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Tumor mutational burden (TMB) has recently been approved as an agnostic biomarker for immune checkpoint inhibitors. However, methods for TMB testing have not yet been standardized. The International Quality Network for Pathology (IQNPath) organized a pilot external quality assessment (EQA) scheme for TMB testing. The aim of this program was the validation of the materials and the procedures for the EQA of this complex biomarker. Five formalin-fixed paraffin-embedded (FFPE) cell lines were selected to mimic the various TMB values observed in clinical practice. The FFPE samples were tested with the FoundationOne CDx (F1CDx) assay as the reference test and three commercially available targeted sequencing panels. Following this internal validation, the five cell lines were sent to 29 laboratories selected on the basis of a previous survey. Nineteen of the 23 laboratories that submitted results (82.6%) used targeted sequencing for TMB estimation. Only two laboratories performed whole exome sequencing (WES) and two assessed TMB by clinical exome. A high variability in the reported TMB values was observed. The variability was higher for samples with the highest TMB value according to the F1CDx test. However, good reproducibility of the TMB score was shown by laboratories using the same panel. The majority of laboratories did not indicate a TMB cut-off value for clinical interpretation. In conclusion, this pilot EQA scheme suggests that it is feasible to run such an EQA program for TMB assessment. However, the results of our pilot highlight the numerous challenges for the standardization of this test.

Increasing evidence suggests that liquid biopsy might play a relevant role in the management of metastatic non-small cell lung cancer (NSCLC) patients. Here, we show how the Molecular Tumor Board (MTB) in our cancer center employed liquid biopsy to support therapeutic decisions in a patient with NSCLC carrying a rare EGFR mutation. A 44-year-old woman, never-smoker with an EGFR, ALK, and ROS1-negative lung adenocarcinoma and multiple brain metastases received systemic therapy and surgery before being referred to our Institute. The MTB suggested NGS testing of tumor biopsy that revealed a rare exon-20 EGFR insertion (p.His773dup; c.2315_2316insCCA) and EGFR amplification. The MTB recommended treatment with erlotinib and follow-up with liquid biopsy, by using both cell-free DNA (cfDNA) and circulating tumor cells (CTCs). An increase of EGFR mutation levels in cfDNA revealed resistance to treatment about 6 months before clinical progression. Extremely low levels of EGFR p.T790M were detected at progression. Based on preclinical data suggesting activity of osimertinib against EGFR exon-20 insertions, the MTB recommended treatment with brain and bone radiotherapy and osimertinib. A dramatic reduction of EGFR mutation levels in the cfDNA was observed after 4 weeks of treatment. The PET scan demonstrated a metabolic partial remission that was maintained for 9 months. This case supports the evidence that liquid biopsy can aid in the management of metastatic NSCLC. It also suggests that treatment with osimertinib might be a therapeutic option in patients with EGFR exon-20 insertions when a clinical trial is not available.

Analysis of plasma-derived cell-free DNA (cfDNA) might allow for the early identification of resistance in metastatic colorectal carcinoma (mCRC) patients receiving anti-EGFR monoclonal antibodies. We tested plasma samples from the Erbitux Metastatic Colorectal Cancer Strategy (ERMES) phase III trial of FOLFIRI+Cetuximab in first-line treatment of RAS/BRAF wild-type mCRC. Samples were collected at baseline (n = 37), at 8 weeks of treatment (n = 32), progressive disease (PD; n = 36) and 3 months after PD (n = 21). cfDNA testing was performed using the Idylla™ ctKRAS and ctNRAS-BRAF tests and the Oncomine Pan-Cancer Cell-Free Assay. Analysis of basal samples revealed RAS/BRAF mutations in 6/37 cases. A transient RAS positivity not associated with PD was observed at 8 weeks in five cases that showed no mutations at baseline and PD. The frequency of mutant cases increased at PD (33.3%) and decreased again at 3 months after PD (9.5%). The median progression-free survival (mPFS) of patients RAS/BRAF mutant at PD was 7.13 months versus 7.71 months in wild-type patients (p = 0.3892). These data confirm that the occurrence of RAS/BRAF mutations in mCRC patients receiving anti-EGFR agents is relatively frequent. However, the cfDNA dynamics of RAS mutations in patients treated with anti-EGFR agents plus polychemotherapy are complex and might not be directly associated with resistance to treatment.

The complex pathophysiologic interplay between SARS-CoV-2 infection and complement activation is the subject of active investigation. It is clinically mirrored by the occurrence of exacerbations of complement mediated diseases during COVID-19 infection. These include complement-mediated hemolytic anemias such as paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA), particularly cold agglutinin disease (CAD), and hemolytic uremic syndrome (HUS). All these conditions may benefit from complement inhibitors that are also under study for COVID-19 disease. Hemolytic exacerbations in these conditions may occur upon several triggers including infections and vaccines and may require transfusions, treatment with complement inhibitors and/or immunosuppressors (i.e., steroids and rituximab for AIHA), and result in thrombotic complications. In this manuscript we describe four patients (2 with PNH and 2 with CAD) who experienced hemolytic flares after either COVID-19 infection or SARS-Cov2 vaccine and provide a review of the most recent literature. We report that most episodes occurred within the first 10 days after COVID-19 infection/vaccination and suggest laboratory monitoring (Hb and LDH levels) in that period. Moreover, in our experience and in the literature, hemolytic exacerbations occurring during COVID-19 infection were more severe, required greater therapeutic intervention, and carried more complications including fatalities, as compared to those developing after SARS-CoV-2 vaccine, suggesting the importance of vaccinating this patient population. Patient education remains pivotal to promptly recognize signs/symptoms of hemolytic flares and to refer to medical attention. Treatment choice should be based on the severity of the hemolytic exacerbation as well as of that of COVID-19 infection. Therapies include transfusions, complement inhibitor initiation/additional dose in the case of PNH, steroids/rituximab in patients with CAD and warm type AIHA, plasma exchange, hemodialysis and complement inhibitor in the case of atypical HUS. Finally, anti-thrombotic prophylaxis should be always considered in these settings, provided safe platelet counts.

