA levels (ng/mL) are measured in every plasmatic sample. Results The NGS results obtained by different panels are similar even though the number of mutations is more concordant for lung pathologies. There are no significant differences in the actionability levels of the identified variants. Most of the molecular profiles of liquid biopsies reflect tissue data. Conclusions Preliminary data from this study confirm the need to clarify the limitations and potential of liquid biopsy beyond the lung setting. Overall, parameters related to cfDNA levels and variant allele frequency could provide important indications for prognosis and disease monitoring.

Lab tests for MPN.

Molecular laboratory investigations for myeloproliferative neoplasm (MPN) can ideally be divided into two distincts groups, those for the detection of the BCR-ABL rearrangement (suspect of chronic myeloid leukemia) and those for the variants determination of the driver genes of the negative Philadelphia forms (MPN Ph neg). The BCR-ABL detection is based on RT-Polymerase Chain Reaction techniques and more recently on droplet digital PCR (ddPCR). For this type of analysis, combined with chromosome banding analysis (CBA) and Fluorescent in situ hybridization (FISH), it is essential to quantify BCR-ABL mutated copies by standard curve method. The investigation on driver genes for MPN Ph neg forms includes activity for erythroid forms such as Polycythemia Vera (test JAK2V617F and JAK2 exon 12), for non-erythroid forms such as essential thrombocythemia and myelofibrosis (test JAK2V617F, CALR exon 9, MPL exon 10), for "atypical" ones such as mastocytosis (cKIT D816V test) and for hypereosinophilic syndrome (FIP1L1-PDGFRalpha test). It's crucial to assign prognosis value through calculating allelic burden of JAK2 V617F variant and determining CALR esone 9 variants (type1/1like, type2/2like and atypical ones). A fundamental innovation for investigating triple negative cases for JAK2, CALR, MPL and for providing prognostic score is the use of Next Generation Sequencing panels containing high molecular risk genes as ASXL1, EZH2, TET2, IDH1/IDH2, SRSF2. This technique allows to detect additional or subclonal mutations which are usually acquired in varying sized sub-clones of hematopoietic progenitors. These additional variants have a prognostic significance and should be indagated to exclude false negative cases.

Experience of Uncommon EGFR Mutation in Lung Scheme Quality Program: Discussing Risks and Opportunities for the Improvement of Laboratory Response.

Background The quality programs can be considered to be a valuable tool for global and individual growth. Each result, obtained by a single laboratory, contributes to define the standardization of the response. In the case of the uncommon epidermal growth factor receptor (EGFR) mutations, the molecular result is sometimes difficult to interpret in terms of biological significance and therapy choosing. The standardization effort in the diagnostic lung setting also consists of active quality program participation. Materials and Methods The quality control analysis, which is defined as a clinical case, was performed by the extraction of DNA from FFPE sections and by RT-PCR on the EGFR (exons 19, 20, 21), BRAF, and KRAS genes. The laboratory performed a validation sequencing of EGFR exon 20 with the help of the Sanger method. Results The laboratory reported positivity for EGFR exon 20 insertions and negative results for BRAF and KRAS. The quality test finished with the redaction of a report containing the recommendation to consider the efficacy of therapy with tyrosine kinase inhibitors (TKI). This specific interpretation has determined poor performance judgment by the quality provider, which explained why most of these mutations are TKI-resistant. Conclusions This experience provides an opportunity to reflect on the critical aspects of this diagnostic setting. The detection of some uncommon EGFR mutations should entail the mutation characterization, especially for the rare exon 20 insertions, of which are not classifiable as "resistant." Moreover, this experience allows reflecting on the quality program design, mandatory actions for the laboratory, and routine activity in the oncologic multidisciplinary team.

Comparison of serologic and molecular SARS-CoV 2 results in a large cohort in Southern Tuscany demonstrates a role for serologic testing to increase diagnostic sensitivity.

