PURPOSE:Prognostic biomarkers remain necessary in sporadic desmoid tumor (DT) because the clinical course is unpredictable. DT location along with gene expression between thoracic and abdominal wall locations was analyzed. METHOD:Sporadic DT patients (GEIS Registry) diagnosed between 1982 and 2018 who underwent upfront surgery were enrolled retrospectively in this study. The primary endpoint was relapse-free survival (RFS). Additionally, the gene expression profile was analyzed in DT localized in the thoracic or abdominal wall, harboring the most frequent CTNNB1 T41A mutation. RESULTS:From a total of 454 DT patients, 197 patients with sporadic DT were selected. The median age was 38.2 years (1.8-89.1) with a male/female distribution of 33.5/66.5. Most of them harbored the CTNNB1 T41A mutation (71.6 %), followed by S45F (17.8 %) and S45P (4.1 %). A significant worse median RFS was associated with males (p = 0.019), tumor size ≥ 6 cm (p = 0.001), extra-abdominal DT location (p < 0.001) and the presence of CTNNB1 S45F mutation (p = 0.013). In the multivariate analysis, extra-abdominal DT location, CTNNB1 S45F mutation and tumor size were independent prognostic biomarkers for worse RFS. DTs harboring the CTNNB1 T41A mutation showed overexpression of DUSP1, SOCS1, EGR1, FOS, LIF, MYC, SGK1, SLC2A3, and IER3, and underexpression of BMP4, PMS2, HOXA9, and WISP1 in thoracic versus abdominal wall locations. CONCLUSION:Sporadic DT location exhibits a different prognosis in terms of RFS favoring the abdominal wall compared to extra-abdominal sites. A differential gene expression profile under the same CTNNB1 T41A mutation is observed in the abdominal wall versus the thoracic wall, mainly affecting the Wnt/β-catenin, TGFβ, IFN, and TNF pathways.

The COVID-19 patients showed hyperinflammatory response depending on the severity of the disease but little have been reported about this response in oncologic patients that also were infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sixty-five circulating cytokines/chemokines were quantified in 15 oncologic patients, just after SARS-CoV-2 infection and fourteen days later, and their levels were compared in patients who required hospitalisation by COVID-19 versus non -hospitalised patients. A higher median age of 72 years (range 61-83) in oncologic patients after SARS-CoV-2 infection was associated with hospitalisation requirement by COVID-19 versus a median age of 49 years (20-75) observed in the non -hospitalised oncologic patients (p = 0.008). Moreover, oncologic patients at metastatic stage or with lung cancer were significantly associated with hospitalisation by COVID-19 (p = 0.044). None of these hospitalised patients required ICU treatment. Higher basal levels of tumour necrosis factor receptor II (TNF-RII), interferon-gamma (IFN gamma)-induced protein 10 (IP-10) and hepatocyte growth factor (HGF) in plasma were significantly observed in oncologic patients who required hospitalisation by COVID-19. Higher TNF-RII, IP-10 and HGF levels after the SARS-CoV-2 infection in oncologic patients could be used as biomarkers of COVID-19 severity associated with hospitalisation requirements.

Background:Alveolar soft-part sarcoma (ASPS) is a rare tumor driven by the ASPSCR1-TFE3 fusion protein, with a propensity for metastasis. Prognostic factors remain poorly understood, and traditional chemotherapies are largely ineffective. Recent interest lies in immune checkpoint inhibitors (ICIs), yet predictive biomarkers for treatment response are lacking. Previous studies have shown promising results with ICIs in ASPS, indicating a need for further investigation into biomarkers associated with immune response. Objectives:To identify prognostic biomarkers in ASPS and to explore the role of immune-related markers, particularly L1CAM, in predicting patient outcomes. Design:A retrospective cohort study of 19 ASPS patients registered in the GEIS database. The study involved the collection of clinical and histopathological data, followed by an analysis of immune markers and gene expression profiles to identify potential prognostic indicators. Methods:Clinical and histopathological data were retrospectively collected from the GEIS-26 study cohort of 19 ASPS patients. Immunohistochemistry was performed to evaluate immune markers programmed death-1 ligand (PD-L1), programmed death-1, FAS, FASL, CD8, CD3, and CD4. An HTG ImmunOncology panel was conducted on formalin-fixed paraffin-embedded samples to explore gene expression. Effects of differentially expressed genes on survival were explored by Kaplan-Meier. Results:PD-L1 positivity was widely observed (63%) in tumors, and CD8+ lymphocytic infiltration was common. High CD8 density correlated with greater overall survival (OS) while not statistically significant. No associations were found for other immune markers. L1CAM was identified as differentially expressed in patients with low CD8 infiltration and correlated negatively with OS. Conclusion:High L1CAM expression correlated with poorer OS, highlighting its potential as a prognostic marker and therapeutic target in ASPS. Immunomodulatory interventions may hold promise, as evidenced by PD-L1 expression and CD8+ infiltration. Further research, including larger cohorts and international collaborations, is needed to validate these findings and explore therapeutic strategies targeting L1CAM in ASPS.