Bortezomib is a first-in-class, potent, selective and reversible proteasome inhibitor approved for the treatment of multiple myeloma (MM) and relapsed/refractory mantle cell lymphoma. In these diseases, bortezomib targets plasma cells and lymphocytes reducing tumor burden. Recently, preclinical evidence highlighted its efficacy in reducing long-lived plasma cells responsible of autoantibodies production in several models of autoimmune conditions. These findings paved the way to a number of experiences of bortezomib use in patients with various autoimmune conditions, including autoimmune hemolytic anemia (AIHA). The latter is a nice model of autoimmunity in hematology and is caused by the production of autoantibodies against erythrocytes resulting in various degrees of hemolytic anemia. AIHA is classified in warm and cold forms according to the thermal characteristics of the autoantibody, and first-line treatment mainly relies on steroids for warm cases and the anti-CD20 rituximab for cold ones. Relapsed/refractory cases are still an unmet need, and bortezomib has been proposed in this setting with intriguing efficacy. In this review, we collected available literature on bortezomib use in AIHA and in other immune-mediated hematologic and non-hematologic diseases. Overall, most experiences highlight bortezomib efficacy even in multi-relapsed/refractory patients and suggest to consider its use in AIHA after rituximab failure.

Large granular lymphocytes (LGL) are lymphoid cells characterized by either a T-cell or a natural killer phenotype whose expansion may be reactive to toxic, infectious, and neoplastic conditions, or result from clonal selection. Recently, the higher attention to LGL clones led to their detection in many clinical conditions including myeloid neoplasms and bone marrow failures. In these contexts, it is still unclear whether LGL cells actively contribute to anti-stem cell autoimmunity or are only a reaction to dysplastic/leukemic myelopoiesis. Moreover, some evidence exists about a common clonal origin of LGL and myeloid clones, including the detection of STAT3 mutations, typical of LGL, in myeloid precursors from myelodysplastic patients. In this article we reviewed available literature regarding the association of LGL clones with myeloid neoplasms (myelodysplastic syndromes, myeloproliferative neoplasms, and acute myeloid leukemias) and bone marrow failures (aplastic anemia and pure red cell aplasia, PRCA) focusing on evidence of pathogenic, clinical, and prognostic relevance. It emerged that LGL clones may be found in up to one third of patients, particularly those with PRCA, and are associated with a more cytopenic phenotype and good response to immunosuppression. Pathogenically, LGL clones seem to expand after myeloid therapies, whilst immunosuppression leading to LGL depletion may favor leukemic escape and thus requires caution.

Myelodysplastic syndromes (MDS) are a very heterogeneous disease, with extremely variable clinical features and outcomes. Current management relies on risk stratification based on IPSS and IPSS-R, which categorizes patients into low (LR-) and high-risk (HR-) MDS. Therapeutic strategies in LR-MDS patients mainly consist of erythropoiesis stimulating agents (ESAs), transfusion support, and luspatercept or lenalidomide for selected patients. Current unmet needs include the limited options available after treatment failure, and the consequent transfusion burden with several hospital admissions and poor quality of life. Therapeutic approaches in HR-MDS patients are aimed at changing the natural course of the disease and hypometylating agents (HMA) are the first choice. The only potentially curative treatment is allogeneic stem cell transplant (allo-HCT), restricted to a minority of young and fit candidates. Patients unfit for or those that relapse after the abovementioned options harbor an adverse prognosis, with limited overall survival and frequent leukemic evolution. Recent advances in genetic mutations and intracellular pathways that are relevant for MDS pathogenesis are improving disease risk stratification and highlighting therapeutic targets addressed by novel agents. Several drugs are under evaluation for LR and HR patients, which differ by their mechanism of action, reported efficacy, and phase of development. This review analyzes the current unmet clinical needs for MDS patients and provides a critical overview of the novel agents under development in this setting.

It is well established that immune thrombocytopenia (ITP) results from increased immune mediated platelet destruction (anti-platelets antibodies, autoreactive T cells, and reduction of regulatory T cells) along with impaired production in the bone marrow.1 The latter has been attributed to both cellular and humoral mediators that cause suppression of megakaryocyte production and maturation.2 Current standard first line therapy consists of corticosteroids, with or without intravenous Ig, achieving about 70-80% response rate. However, a consistent proportion of patients would relapse after corticosteroid discontinuation or tapering, and requires further therapy. ...

The introduction in the clinic of immune checkpoint inhibitors (IOs) has represented an important improvement for the treatment of patients with advanced non-small cell lung cancer (NSCLC). These drugs have shown a higher activity as compared with chemotherapy in both first- and second-line of treatment, with some patients experiencing a long-lasting response. More recently, combinations of IOs have entered clinical trials in different tumor types including NSCLC. Nevertheless, IOs are active only in a subgroup of patients and biomarkers for appropriate patients' selection are urgently needed to offer the patients an effective therapy, and also to manage the costs. Tumor mutation burden (TMB) has powerfully emerged as a potential biomarker for immunotherapy and might enter the clinic in the next months, although different challenges are still unsolved. Different methods exist to evaluate TMB in tissue, ranging from whole exome sequencing (WES) to targeted sequencing of smaller sets of genes, which need to be fully standardized to ensure that patients receive an appropriate TMB test with clear clinical interpretation. In addition, as already happened for the implementation of liquid biopsy testing from NSCLC patients to identify targetable alterations, researchers are also evaluating the possibility to calculate TMB in blood, to further enlarge the number of NSCLC patients who may benefit from immunotherapy. Preliminary data highlight the difficulty to develop targeted sequencing panels for the assessment of TMB starting from the circulating cell free DNA (cfDNA). The applicability of TMB testing on liquid biopsy needs further investigation and may be clarified within the ongoing clinical trials.

Several immune checkpoint inhibitors (ICIs) have already been introduced into clinical practice or are in advanced phases of clinical experimentation. Extensive efforts are being made to identify robust biomarkers to select patients who may benefit from treatment with ICIs. Tumor mutation burden (TMB) may be a relevant biomarker of response to ICIs in different tumor types; however, its clinical use is challenged by the analytical methods required for its evaluation. The possibility of using targeted next-generation sequencing panels has been investigated as an alternative to the standard whole exome sequencing approach. However, no standardization exists in terms of genes covered, types of mutations included in the estimation of TMB, bioinformatics pipelines for data analysis, and cut-offs used to discriminate samples with high, intermediate or low TMB. Bioinformatics serve a relevant role in the analysis of targeted sequencing data and its standardization is essential to deliver a reliable test in clinical practice. In the present study, cultured and formalin-fixed, paraffin-embedded cell lines were analyzed using a commercial panel for TMB testing; the results were compared with data from the literature and public databases, demonstrating a good correlation. Additionally, the correlation between high tumor mutation burden and microsatellite instability was confirmed. The bioinformatics analyses were conducted using two different pipelines to highlight the challenges associated with the development of an appropriate analytical workflow.