BACKGROUND: Since February 2020, Italian hospitals registered COVID-19 (COronaVIrus Disease 19) cases more often than the rest of the Europe. During this epidemic, health authorities requested swab tests, while seeking new patient paths. METHODS: A dual laboratory approach was evaluated, consisting of patient care reports for viral RNA detection on swabs and rapid serological tests in 516 patients (192 symptomatic or paucisymptomatic and 324 asymptomatic). RESULTS: We found the molecular positive fraction equal to 12% (23/192) among symptomatic/paucisymptomatic (S/P) and 15.4% (50/324) in asymptomatic (As) sets. Among subsets, we observed serologically positive results, corresponding to 35% (8/23) for S/P and 38% (19/50) for As. Among molecular negative cases, we detected specific Immunoglobulin G or M (Ig G or Ig M) positivity in the S/P cohort equal to 6.6% (11/167) and 6% (15/246) in As cases. For indeterminate molecular results, we found S/P serological positivity equal to 100% (1/1) and 54% (13/24) in As patients. We found higher (p < 0.05) seropositivity in older patients (n = 8) among symptomatic and positives for viral RNA (n.23). CONCLUSIONS: It has been observed that a dual approach of serological and molecular tests detects a higher absolute number of disease cases in a pandemic context,which could improve monitoring and health surveillance efficacy. The age-related seropositivity frequency in this study, if confirmed, could enhance the validity of serological tests, especially in older patients.In these subjects, molecular positivity accompanied by serological positivity (distinct for M and G immunoglobulins) should help determine disease status and support decisions related to patient management.

Elevated monocyte distribution width in COVID-19 patients: The contribution of the novel sepsis indicator.

INTRODUCTION: Interesting results regarding the contribution of MDW (Monocyte Distribution Width) in the Infectious Disease Unit have been reported. An observational study is ongoing at San Donato Hospital with the aim to evaluate the contribution of MDW in the diagnostic pathway in adult patients entering in the ED setting and tested for SARS-CoV-2. MATERIAL AND METHOD: COVID-19 symptomatic and paucisymptomatic patients presenting to ED (Emergency Department), have been enrolled consecutively. Whole blood venous samples have been collected on K2 EDTA for MDW determination, at the same time a nasopharyngeal swab for SARS-CoV-2 RNA detection have been collected. RESULTS: One hundred six patients were negative for SARS-CoV-2 with MDW mean value of 20.3 ± 3.3, while forty-one were positive for SARS-CoV-2 with higher MDW mean value of 27.3 ± 4.9 (P < 0.005). The ROC curve analysis has been evaluated showing MDW AUC of 0.91. Finally twenty-three patients hospitalized in high-intensity care unit showed an MDW value higher than the eighteen patients presenting few symptoms [28.8 ± 5.3 vs 25.4 ± 3.6 respectively, P < 0.05]. DISCUSSION: Monocytic population, in Covid19 disease, are the first elements of innate immunity to be involved, these changes are the basis of the modification of the MDW, with evident efficacy in term of sensitivity, particularly in the studied Covid19 patients. Moreover the patients hospitalized in high-intensity care unit showed significantly elevated MDW respects to middle or low symptomatic one, suggest including this parameter as prognostic marker or of therapy efficacy, integrated with other laboratory findings.

International external quality assurance of JAK2 V617F quantification.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

Mutation landscape in patients with myelofibrosis receiving ruxolitinib or hydroxyurea.

Refractoriness to ruxolitinib in patients with myelofibrosis (MF) was associated with clonal evolution; however, whether genetic instability is promoted by ruxolitinib remains unsettled. We evaluated the mutation landscape in 71 MF patients receiving ruxolitinib (n = 46) and hydroxyurea (n = 25) and correlated with response. A spleen volume response (SVR) was obtained in 57% and 12%, respectively. Highly heterogenous patterns of mutation acquisition/loss and/or changes of variant allele frequency (VAF) were observed in the 2 patient groups without remarkable differences. In patients receiving ruxolitinib, driver mutation type and high-molecular risk profile (HMR) at baseline did not impact on response rate, while HMR and sole ASXL1 mutations predicted for SVR loss at 3 years. In patients with SVR, a decrease of ≥ 20% of JAK2V617F VAF predicted for SVR duration. VAF increase of non-driver mutations and clonal progression at follow-up correlated with SVR loss and treatment discontinuation, and clonal progression also predicted for shorter survival. These data indicate that (i) ruxolitinib does not appreciably promote clonal evolution compared with hydroxyurea, (ii) VAF increase of pre-existing and/or (ii) acquisition of new mutations while on treatment correlated with higher rate of discontinuation and/or death, and (iv) reduction of JAK2V617F VAF associated with SVR duration.

Recommendations for molecular testing in classical Ph1-neg myeloproliferative disorders-A consensus project of the Italian Society of Hematology.