HMGA1 is a structural epigenetic chromatin factor that has been associated with tumor progression and drug resistance. Here, we reported the prognostic/predictive value of HMGA1 for trabectedin in advanced soft-tissue sarcoma (STS) and the effect of inhibiting HMGA1 or the mTOR downstream pathway in trabectedin activity. The prognostic/predictive value of HMGA1 expression was assessed in a cohort of 301 STS patients at mRNA (n = 133) and protein level (n = 272), by HTG EdgeSeq transcriptomics and immunohistochemistry, respectively. The effect of HMGA1 silencing on trabectedin activity and gene expression profiling was measured in leiomyosarcoma cells. The effect of combining mTOR inhibitors with trabectedin was assessed on cell viability in vitro studies, whereas in vivo studies tested the activity of this combination. HMGA1 mRNA and protein expression were significantly associated with worse progression-free survival of trabectedin and worse overall survival in STS. HMGA1 silencing sensitized leiomyosarcoma cells for trabectedin treatment, reducing the spheroid area and increasing cell death. The downregulation of HGMA1 significantly decreased the enrichment of some specific gene sets, including the PI3K/AKT/mTOR pathway. The inhibition of mTOR, sensitized leiomyosarcoma cultures for trabectedin treatment, increasing cell death. In in vivo studies, the combination of rapamycin with trabectedin downregulated HMGA1 expression and stabilized tumor growth of 3-methylcholantrene-induced sarcoma-like models. HMGA1 is an adverse prognostic factor for trabectedin treatment in advanced STS. HMGA1 silencing increases trabectedin efficacy, in part by modulating the mTOR signaling pathway. Trabectedin plus mTOR inhibitors are active in preclinical models of sarcoma, downregulating HMGA1 expression levels and stabilizing tumor growth.

Background and objectives: Social distancing and quarantine implanted during the COVID-19 outbreak could have delayed the accession of oncologic patients to hospitals and treatments. This study analysed the management of sarcoma patients during this period in five Spanish hospitals. Design and methods: Clinical data from adult sarcoma patients, soft tissue and bone sarcomas, managed during the COVID-19 outbreak, from 15 March to 14 September 2020 (Covid cohort), were retrospectively collected and time for diagnosis, surgery and active treatments were compared with sarcoma patients managed during the same pre-pandemic period in 2018 (Control cohort). Results: A total of 126 and 182 new sarcoma patients were enrolled in the Covid and Control cohorts, respectively, who were mainly diagnosed as soft tissue sarcomas (81.0% and 80.8%) and at localized stage (80.2% and 79.1%). A diagnostic delay was observed in the Covid cohort with a median time for the diagnosis of 102.5 days (range 6–355) versus 83 days (range 5–328) in the Control cohort ( p = 0.034). Moreover, a delay in surgery was observed in cases with localized disease from the Covid cohort with a median time of 96.0 days (range 11–265) versus 54.5 days (range 2–331) in the Control cohort ( p = 0.034). However, a lower delay for neoadjuvant radiotherapy was observed in the Covid cohort with a median time from the diagnosis to the neoadjuvant radiotherapy of 47 days (range 27–105) versus 91 days (range 27–294) in the Control cohort ( p = 0.039). No significant differences for adjuvant radiotherapy, neoadjuvant/adjuvant chemotherapy and neoadjuvant/adjuvant palliative chemotherapy were observed between both cohorts. Neither progression-free survival (PFS) nor overall survival (OS) was significantly different. Conclusion: Delays in diagnosis and surgery were retrospectively observed in sarcoma patients during the COVID-19 outbreak in Spain, while the time for neoadjuvant radiotherapy was reduced. However, no impact on the PFS and OS was observed.