The co-occurrence of myeloid neoplasms and lymphoproliferative diseases (LPDs) has been epidemiologically described, particularly in myeloproliferative neoplasms (MPNs). However, the clinical features of these patients are poorly known. In this study, we evaluated a single-center cohort of 44 patients with a diagnosis of myeloid and LPD focusing on clinical features, therapy requirement, and outcome. The two diagnoses were concomitant in 32% of patients, while myeloid disease preceded LPD in 52% of cases (after a median of 37 months, 6–318), and LPD preceded myeloid neoplasm in 16% (after a median of 41 months, 5–242). The most prevalent LPD was non-Hodgkin lymphoma (50%), particularly lymphoplasmacytic lymphoma (54.5%), followed by chronic lymphocytic leukemia (27%), plasma cell dyscrasias (18.2%), and rarer associations such as Hodgkin lymphoma and Erdheim–Chester disease. Overall, 80% of BCR-ABL1-negative MPN patients required a myeloid-specific treatment and LPD received therapy in 45.5% of cases. Seven subjects experienced vascular events, 13 a grade >/= 3 infectious episode (9 pneumonias, 3 urinary tract infection, and 1 sepsis), and 9 developed a solid tumor. Finally, nine patients died due to solid tumor (four), leukemic progression (two), infectious complications (two), and brain bleeding (one). Longer survival was observed in younger patients (p = 0.001), with better performance status (p = 0.02) and in the presence of driver mutations (p = 0.003). Contrarily, a worse survival was significantly associated with the occurrence of infections (p < 0.0001). These data suggest that in subjects with co-occurrence of myeloid and lymphoid neoplasms, high medical surveillance for infectious complications is needed, along with patient education, since they may negatively impact outcome.

BackgroundSince the first complete genome sequencing of SARS-CoV-2 in December 2019, more than 550,000 genomes have been submitted into the GISAID database. Sequencing of the SARS-CoV-2 genome might allow identification of variants with increased contagiousness, different clinical patterns and/or different response to vaccines. A highly automated next generation sequencing (NGS)-based method might facilitate an active genomic surveillance of the virus.MethodsRNA was extracted from 27 nasopharyngeal swabs obtained from citizens of the Italian Campania region in March–April 2020 who tested positive for SARS-CoV-2. Following viral RNA quantification, sequencing was performed using the Ion AmpliSeq SARS-CoV-2 Research Panel on the Genexus Integrated Sequencer, an automated technology for library preparation and sequencing. The SARS-CoV-2 complete genomes were built using the pipeline SARS-CoV-2 RECoVERY (REconstruction of COronaVirus gEnomes & Rapid analYsis) and analysed by IQ-TREE software.ResultsThe complete genome (100%) of SARS-CoV-2 was successfully obtained for 21/27 samples. In particular, the complete genome was fully sequenced for all 15 samples with high viral titer (> 200 copies/µl), for the two samples with a viral genome copy number ConclusionsThe Genexus system resulted successful for SARS-CoV-2 complete genome sequencing, also in cases with low viral copies. The use of this highly automated system might facilitate the standardization of SARS-CoV-2 sequencing protocols and make faster the identification of novel variants during the pandemic.

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Background Sequencing artifacts, clonal hematopoietic mutations of indeterminate potential (CHIP) and tumor heterogeneity have been hypothesized to contribute to the low concordance between tissue and cell-free DNA (cfDNA) molecular profiling with targeted sequencing. Methods We analyzed by targeted sequencing cfDNA from 30 healthy individuals, and cfDNA and matched tumor samples from 30 EGFR-mutant and 77 EGFR wild-type metastatic non-small-cell lung cancer (mNSCLC) patients. Discordant cases were solved by droplet digital PCR (ddPCR). Results By testing cfDNA from healthy donors, we developed an algorithm to recognize sequencing artifacts. Applying this method to cfDNA from mNSCLC patients, EGFR mutations were detected with a good sensitivity (76.7%) and specificity (97.4%). In contrast, sensitivity and specificity for KRAS variants were 61.5% and 93.8%, respectively. All EGFR and KRAS variants detected in plasma but not in tissue were confirmed by ddPCR, thus excluding sequencing artifacts. In a fraction of cases, KRAS mutations found in plasma samples were confirmed in tumor tissue suggesting tumor heterogeneity. KRAS variants were found to be more likely sub-clonal as compared with EGFR mutations, and a correlation between clonal origin and frequency of detection in plasma was found. In a case with both EGFR and KRAS variants in cfDNA, we could demonstrate the presence of the KRAS variant in tumor tissue associated with lack of response to tyrosine kinase inhibitors (TKIs). Conclusions Although sequencing artifacts can be identified in targeted sequencing of cfDNA, tumor heterogeneity and CHIP are likely to influence the concordance between plasma and tissue testing.

The relationship between chronic lymphocytic leukaemia (CLL) and qualitative/quantitative gammaglobulin abnormalities is well established. Nevertheless, in order to better understand this kind of connection, we examined 1505 patients with CLL and divided them into four subgroups on the basis of immunoglobulin (Ig) aberrations at diagnosis. A total of 73 (4·8%), 149 (10%), 200 (13·2%) and 1083 (72%) patients were identified with IgM monoclonal gammopathy (IgM/CLL), IgG monoclonal gammopathy (IgG/CLL), hypogammaglobulinaemia (hypo-γ) and normal Ig levels (γ-normal) respectively. IgM paraprotein was significantly associated with a more advanced Binet/Rai stage and del(17p)/TP53 mutation, while IgG abnormalities correlated with a higher occurrence of trisomy 12. Patients with any type of Ig abnormality had shorter treatment-free survival (TFS) but no significant impact affecting overall survival (OS) compared to those with normal Ig levels.

Circulating cell-free DNA (cfDNA) testing has emerged as an alternative to tumor tissue analyses for the management of metastatic non-small-cell lung cancer (NSCLC) patients. Analysis of cfDNA is a minimally invasive procedure that might better reflect tumor heterogeneity and allows repeated testing over the time. Areas covered: This review article covers the different applications of cfDNA testing in NSCLC: early diagnosis of the disease; detection of minimal residual disease in early lung cancer; identification of predictive and prognostic markers in advanced NSCLC patients; monitoring the response to therapy; assessment of tumor mutation burden. Expert commentary: The use of liquid biopsy is rapidly expanding to different applications. The combination of different circulating biomarkers (cfDNA, protein, miRNA) might improve the sensitivity and specificity of this approach in patients with low tumor burden. cfDNA testing is representing a valid source for molecular profiling in management of metastatic NSCLC patients and is providing important knowledge on tumor heterogeneity. Clinical trials are needed in order to transfer the information deriving from liquid biopsy testing in new therapeutic strategies.