The discovery that Philadelphia-negative classical myeloproliferative neoplasms (MPNs) present with several molecular abnormalities, including the mostly represented JAK2V617F mutation, opened new horizons in the diagnosis, prognosis, and monitoring of these disorders. However, the great strides in the knowledge on molecular genetics need parallel progresses on the best approach to methods for detecting and reporting disease-associated mutations, and to shape the most effective and rationale testing pathway in the diagnosis, prognosis and monitoring of MPNs. The MPN taskforce of the Italian Society of Hematology (SIE) assessed the scientific literature and composed a framework of the best, possibly evidence-based, recommendations for optimal molecular methods as well as insights about the applicability and interpretation of those tests in the clinical practice, and clinical decision for testing MPNs patients. The issues dealt with: source of samples and nucleic acid template, the most appropriate molecular abnormalities and related detection methods required for diagnosis, prognosis, and monitoring of MPNs, how to report a diagnostic molecular test, calibration and quality control. For each of these issues, practice recommendations were provided.

Presentation and outcome of patients with 2016 WHO diagnosis of prefibrotic and overt primary myelofibrosis.

The 2016 revision of the World Health Organization (WHO) classification of myeloproliferative neoplasms defines 2 stages of primary myelofibrosis (PMF): prefibrotic/early (pre-PMF) and overt fibrotic (overt PMF) phase. In this work, we studied the clinical and molecular features of patients belonging to these categories of PMF. The diagnosis of 661 PMF patients with a bone marrow biopsy at presentation was revised according to modern criteria; clinical information and annotation of somatic mutations in both driver and selected nondriver myeloid genes were available for all patients. Compared with pre-PMF, overt PMF was enriched in patients with anemia, thrombocytopenia, leukopenia, higher blast count, symptoms, large splenomegaly, and unfavorable karyotype. The different types of driver mutations were similarly distributed between the 2 categories, whereas selected mutations comprising the high mutation risk (HMR) category (any mutations in ASXL1, SRSF2, IDH1/2, EZH2) were more represented in overt PMF. More patients with overt PMF were in higher International Prognostic Scoring System risk categories at diagnosis, and the frequency increased during follow-up, suggesting greater propensity to disease progression compared with pre-PMF. Median survival was significantly shortened in overt PMF (7.2 vs 17.6 years), with triple negativity for driver mutations and presence of HMR mutations representing independent predictors of unfavorable outcome. The findings of this "real-life" study indicate that adherence to 2016 WHO criteria allows for identification of 2 distinct categories of patients with PMF where increased grades of fibrosis are associated with more pronounced disease manifestations, adverse mutation profile, and worse outcome, overall suggesting they might represent a phenotypic continuum.

Prognostic impact of bone marrow fibrosis in primary myelofibrosis. A study of the AGIMM group on 490 patients.

The prognostic significance of bone marrow (BM) fibrosis grade in patients with primary myelofibrosis (PMF) is still debated. A fibrosis grade greater than 1 was shown to associate with higher risk of death, and addition of fibrosis grade to IPSS score resulted in a more accurate prediction of survival. The aim of this study was to analyze the prognostic impact of BM fibrosis in 490 patients with PMF, evaluated at diagnosis, molecularly annotated and with extensive follow-up information. We found that fibrosis grade 2 and greater on a 0-3 scale was associated with clinical characteristics indicative of a more advanced disease, such as anemia, leukopenia, thrombocytopenia, constitutional symptoms, larger splenomegaly and a higher IPSS risk category. Patients with higher grade of fibrosis were also more likely to have additional somatic mutations in ASXL1 and EZH2, that are prognostically adverse. Median survival was significantly reduced in patients with grade 2 and 3 fibrosis as compared with grade 1; this effect was maintained when analysis was restricted to younger patients. In multivariate analysis, fibrosis grade independently predicted for survival regardless of IPSS variables and mutational status; the adverse impact of fibrosis was noticeable especially in lower IPSS risk categories. Overall, results indicate that higher grades of fibrosis correlate with unique clinical and molecular aspects and represent an independent adverse variable in patients with PMF; these observations deserve confirmation in prospectively designed series of patients. Am. J. Hematol. 91:918-922, 2016. © 2016 Wiley Periodicals, Inc.

Epidemiology and clinical relevance of mutations in postpolycythemia vera and postessential thrombocythemia myelofibrosis: A study on 359 patients of the AGIMM group.