In the present study, we screened 84 Follicular Lymphoma patients for somatic mutations suitable as liquid biopsy MRD biomarkers using a targeted next-generation sequencing (NGS) panel. We found trackable mutations in 95% of the lymph node samples and 80% of the liquid biopsy baseline samples. Then, we used an ultra-deep sequencing approach with 2 · 10−4 sensitivity (LiqBio-MRD) to track those mutations on 151 follow-up liquid biopsy samples from 54 treated patients. Positive LiqBio-MRD at first-line therapy correlated with a higher risk of progression both at the interim evaluation (HRINT 11.0, 95% CI 2.10–57.7, p = 0.005) and at the end of treatment (HREOT, HR 19.1, 95% CI 4.10–89.4, p < 0.001). Similar results were observed by PET/CT Deauville score, with a median PFS of 19 months vs. NR (p < 0.001) at the interim and 13 months vs. NR (p < 0.001) at EOT. LiqBio-MRD and PET/CT combined identified the patients that progressed in less than two years with 88% sensitivity and 100% specificity. Our results demonstrate that LiqBio-MRD is a robust and non-invasive approach, complementary to metabolic imaging, for identifying FL patients at high risk of failure during the treatment and should be considered in future response-adapted clinical trials.

Rhabdomyosarcoma (RMS) in adults is a rare and aggressive disease, which lacks standard therapies for relapsed or advanced disease. This retrospective study aimed to describe the activity of BOMP-EPI (bleomycin, vincristine, methotrexate and cisplatin alternating with etoposide, cisplatin and ifosfamide), an alternative platinum-based regimen, in adult patients with relapsed/metastatic RMS. In the study, 10 patients with RMS with a median age at diagnosis of 20.8 years and a female/male distribution of 6/4 received a mean of 2.5 cycles of BOMP-EPI. The best RECIST response was a complete response in 1/10 (10%) patients, a partial response in 5/10 (50%), stable disease in 3/10 (30%) and progression in 1/10 (10%). With a median follow-up in the alive patients from the start of therapy of 30.5 months (15.7–258), all patients progressed with a median progression-free survival of 8.47 months (95% CI 8.1–8.8), and 7/10 patients died with a median overall survival of 24.7 months (95% CI 13.7–35.6). BOMP-EPI was an active chemotherapy regimen in adults with pediatric-type metastatic RMS, with outcomes in terms of survival that seem superior to what was expected for this poor-prognosis population. Low HMGB1 expression level was identified as a predictive factor of better response to this treatment.

11546 Background: There is an urgent clinical need to identify novel treatments for advanced STS, since all the clinical trials testing chemotherapy combinations have failed to improve survival compared to doxorubicin (dox) alone. AS deregulation has been associated with cancer initiation, progression and resistance to therapy. This deregulation can occur due to mutations or imbalanced expression or activity of splicing factors (SF). The alteration SF can alter thousands of pre-mRNAs, increasing cellular complexity and facilitating tumour progression. CDC-like kinases (CLK) that phosphorylate serine-arginine-rich SF (SRSFs), are key regulators of AS. SM09419 is an oral pan CLK/DYRK inhibitor that has been shown to inhibit SRSF phosphorylation, selectively modify spliceosome activity and decrease tumour growth by multiple mechanisms including the downregulation of Wnt/β-catenin signalling, a pathway that we reported to be important in sarcoma. Methods: A panel of 9 STS cell lines, including LMS (CP0024, IEC005, AA, SK-UT-1), LPS (93T449), synovial sarcoma (MCP037) and UPS (MCP016, MCP021, MCP025) were used to analyse the effect of SM09419 (provided by Biosplice) or PRI-724 (Wnt/β-catenin inhibitor) on cell viability (MTS assays). TOP flash β-catenin/TCF-responsive reporter assay was performed to evaluate the inhibition of the Wnt/β-catenin pathway in response to SM09419. In vivo experiments tested the combination of SM09419 (25mg/kg 5 days on and 2 off, for 21 days cycle) plus IP dox (5mg/kg once a week for 21 days cycle) in LMS (IEC005) and synovial sarcoma (FJD-SS-001) PDX models. Results: Our results showed that SM09419 had superior activity in STS cells compared to specific Wnt/β-catenin inhibitors with IC 50 values starting at 29.8 nM in SK-UT-1 cell line, whereas PRI-724 IC 50 values were clearly higher, starting from 5.18 µM in 93T449 cells. SM09419 treatment significantly inhibited the transcriptional activity of β-catenin by 40% in the MCP021 cells, demonstrating the regulatory activity of AS inhibitors on this pathway. I n vivo studies confirmed the higher efficacy of SM09419 in monotherapy compared to dox, in inhibiting tumour growth (p < 0.05), which was potentiated when combined with dox. The combination was increasingly active in synovial sarcoma with tumour regressions in 3 out of 4 mice, including one complete response. Treatment schedule was adapted to a single IP administration of dox (5mg/kg) and 5 administrations of SM09419 (25mg/kg), to improve the tolerability of the combination. Conclusions: The effects of pan CLK/DYRK inhibition on growth and survival of STS cancer cell lines and PDX models, indicate that can STS may be therapeutically addressed with these AS-modulators. The combination of SM09419 with dox was active in STS preclinical models known for their resilience toward treatment. Based on these results, the exploration of such combinations in clinical trials is warranted.