Simple Summary This review analyzes the occurrence, clinical characteristics, and prognostic impact and treatment of autoimmune hemolytic anemia (AIHA) in chronic lymphocytic leukemia (CLL). Autoimmune hemolytic anemia is observed in about 10% of CLL. Pathogenesis is multifactorial involving humoral, cellular, and innate immunity, so the different mechanisms are well described in this review which also focuses on drugs associated to CLL-AIHA and on difficulties to diagnose it. There is a comprehensive revision of the main published casistics and then of the treatments; in particular the paper analyzes the main chemo-immunotherapeutic agents used in this setting. Since the therapy depends on the presence and severity of clinical symptoms, disease status, and comorbidities, treatment is nowadays more individualized in CLL and also in CLL-AIHA. Patients not responding to corticosteroids and rituximab are treated with CLL-specific drugs as per current guidelines according to age and comorbidities and new targeted agents against BCR and BCL-2 which can be given orally and have few side effects, are very effective both in progressive CLL and in situations such as AIHA. Abstract Chronic lymphocytic leukemia (CLL) patients have a greater predisposition to develop autoimmune complications. The most common of them is autoimmune hemolytic anemia (AIHA) with a frequency of 7–10% of cases. Pathogenesis is multifactorial involving humoral, cellular, and innate immunity. CLL B-cells have damaged apoptosis, produce less immunoglobulins, and could be responsible for antigen presentation and releasing inflammatory cytokines. CLL B-cells can act similar to antigen-presenting cells activating self-reactive T helper cells and may induce T-cell subsets imbalance, favoring autoreactive B-cells which produce anti-red blood cells autoantibodies. Treatment is individualized and it depends on the presence and severity of clinical symptoms, disease status, and comorbidities. Corticosteroids are the standardized first-line treatment; second-line treatment comprises rituximab. Patients not responding to corticosteroids and rituximab should be treated with CLL-specific drugs as per current guidelines according to age and comorbidities. New targeted drugs (BTK inhibitors and anti BCL2) are recently used after or together with steroids to manage AIHA. In the case of cold agglutinin disease, rituximab is preferred, because steroids are ineffective. Management must combine supportive therapies, including vitamins; antibiotics and heparin prophylaxis are indicated in order to minimize infectious and thrombotic risk.

Occult hepatitis B virus Infection (OBI) is defined as the presence of HBV-DNA in the liver and/or peripheral blood mononuclear cells (PBMCs) (with detectable or undetectable HBV-DNA in the serum) ...

The screening for BRAF V600E mutation is employed in clinical practice for its prognostic and potentially predictive role in patients with metastatic colorectal carcinoma (mCRC). Little information is available on the sensitivity and specificity of the testing methods to detect this mutation in CRC. By using serial dilution of BRAF mutant DNA with wild type DNA, we found that the sensitivity of allelic discrimination-Real Time PCR was higher than PCR-Sequencing (10% vs 20%). In agreement, the Real Time PCR assay displayed increased analytical sensitivity in detecting the BRAF V600E mutation as compared with PCR-Sequencing in a cohort of 510 consecutive CRCs (21 vs 16 cases). Targeted resequencing demonstrated that all cases negative by PCR-Sequencing had an allelic frequency of the BRAF mutation

Elderly patients with chronic lymphocytic leukemia (CLL) or patients with comorbidities are often treated with chlorambucil (Chl) as front-line therapy despite relatively low response rates. The addition of a monoclonal anti-CD20 antibody to Chl substantially increases response rates and prolongs progression-free survival (PFS) in these patients, without increasing toxicity. As a result, the ESMO guidelines recommend that previously untreated CLL patients with relevant co-morbidity, but without TP53 deletion/mutation, should be treated with the combination of Chl plus an anti-CD20 antibody (rituximab, ofatumumab or obinutuzumab). Areas covered: This review focuses on the treatment approach of elderly and unfit patients with untreated chronic lymphocytic leukemia. Expert commentary: The addition of a monoclonal anti-CD20 antibody to Chl is currently the suggested treatment in this subset of CLL patients. The choice of the anti-CD20 antibody remains an open question, although obinutuzumab was found to be superior to rituximab, in a head-to-head comparison of Chl-based combinations, in untreated CLL patients with comorbidities, with higher progression free survival, complete remission rates and minimal residual disease-negative remissions. Because patients with a TP53 deletion/mutation are resistant to chemo-immunotherapy, treatment with the BTK inhibitor ibrutinib is recommended in this setting.

Breast cancer is the second most common cause of mortality in women; therefore, the identification of novel putative markers is required to improve its diagnosis and prognosis. Selenium is known to protect mammary epithelial cells from oxidative DNA damage, and to inhibit the initiation phase of carcinogenesis by stimulating DNA repair and apoptosis regulation. Consequently, the present study has focused attention on the selenoprotein family and their involvement in breast cancer. The present study performed a global analysis of the seleno-transcriptome expression in human breast cancer MCF-7 and MDA-MB231 cell lines compared with healthy breast MCF-10A cells using reverse transcription-quantitative polymerase chain reaction. The present data revealed the presence of differently expressed genes in MCF-7 and MDA-MB231 cells compared with MCF-10A cells: Four downregulated [glutathione peroxidase (GPX)1, GPX4, GPX5 and GPX7] and three upregulated (deiodinase iodothyronine, type II, GPX2 and GPX3) genes. Additionally, interactomic investigation were performed by the present study to evaluate the association between the downregulated and upregulated genes, and to identify putative HUB nodes, which represent the centers of association between the genes that are capable of direct control over the gene networks. Network analysis revealed that all differentially regulated genes, with the exception of selenoprotein T, are implicated in the same network that presents three HUB nodes interconnected to the selenoprotein mRNAs, including TP53, estrogen receptor 1 and catenin-β1 (CTNNB1). Overall, these data demonstrated for the first time, a profile of seleno-mRNAs specific for human breast cells, indicating that these genes alter their expression on the basis of the ER-positivity or negativity of breast cancer cells.