Transformation to secondary myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PPV-MF) and essential thrombocythemia (PET-MF). Although primary (PMF) and secondary MF are considered similar diseases and managed similarly, there are few studies specifically focused on the latter. The aim of this study was to characterize the mutation landscape, and describe the main clinical correlates and prognostic implications of mutations, in a series of 359 patients with PPV-MF and PET-MF. Compared with PV and ET, the JAK2V617F and CALR mutated allele burden was significantly higher in PPV-MF and/or PET-MF, indicating a role for accumulation of mutated alleles in the process of transformation to MF. However, neither the allele burden nor the type of driver mutation influenced overall survival (OS), while absence of any driver mutation (triple negativity) was associated with significant reduction of OS in PET-MF, similar to PMF. Of the five interrogated subclonal mutations (ASXL1, EZH2, SRSF2, IDH1, and IDH2), that comprise a prognostically detrimental high molecular risk (HMR) category in PMF, only SRSF2 mutations were associated with reduced survival in PET-MF, and no additional mutation profile with prognostic relevance was highlighted. Overall, these data indicate that the molecular landscape of secondary forms of MF is different from PMF, suggesting that unknown mutational events might contribute to the progression from chronic phase disease to myelofibrosis. These findings also support more extended genotyping approaches aimed at identifying novel molecular abnormalities with prognostic relevance for patients with PPV-MF and PET-MF. Am. J. Hematol. 91:681-686, 2016. © 2016 Wiley Periodicals, Inc.

Targeted deep sequencing in polycythemia vera and essential thrombocythemia.

Polycythemia vera (PV) is characterized by JAK2 and essential thrombocythemia (ET) by JAK2, calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) mutations; we describe the occurrence and prognostic relevance of DNA sequence variants/mutations other than JAK2/CALR/MPL. A myeloid neoplasm-relevant 27-gene panel was used for next-generation sequencing of bone marrow or whole blood DNA and conventional tools were used for analysis. "Adverse variants/mutations" were identified by age-adjusted multivariable analysis of impact on overall, leukemia-free, or myelofibrosis-free survival. Fifty-three percent of 133 Mayo Clinic patients with PV and 53% of 183 with ET harbored 1 or more sequence variants/mutations other than JAK2/CALR/MPL; the most frequent were TET2 and ASXL1. "Adverse variants/mutations" in PV included ASXL1, SRSF2, and IDH2 and in ET SH2B3, SF3B1, U2AF1, TP53, IDH2, and EZH2; combined prevalence was 15% and 15%, respectively. Adverse variants/mutations were associated with inferior survival in both PV (median, 7.7 vs 16.9 years) and ET (median, 9 vs 22 years) and the effect was independent of conventional prognostic models with respective hazard ratio (95% confidence interval) of 2.8 (1.5-5.1) and 2.6 (1.4-4.8); these observations were validated in 215 Italian patients with PV and 174 with ET. In both Mayo Clinic and Italian cohorts, leukemic or fibrotic progression was also predicted by adverse variants/mutations. Number of mutations did not provide additional prognostic information. We conclude that targeted deep sequencing in PV and ET allows for genetic risk stratification that is independent of clinically derived prognostic models.

Impact of JAK2(V617F) mutation status on treatment response to anagrelide in essential thrombocythemia: an observational, hypothesis-generating study.

A JAK2(V617F) mutation is found in approximately 55% of patients with essential thrombocythemia (ET), and represents a key World Health Organization diagnostic criterion. This hypothesis-generating study (NCT01352585) explored the impact of JAK2(V617F) mutation status on treatment response to anagrelide in patients with ET who were intolerant/refractory to their current cytoreductive therapy. The primary objective was to compare the proportion of JAK2-positive versus JAK2-negative patients who achieved at least a partial platelet response (≤600×10(9)/L) after anagrelide therapy. Of the 47 patients enrolled, 46 were included in the full analysis set (JAK2-positive, n=22; JAK2-negative, n=24). At 12 months, 35 patients (n=14 and n=21, respectively) had a suitable platelet sample; of these, 74.3% (n=26) achieved at least a partial response. The response rate was higher in JAK2-positive (85.7%, n=12) versus JAK2-negative patients (66.7%, n=14) (odds ratio [OR] 3.00; 95% confidence interval [CI] 0.44, 33.97). By using the last observation carried forward approach in the sensitivity analysis, which considered the imbalance in patients with suitable samples between groups, the overall response rate was 71.7% (n=33/46), with 77.3% (n=17/22) of JAK2-positive and 66.7% (n=16/24) of JAK2-negative patients achieving at least a partial response (OR 1.70; 95% CI 0.39, 8.02). There was no significant change in median allele burden over 12 months in the 12 patients who achieved a response. In conclusion, the overall platelet response rate was high in both JAK2-positive and JAK2-negative patients; however, a larger study would be required to confirm the differences observed according to JAK2(V617F) mutation status.