The screening of the BCR::ABL1 kinase domain (KD) mutation has become a routine analysis in case of warning/failure for chronic myeloid leukemia (CML) and B-cell precursor acute lymphoblastic leukemia (ALL) Philadelphia (Ph)-positive patients. In this study, we present a novel DNA-based next-generation sequencing (NGS) methodology for KD ABL1 mutation detection and monitoring with a 1.0E−4 sensitivity. This approach was validated with a well-stablished RNA-based nested NGS method. The correlation of both techniques for the quantification of ABL1 mutations was high (Pearson r = 0.858, p < 0.001), offering DNA-DeepNGS a sensitivity of 92% and specificity of 82%. The clinical impact was studied in a cohort of 129 patients (n = 67 for CML and n = 62 for B-ALL patients). A total of 162 samples (n = 86 CML and n = 76 B-ALL) were studied. Of them, 27 out of 86 harbored mutations (6 in warning and 21 in failure) for CML, and 13 out of 76 (2 diagnostic and 11 relapse samples) did in B-ALL patients. In addition, in four cases were detected mutation despite BCR::ABL1 < 1%. In conclusion, we were able to detect KD ABL1 mutations with a 1.0E−4 sensitivity by NGS using DNA as starting material even in patients with low levels of disease.

Pazopanib was assessed prospectively in the GEIS-32 phase II study (NCT02066285) on advanced solitary fibrous tumour (SFT), resulting in a longer progression-free survival (PFS) and overall survival (OS) compared with historical controls treated with chemotherapy. A retrospective analysis of peripheral inflammatory indexes in patients enrolled into GEIS-32 was performed to evaluate their prognostic and predictive value. Patients received pazopanib 800 mg/day as the first antiangiogenic line. The impacts of baseline neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), and red cell distribution width (RDW) on PFS, OS, and Choi response were evaluated by univariate and multivariate analysis. Metastasis-free interval (MFI), mitotic count, and ECOG were also included as potential prognostic factors. Sixty-seven SFT patients, enrolled in this study, showed a median age of 63 years and a female/male distribution of 57/43. The median follow-up from treatment initiation was 16.8 months. High baseline NLR, PLR, and standardised RDW were significantly associated with worse PFS and OS. NLR, RDW, MFI, and mitotic count were independent variables for PFS, while RDW and ECOG were independent for OS. Further, NLR and mitotic count were independent factors for Choi response. High baseline NLR and RDW values were independent prognostic biomarkers for worse outcome in advanced SFT patients treated with pazopanib.

The screening of the BCR::ABL1 kinase domain (KD) mutation has become a routine analysis in case of warning/failure for chronic myeloid leukemia (CML) and B-cell precursor acute lymphoblastic leukemia (ALL) Philadelphia (Ph)-positive patients. In this study, we present a novel DNA-based next-generation sequencing (NGS) methodology for KD ABL1 mutation detection and monitoring with a 1.0E−4 sensitivity. This approach was validated with a well-stablished RNA-based nested NGS method. The correlation of both techniques for the quantification of ABL1 mutations was high (Pearson r = 0.858, p < 0.001), offering DNA-DeepNGS a sensitivity of 92% and specificity of 82%. The clinical impact was studied in a cohort of 129 patients (n = 67 for CML and n = 62 for B-ALL patients). A total of 162 samples (n = 86 CML and n = 76 B-ALL) were studied. Of them, 27 out of 86 harbored mutations (6 in warning and 21 in failure) for CML, and 13 out of 76 (2 diagnostic and 11 relapse samples) did in B-ALL patients. In addition, in four cases were detected mutation despite BCR::ABL1 < 1%. In conclusion, we were able to detect KD ABL1 mutations with a 1.0E−4 sensitivity by NGS using DNA as starting material even in patients with low levels of disease.