Introduction: Chronic Lymphocytic Leukemia (CLL) is a clinically and biologically heterogeneous disorder that can be associated with immunological dysregulation. Several prognostic factors, such as IGHV status and chromosomal aberrations as trisomy 12, del11q, del13q or del17p have been detected so far. More recent genetic mutations, such as BIRC3, SF3B1, NOTCH1 and TP53 stratify even further the prognosis and the outcome in CLL patients (pts). However all data above reported, because of expensive techniques and experience typical of big laboratories, are not available to all medical centers. Therefore we considered necessary to identify more accessible and easy to perform parameters in all laboratories, as data concerning the presence of hypogammaglobulinemia, IgM or IgG monoclonal gammopathy, whose prevalence and impact on natural history of CLL in the medical literature are controversial, contradictory and not very significant because of few numbers in cohorts. Therefore the first aim of the study was to evaluate the prevalence of monoclonal IgM/CLL, IgG/CLL and hypogammaglobulinemia compared with CLL pts with normal immunoglobulins (Ig) levels and to establish the impact on clinical outcomes based on Ig levels. The second aim was to find a correlation between each group of pts and biological data. Methods: From four different Italian centers, we collected data from 1505 pts diagnosed with typical CLL from 1987 to 2017 and we divided them in four groups based on the presence of qualitative and quantitative alterations of serum immunoglobulins. Baseline assessment and levels of serum Ig, immunofixation, chromosomal aberrations and clinical features were collected. [Table 1]. We evaluated the impact of each immunoglobulin subset on progression free survival (PFS) and overall survival (OS). Results: Once assessment was made, we included in our study 1505 pts. Median age of 65.5 was similar in all groups and data gathered from the four centers showed an overlapping rate about prevalence and time-dependent parameters in CLL pts and their related subclasses. The overall prevalence of monoclonal gammopathy was 15%, of whom 149 pts (10%) with IgG/CLL and 73 pts (5%) with IgM/CLL. Hypogammaglobulinemia was detected in 200 pts (13%), while 1083 pts (72%) had no evidence of paraprotein or hypogammaglobulinemia. The worst outcome group was identified among CLL with IgM paraprotein; moreover, this group of pts showed more advanced stages at diagnosis according to Binet staging (p To evaluate if the specific deficit of IgG or IgM could have any unfavourable impact on each group's prognosis, we measured the serum levels of IgM and IgG in all pts. No statistically significant differences were observed among time-dependent parameters in IgG/CLL, IgM/CLL and hypogamma groups, based on IgM and IgG levels, if deficient or normal.Finally, in the group of pts who had normal gamma-level, median PFS and TTT was reached at 79 months (p=0.006 and 0.002, respectively) in the subgroup with IgM deficiency, while no statistical difference was observed regarding OS (p=0.785). Conclusions: Our study provides data about prevalence and impact of monoclonal gammopathy and hypogammaglobulinemia on the outcomes of CLL patients. In this study, we observed that the 28% pts had any qualitative or quantitative immunoglobulins alteration, of whom 15% had monoclonal gammopathy and 13% had hypogammaglobulinemia. The worst outcomes were observed in the IgM/CLL pts in terms of PFS and OS. In conclusion, the laboratory data of serum Ig could be used as predictive markers of clinical outcomes because, for their easy availability, they could direct the prognosis of CLL pts. As a future perspective, we would like to characterize pts with monoclonal IgM component from a biological view, to correlate the subset of somatic mutations of the heavy chains of surface immunoglobulins with the monoclonal component and its prognostic significance. Disclosures Varettoni: ABBVIE: Other: travel expenses; Roche: Consultancy; Gilead: Other: travel expenses; Janssen: Consultancy. Trentin:ABBVIE: Honoraria, Other: board; Janssen: Consultancy, Honoraria, Other: Board; gilead: Consultancy; Roche: Honoraria, Other: Board.

Background The role of tumour mutation burden (TMB) as predictive biomarker of response to immune checkpoint inhibitors (ICI) is being explored in colorectal cancer (CRC). Microsatellite instability status (MSI) is currently used to identify CRC patients who may benefit from ICI. However, TMB values vary significantly among MSI CRC. In addition, selected microsatellite stable (MSS) with high TMB might also benefit treatment with ICI. Methods TMB evaluation was performed with the Oncomine Tumor Mutation Load Assay (OTML, Thermofisher) on the Ion S5XL platform. Data analysis was carried out using Ion Reporter Software v5.10. TMB was calculated as the total number of non-synonymous somatic single nucleotide variants (SNVs) and indels divided by number of bases sequenced. MSI status was analysed by means of the Idylla MSI assay (Biocartis), which evaluates the presence of mutations in 7 novel MSI loci. Results TMB analysis was performed on 106 formalin-fixed paraffin embedded CRC samples and the data were compared with the MSI results. The Idylla assay classified 68 samples as MSS and 38 samples as microsatellite instable (MSI-H). The TMB values ranged from zero to 21.22 (median: 5.005) in the MSS and from 13.42 to 204.8 in the MSI-H group (median: 25.66), with a significant difference in median values (P Conclusions The TMB values assessed with the OTML assay strongly correlated with MSI status in CRC. However, significant heterogeneity in TMB levels were detected among both MSI and MSS tumors, suggesting that TMB testing might provide additional information on sensitivity to ICI in CRC. The correlations with driver gene alterations might help in selecting tumors to be tested for TMB. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

The ICARUS trial is a phase II, open label, multicenter, single arm study conducted to investigate the efficacy, safety, and tolerability of a rechallenge treatment with the first-generation tyrosine kinase inhibitor (TKI) gefitinib in advanced non-small-cell lung cancer (NSCLC) patients carrying activating mutations of the epidermal growth factor receptor (EGFR). The ICARUS trial enrolled 61 patients who were rechallenged with gefitinib at progression after second-line chemotherapy. Serum-derived circulating cell-free DNA (cfDNA) collected before the rechallenge from a cohort of 29 patients, was retrospectively analyzed for the EGFR exon 19 deletions and for the p.L858R and p.T790M single nucleotide variants (SNV). The analysis of cfDNA detected the same EGFR activating mutation reported in the tumor tissue in 20/29 patients, with a sensitivity of 69%. Moreover, a p.T790M variant was found in 14/29 patients (48.3%). The median progression-free survival (PFS) was 2.7 months for p.T790M positive patients (CI 95% 1.4&ndash, 3.1 months) versus 3.5 months for the p.T790M negative patients (CI 95% 1.6&ndash, 5.3 months), resulting in a statistically significant difference (Long rank test p = 0.0180). These findings confirmed the role of the p.T790M mutation in the resistance to first-generation TKIs. More importantly, our data suggest that TKI rechallenge should be guided by biomarker testing.