Neutrophilic progression in a case of polycytemia vera mimicking chronic neutrophilic leukemia: clinical and molecular characterization.

BACKGROUND: In a small subset of polycytemia vera (PV), neutrophilia not secondary to reactive conditions or treatment can develop and persist. Clinical significance and morphogenetic alterations associated with this uncommon phenomenon are not well defined. CASE REPORT: An 81-year-old Caucasian woman, affected by polycytemia vera lasting 17 years, presented in March 2012 with hyperleukocytosis, absolute neutrophilia, and thrombocytosis despite hydroxyurea treatment. All other laboratory parameters were normal, except for an increased neutrophil alkaline phosphatase and lactate dehydrogenase. Reactive neutrophilia due to infection or neoplasia have been ruled out by a total body computerized tomography scan, and by low levels of C reactive protein. Re-evaluation of bone marrow showed hypercellular smears with expansion of granulopoiesis while immature granulocytes were <10% and myeloblasts were <1%. Bone marrow trephine biopsy showed hypercellular marrow, with panmyelosis, increased myeloid/erithroid ratio, polymorphic clusters of megakaryocytes. A loose network of reticulin fibers with many intersections was identified by means of Gomori's silver impregnation. There were no hybrid BCR/ABL gene transcripts of p210, p190 and p230, no mutations in platelet derived growth factor receptors alpha and beta. Flow cytometry on the aspirate showed that CD34+ CD117+ myeloblasts constituted less than 1% of total marrow nucleated cells, mature granulocytes demonstrated persistent expression of CD33. Mutational analysis of the gene CSF3R by PCR amplification revealed no alterations in exons 14-17, including codons 615 and 618. The case presented here represents a possible evolution of PV, albeit very rare. CONCLUSIONS: The condition described here differs from the CNL for the persistence of morphological pictures typical of myeloproliferative diseases, for absence of CSF3R gene mutations and for the hyper expansion of the mature granulopoietic series. The clinical significance and morphogenetic alterations associated with this uncommon phenomenon are not well defined.

Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia.

Mutations in the calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia (ET) lacking the JAK2V617F and MPLW515 mutations, but no information is available on the clinical correlates. In this series, CALR mutations were found in 15.5% of 576 World Health Organization-defined ET patients, accounting for 48.9% of JAK2 and MPL wild-type (wt) patients. CALR-mutated patients were preferentially male and showed higher platelet count and lower hemoglobin and leukocyte count compared with JAK2- and MPL-mutated patients. Patients carrying the CALR mutation had a lower risk of thrombosis than JAK2- and MPL-mutated patients; of interest, their risk was superimposable to patients who were wt for the above mutations. CALR mutation had no impact on survival or transformation to post-ET myelofibrosis. Genotyping for CALR mutations represents a novel useful tool for establishing a clonal myeloproliferative disorder in JAK2 and MPL wt patients with thrombocytosis and may have prognostic and therapeutic relevance.

Acquired copy-neutral loss of heterozygosity of chromosome 1p as a molecular event associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms.

We studied mutations of MPL exon 10 in patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF), first investigating a cohort of 892 consecutive patients. MPL mutation scanning was performed on granulocyte genomic DNA by using a high-resolution melt assay, and the mutant allele burden was evaluated by using deep sequencing. Somatic mutations of MPL, all but one involving codon W515, were detected in 26/661 (4%) patients with ET, 10/187 (5%) with PMF, and 7/44 (16%) patients with post-ET myelofibrosis. Comparison of JAK2 (V617F)-mutated and MPL-mutated patients showed only minor phenotypic differences. In an extended group of 62 MPL-mutated patients, the granulocyte mutant allele burden ranged from 1% to 95% and was significantly higher in patients with PMF or post-ET myelofibrosis compared with those with ET. Patients with higher mutation burdens had evidence of acquired copy-neutral loss of heterozygosity (CN-LOH) of chromosome 1p in granulocytes, consistent with a transition from heterozygosity to homozygosity for the MPL mutation in clonal cells. A significant association was found between MPL-mutant allele burden greater than 50% and marrow fibrosis. These observations suggest that acquired CN-LOH of chromosome 1p involving the MPL location may represent a molecular mechanism of fibrotic transformation in MPL-mutated myeloproliferative neoplasms.