Introduction In colorectal cancer (CRC), the use of immunotherapy is currently based on the evaluation of microsatellite instability (MSI) for the identification of patients who may benefit from this treatment. Recently, tumour mutation burden (TMB) has been evaluated as a useful biomarker to predict response to immune checkpoint (IO) therapy. TMB can be measured by means of targeted next generation sequencing with panels covering approximately 1 Mb of the human genome. We performed TMB analysis on tumor cell lines and CRC samples and compared the results with MSI status to explore the relationship between these two biomarkers. Materials and methods MSI status was evaluated by means of routine immunohistochemistry (IHC), the Idylla MSI assay (Biocartis) and with fluorescence capillary electrophoresis-based DNA fragment size analysis of a T17 mononucleotide sequences within intron 8 of heat shock protein 110 kDa HSP110 (17 polythymine, T17). TMB evaluation was performed with the Oncomine Tumor Mutation Load Assay (Thermofisher) on the Ion S5XL platform. Data analysis was carried out using Ion Reporter Software v5.6. TMB was calculated as the total number of somatic single nucleotide variants (SNVs) divided by number of bases with sufficient coverage. Results We first evaluated the TMB in 13 tumor cell lines and correlated the results with massively parallel sequencing data from >1600 genes for the same cell lines available on cBioPortal, demonstrating an high correlation between the Oncomine Tumor Mutation Load Assay and the TMB value obtained by wide genetic profiling (R² = 0.976). We next analyzed a cohort of 23 MMR deficient (MMR-D) and 32 MMR-proficient (MMR-P) formalin fixed paraffin embedded (FFPE) CRC, as classified by IHC. The MMR data were compared to TMB and revealed TMB values not in line with the expected results, since some samples in the MMR-D group had low TMB values (TMB range: 5.5-134.4). We re-evaluated the 23 MMR-D samples by means of the Idylla System. The Idylla MSI assay re-classified 7 samples as microsatellite stable (MSS) and categorized the remaining 16 samples as MSI-High (MSI-H). The results were confirmed by the T17 analysis. In the MSS group (n. 39), the mean and median TMB value were 11.48 and 10.39, with TMB ranging from 4.88 to 33.29. The mean and median TMB for the MSI-H group (n. 16) were 43.89 and 38.63, and the TMB values ranged from 19.63 to 134.4. While the medians of the two groups were significantly different (Mann-Whitney test P Conclusions These data suggest that routine IHC methods for MSI evaluation can lead to a wrong classification of CRC. On the other hand, TMB assessment might better identify patients who may benefit from IOs within both the MSI-H and the MSS subgroups. Citation Format: Francesca Fenizia, Raffaella Pasquale, Cristin Roma, Francesca Bergantino, Anna Maria Rachiglio, Nicoletta Chicchinelli, Paolo Graziano, Gerardo Botti, Fabiana Tatangelo, Giosuè Scognamiglio, Christopher Allen, Matilde Lambiase, Alessia Iannaccone, Nicola Normanno. Tumor mutation burden and microsatellite instability in colorectal cancer [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 3975.

The activity of the epidermal growth factor receptor (EGFR) antibodies cetuximab and panitumumab in metastatic colorectal carcinoma (mCRC) is significantly limited by molecular mechanisms leading to intrinsic or acquired resistance. The S492R mutation of the EGFR, which is caused by either the 1476C>A or the 1474A>C substitution, interferes with binding to cetuximab but not to panitumumab, and has been detected in mCRC with acquired resistance to cetuximab. Since mechanisms of acquired and intrinsic resistance to EGFR monoclonal antibodies in CRC significantly overlap, we evaluated the frequency of the S492R mutation in a series of KRAS-exon 2 wild-type CRC patients. Genomic DNA was extracted from formalin fixed paraffin embedded (FFPE) tissues that were obtained from 505 systemic therapy-naïve CRC patients. A PCR/sequencing method for the detection of the S492R mutation was developed, by using as positive control a plasmid in which the 1474A>C mutation was generated by site directed mutagenesis. The lowest level of detection of this assay was approximately 10% mutant DNA in a background of wild-type DNA. PCR sequencing analysis revealed no S492R mutations in any of the analyzed 505 CRC specimens. Our findings suggest that the S492R mutation is not involved in primary resistance to cetuximab in CRC. Therefore, patients with mCRC should not be routinely screened for this mutation prior therapy with cetuximab.

An ideal target-based agent for the treatment of cancer patients should fulfil a number of requirements, including the availability of biomarkers to select the target population, superiority over existing treatments and specific advantages in terms of pharmacokinetics and/or metabolism. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, erlotinib and afatinib, have been investigated in the treatment of non-small cell lung cancer (NSCLC), and to date a large amount of clinical data are available. The activity of EGFR-TKIs was initially investigated in unselected patients leading to unsatisfactory results. However, the discovery that response to EGFR-TKIs is associated with the presence of activating EGFR mutations in NSCLC, has led to the design of clinical trials in which patients were selected on the basis of the EGFR mutational status or of clinical and pathological features that are highly associated with the presence of EGFR mutations. In this respect, several phase III randomized trials have demonstrated that first-line EGFR-TKIs, compared to chemotherapy, is associated with longer progression-free survival, higher response rate, better toxicity profile and quality of life in patients carrying EGFR mutations. Although no survival advantage was demonstrated, all the trials suffered of a high post-progression treatment cross-over, which predictably undermined the results. This review will summarize the current evidence that strongly support the hypothesis that gefitinib, erlotinib and afatinib are ideal drugs for NSCLC patients carrying EGFR mutations.

To the editor: Recently, an international collaboration collected information from ∼3500 chronic lymphocytic leukemia (CLL) patients to develop a comprehensive tool for predicting overall survival (OS) (the international prognostic index for patients with chronic lymphocytic leukemia [CLL-IPI]).[

Introduction. Elderly patients with chronic lymphocytic leukemia (CLL) and younger patients with comorbidities are often treated with chlorambucil (Chl), despite the relatively low response rates. The addition of anti-CD20 monoclonal antibodies to Chl substantially increases the response rates, without negatively affecting tolerability. Overall response rates (ORR) between 66% to 84% have been reported with these combinations, with complete responses (CR)ranging from 8% to 26%. Methods. We conducted a retrospective analysis on the use of the Chl-rituximab (R) combination as frontline treatment for elderly (≥65 years) and/or unfit (CIRS >6) CLL patients treated at 15 different Italian hematologic centers. The main aim of the study was to further establish the safety and efficacy of the Chl-R protocol and investigate whether certain CLL patients for whom this protocol could be particularly effective could be identified. To this end, we performed a subgroup analysis stratifying patients according to FISH and IGHV results: high risk group (HR) included patients with 17p deletion, intermediate risk group (IR) patients with 11q deletion and/or unmutated IGHV, low risk group (LR) patients without 11q or 17p deletion and/or unmutated IGHV. Results. One hundred and two patients who underwent treatment between 2009 and 2011 were enrolled in the study. Patients' clinical and biologic characteristics are summarized in Table 1. Three patients discontinued treatment earlier than planned: 1 for an episode of autoimmune hemolytic anemia (AIHA) that developed after the 2nd cycle of Chl and before starting R treatment and 2 patients for disease progression after the 3rd and 5th cycle of Chl-R, respectively. The median number of Chl and R cycles administered in the 102 patients was 8 (range 2-12) and 6 (range 1-9), respectively. The planned treatment schedule was different among centers: the two main schedules used were Chl administered at 1 mg/kg for each cycle every 28 days, given at a fixed daily dose of 10 mg starting from day 1 and repeated for 8 cycles, and Chl administered at 8 mg/m2/day for seven days of each of eight 28-day-cycles. R was added to Chl from the 3rd cycle onwards and was administered on day 1 of each cycle at a dose of 375 mg/m2 during the first administration and 500 mg/m2 for the subsequent 5 cycles. On an intention to treat basis, the ORR was 87.1%. Thirty-two patients (31.7%) obtained a CR and 56 patients (55.4%) obtained a partial response (PR). Nostatistically significant differences were noted in terms of ORR for age above or below 70 years, fitness status, ECOG, bulky disease, cytogenetic risk abnormalities, IGHV mutational status, ZAP-70 or CD38 expression.Median progression-free survival (PFS) and time to retreatment (TTR) were reached at 43.7 and 72.3 months, respectively. Median overall survival (OS) was not reached; 86.1% and 81.2% of patients were alive at 48 and 60 months, respectively. The most frequent serious adverse event was grade 3-4 neutropenia, occurring in 13.7% of patients. Grade 3-4 extra-hematologic side effects were uncommon (9.8%). Subgroup analysis of the LR and IR patients (no HR patients were enrolled) showed that LR patients had a significantly better PFS than IR patients (65.8 months vs 35.2 months, p=0.001; Fig. 1),with 54.9% of patients remaining free from progression 60 months after treatment. Conclusions. Treatment of elderly and/or unfit CLL patients with the Chl-R regimen is associated with low toxicity, a high ORR and durable PFS. Particularly good results are achieved in CLL patients with a mutated IGHV profile and not carrying both 17p and 11q deletion, suggesting that in this low-risk subset of unfit patients Chl-R could represent the optimal therapeutic option, in consideration of safety, efficacy and costs. Disclosures D'Arena: Janssen-Cilag: Honoraria. Coscia:Gilead: Honoraria; ROCHE: Honoraria, Other: Advisory board; Janssen: Honoraria; Mundipharma: Honoraria; Karyopharm: Research Funding. Molica:Jansen: Membership on an entity's Board of Directors or advisory committees; Roche Italy: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Speakers Bureau. Efremov:Gilead: Honoraria.

Background: The role of bone marrow response in patients with immune thrombocytopenia (ITP) has gained paramount importance since the last 10 years, with the demonstrations that marrow megakaryocytes (MGK) are unable to properly compensate platelets peripheral destruction. TPO receptor agonists (TPOa), namely romiplostim (ROMI) and eltrombopag (EPAG), by stimulating megakaryopoiesis are able to induce a response in 74% to 94% of cases in clinical trials. However, real world use of these drugs has shown frequent changes in individual dose requirement, the possibility of treatment discontinuation, and their effectiveness outside registered indications; moreover, nothing is known about predictors of response. Aim: To evaluate clinical and morphologic predictors of response in a real world cohort of ITP patients treated with TPOa. Methods: ITP patients treated with EPAG and ROMI from September 2009 to May 2018 at seven Italian Centers were evaluated. Clinical and hematologic parameters including treatment response and marrow characteristics were retrospectively collected. Results: Table 1 shows baseline clinical and morphologic characteristics for the 86 cases enrolled, altogether and divided according to the TPOa used: patients were mainly middle-aged females presenting with severe thrombocytopenia; anti-PLT autoantibodies were positive in 32.6% of cases, and 58.1% of cases presented with bleeding, 22% of grade III-IV. All cases had received 1st line treatment with steroids and 43% at list a 2nd line among those listed in Tab1. Pre-TPOa marrow evalutation showed hypocellularity in 30.2% of cases, reticulinic fibrosis in 33.7%, a polyclonal lymphoid infiltrate in 43% (mostly mixed or T-cell), and reduced MGK in 4.7% of patients. Some dysplastic features were present in about 50% of cases, either dysmegakaryopoiesis (46.5%) or dyserythropoiesis (25.6%). Median time from diagnosis to TPOa was 2.4 years (0.1-28.8). Patients were treated for a median of 1.4 years (0.3-10.8), and ORR at 3 months and 9 months were 75.6% (CR 44.2 and PR 31.4%) and 65.1% (CR 36 and PR 29.1%), respectively. Response rates to EPAG and ROMI were comparable. Regarding predictors of response, bone marrow hypocellularity (40 NR vs 21% ORR, p=0.05) and megakaryocytopenia (33 vs 6%, p=0.06) were significantly more frequent among NRs. Other factors associated with poor response were dyserythropoiesis (58 vs 26%, p=0.04) and erythroid hyperplasia (18 vs 8%, p=0.03), and presence of a T cell infiltrate (66 vs 18.9%, p=0.03). Finally, NRs cases showed significantly lower neutrophil counts at baseline (1.9 vs 2.3x103/mmc in ORR, p=0.01), and had been more frequently exposed to cyclosporine or azathioprine (50 vs 18% in ORR, p=0.01). Fifty-five patients are still on treatment, whereas 31 (20 EPAG/11 ROMI) discontinued because of NR or relapse (17), adverse events or intolerance (2); of note, 12 patients with ORR discontinued the drug because of sustained CR, and 7 of them are still in remission. 14/65(21.5%) responding cases (10 EPAG/4 ROMI) lost the response after a median of 6.2 months (1.8-60) and were variably managed (3 splenectomized, 1 switched from ROMI to EPAG, 1 received danazol, 5 were re-treated with EPAG, and the remaining were managed with steroids and supportive treatment). Median RFS was 2.3 years (0.1-10), longer in patients without megakaryocytopenia (9.9+0.5 vs 4.1+0.6, p=0.06), dyserythropoiesis (mean 9.1+0.5 vs 4.9+0.7, p=0.2), and reticular fibrosis (9.6+0.5 vs 5.5+0.6, p=0.08). During EPAG treatment 7 grade adverse events occurred: 2 grade IV (1 stroke with PLT counts of about 30x103/mmc, and 1 NSTEMI 1 month after EPAG discontinuation for sustained CR), 1 grade III pneumonia, and 4 grade I/II transaminase elevation. No events occurred under ROMI. Conclusions: TPOa use in the real world setting confirms their reported efficacy, the option to switch and/or re-treat with either EPAG or ROMI, and the possibility to discontinue the drugs. The presence of hypocellularity and megakaryocytopenia, along with dysplastic features and of a lymphoid T cell infiltrate are associated with a reduced response to TPOa and a shorter RFS. Pre-treatment bone marrow evaluation may give hints to unravel the physiopathologic mechanisms underlying TPOa refractoriness. Disclosures Rossi: MUNDIPHARMA: Honoraria; JANNSEN: Other; AMGEN: Other: ADVISORY BOARD; PFIZER: Other: ADVISORY BOARD; BMS: Honoraria; NOVARTIS: Honoraria; ROCHE: Other: Advisory Board; ABBVIE: Other: ADVISORY BOARD; TEVA: Other: ADVISORY BOARD; CELGENE: Other: ADVISORY BOARD; JAZZ: Other: ADVISORY BOARD; SANOFI: Other: ADVISORY BOARD; GILEAD: Other: ADVISORY BOARD; SANDOZ: Honoraria.

Introduction. Chronic Lymphocytic Leukemia (CLL) is an indolent B-cell lymphoproliferative disorder. Several prognostic factors such as IGHV mutation status and chromosomal aberrations as trisomy 12, del11q, del13q or del17p have been detected so far. More recent genetic mutations such as BIRC3, SF3B1, NOTCH1 and TP53 stratifies the prognosis and outcome in CLL patients (pts). However all data above reported, because of expensive techniques and experience typical of big laboratories, are not available to all medical centers. Data concerning the prevalence of hypogammaglobulinemia, IgM or IgG monoclonal gammopathy, and the impact on natural history of CLL pts are controversial and contradictory. The aim of the study is to evaluate the prevalence and the outcome of monoclonal IgM/CLL, IgG/CLL and hypogammaglobulinemia compared with CLL pts with normal immunoglobulin (Ig) levels. Patients and methods. We collected from three different Italian centers 902 pts diagnosed with typical CLL from 1987 to 2017 with a baseline assessment of serum Ig, immunofixation, chromosomal aberrations and clinical features; evaluating their impact on time to progression (TTP), time to treatment (TTT). Results. Once assessment was made, pts included in our study who met eligibility criteria were 902. Fifty-one patients showed IgM monoclonal gammopaty (5,6%); 89 showed IgG (9,9%), 103 patients had hypogammaglobulinemia (11,4%), 659 patients (73%) showed normal gamma level. In the Padova group, the overall prevalence of monoclonal gammopathy is 15.5% of whom 35 pts (10.5%) with IgG/CLL, 17 pts (5%) with IgM/CLL, 45 pts (13.5%) with hypogamma/CLL and 238 pts (71%) with no evidence of paraprotein. Data from the Rome group showed similar results: monoclonal gammopathy is 15.4% of whom 37 pts (9%) with IgG/CLL, 26 pts (6.4%) with IgM/CLL, 42 pts (10.3%) with hypogamma/CLL and 304 pts (74.3%) with no monoclonal gammopathy revealed. In the group from Milano, the prevalence of monoclonal gammopathy is 15.7%; 17 pts (10.7%) with IgG/CLL, 8 pts (5%) with IgM/CLL, 16 pts (10%) with hypogamma/CLL and 118 pts (74.3%) with negativity at immunofixation. Median age was similar in all the groups (66years, range 26-89years) and data gathered from the 3 centers showed an overlapping rate in prevalence of monoclonal gammopathy in CLL and their related subclasses. Median TTP was 75 months (91 months for patients with normal gamma levels and 54 months for patients with abnormal gamma globulin, P Median TTT was 79 months (93 months for patients with normal gamma levels and 54 months for patients with abnormal gamma globulin, P Summary. Our study provided data about incidence and prognosis of monoclonal gammopaty and hypogammaglobulinemia in CLL patients. Twenty-seven percent of patients with CLL showed abnormal gammopaty; these alteration have a negative impact on TTP and TTT in these setting of patients. Finally our data also detected a particular subset of patients with IgM/CLL characterized by a worst TTP and TTT. Disclosures Visentin: janssen: Consultancy, Honoraria. Reda:ABBVIE: Consultancy; Gilead: Consultancy; Janssen and Cilag: Consultancy; Celgene: Consultancy. Trentin:Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria; Gilead: Research Funding; Janssen: Research Funding.

e15507Background: Liquid biopsy is an alternative to tissue biopsy for biomarker testing. In addition, it can be used to monitor the response to therapy and the molecular evolution of the disease. ...

Aim: We assessed the ability of the Therascreen® kit (plasma-Therascreen) and of a peptide nucleic acids (PNA)-clamp approach to detect EGFR mutations in plasma-derived circulating-free tumor DNA (cftDNA) from non-small-cell lung cancer patients. Materials & methods: cftDNA from 96 patients was analyzed for exon 19 deletions and the p.L858R mutation, using both plasma-Therascreen and PNA-clamp-based assays. Results: None of the 70 EGFR wild-type patients showed EGFR mutations in cftDNA with both techniques (specificity: 100%). In 17/26 EGFR-mutant patients, plasma-Therascreen analysis confirmed the mutation identified in the primary tumor (analytical sensitivity: 65.4%). Similar results were obtained with the PNA-clamp method. Conclusion: Both approaches were specific and sensitive for EGFR mutational analysis of cftDNA in non-small-cell lung cancer patients.

ABSTRACT The presence of EGFR mutations predicts the sensitivity to EGF receptor (EGFR)-tyrosine kinase inhibitors in a molecularly defined subset of non-small-cell lung carcinoma (NSCLC) patients. For this reason, EGFR testing of NSCLC is required to provide personalized treatment options and better outcomes for NSCLC patients. As surgery specimens are not available in the majority of NSCLC, other currently available DNA sources are small biopsies and cytological samples, providing however limited and low-quality material. In order to address this issue, the use of surrogate sources of DNA, such as blood, serum and plasma samples, which often contains circulating free tumor DNA or circulating tumor cells, is emerging as a new strategy for tumor genotyping.

Mounting evidence supports the role of p53 in metabolic processes involved in breast carcinogenesis. We investigated whether p53 status affects the association of pre-treatment fasting glucose with treatment outcomes in 106 non diabetic, HER2 positive breast cancer patients treated with trastuzumab. p53 status was validated against gene sequencing of selected codons in 49 patients. The Kaplan–Meier method and log rank test were used to compare survival by categories of fasting glucose in the overall population and separate settings. Cox models included age and body mass index. Direct sequencing confirmed the lack of mutations in 73.7% of p53 negative patients and their presence in 53.3% of p53 positive cases. At 66 months, 88.3% of patients with glucose ≤ 89.0 mg/dl (median value) did not experiment disease progression compared with 70.0% in the highest category (p=0.034), with glucose being an independent predictor (p=0.046). Stratified analysis confirmed this association in p53 negative patients only (p=0.01). In the early setting, data suggested longer disease free survival in p53 negative patients in the lowest glucose category (p=0.053). In our study, p53 status acted as effect modifier of the investigated association. This may help differentiate target sub-groups and affect outcomes interpretation in similarly characterized patients.