Background Solitary fibrous tumour (SFT) is a rare mesenchymal malignancy that lacks robust prognostic and predictive biomarkers. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier, associated with tumour progression, and with poor survival of SFT patients, as previous published by our group. Here, we describe the role of ISG15 in the biology of this rare tumour. Methods ISG15 expression was assessed by immunohistochemistry in tissue microarrays from SFT patients and tested for correlation with progression-free survival and overall survival (OS). The effects of ISG15 knockdown or induction were investigated for cancer stem cell (CSC) characteristics and for drug sensitivity in unique in vitro models of SFT. Results The prognostic value of ISG15 for OS was validated at protein level in malignant SFT patients, prospectively treated with pazopanib and enrolled in GEIS-32 trial. In SFT in vitro models, ISG15 knockdown lead to a decrease in the expression of CSC-related genes, including SOX2, NANOG, ALDH1A1, ABCB1 and ABCC1 . Likewise, ISG15 downregulation decreased the clonogenic/ tumoursphere-forming ability of SFT cells, while enhancing apoptotic cell death after doxorubicin, pazopanib or trabectedin treatment in 3D cell cultures. The regulation of CSC-related genes by ISG15 was confirmed after inducing its expression with interferon-β1; ISG15 induction upregulated 1.28- to 451-fold the expression of CSC-associated genes. Conclusions ISG15 is a prognostic factor in malignant SFT, regulating the expression of CSC-related genes and CSCs maintenance. Our results suggest that ISG15 could be a novel therapeutic target in SFT, which could improve the efficacy of the currently available treatments.

Follicular lymphoma (FL) is considered an indolent disorder with a relatively favourable course. With modern day treatments, long remissions are often achieved both in front-line and relapsed settings. However, a subset of patients has a more aggressive course and a poorer outcome. Both, PET-CT and minimal residual disease (MRD) evaluation by PCR defines groups of patients with different prognoses. MRD measurement by NGS is being studied to predict relapse prior to diagnostic imaging in large B cell lymphoma; however, it has never been used in FL. The aim of the present study is to validate a sensitive and standardizable approach to measure liquid Biopsy MRD (LiqBio-MRD) by NGS with >90% applicability and 2E-4 resolution, and to analyse its prognostic impact in FL patients. Firstly, the best source to identify genetic MRD markers was determined. Genomic DNA from paraffin embedded (FFPE) lymph node biopsies and/or cell free DNA (cfDNA) from peripheral blood (PB) was obtained from 29 FL cases at diagnosis and sequenced with a short length Ampliseq Custom Panel (Thermo-Fisher). This panel was designed to cover all coding regions of 56 lymphoma specific genes in FFPE and cfDNA samples. By applying this panel with an average depth of 700X, a total of 122 somatic mutations were detected in 37 baseline samples. 15 of these lymph node samples presented 65 mutations (average of 4 mutations per patient, rank 1-9), with a mean Variant Read Frequency (VRF) of 0.33 (0.06-0.77). On the other hand, the 21 cfDNA samples presented 71 mutations (average of 3 mutations per patient, rank 0-8), with a mean VRF of 0.21 (0.02-1.0). Notably, in 3 cases the mutations were only detected in the lymph node. Paired samples were available for 13 cases. Of the 72 somatic mutations identified in these cases, only 14 were present in both samples (Figure 1A, left). Besides the higher number of mutations in the lymph nodes, a mean decrease of 0.14 VRF was observed in cfDNA (0.21; 0.03-0.53) compared to lymph nodes (0.35; 0.09-0.76) (Figure 1A, Right). From the initial 29 FL cases, 16 had PB sequential samples available. Three patients were put under observation and the rest received an anthracycline based regimen plus R-maintenance. In treated patients, PET-CT was carried out at diagnosis and after 4 and 6 cycles of treatment. During follow-up, cfDNA was available after 4 (n=10) and 6 cycles of treatment (n=10). Median follow up was 18 months. To quantify LiqBio-MRD in the 31 follow-up samples, we defined an approach involving the sequencing of 12 NGS data points per mutation identified at diagnosis. Three of these data points were tumour sample replicates. The other 9 points were obtained from healthy control donor DNA. All LiqBio-MRD samples were sequenced with at least 100.000x and analysed applying the NGS-MRD algorithm described elsewhere (Onecha, E et al. Hematologica 2019). The mean mutation rate (noise) in controls for the studied mutations was 1.4E-5 (0 - 8E-5) below the targeted sensibility of 2E-4. The LOD was defined for every follow-up sample based on the initial amount of cfDNA used in the test. On average 39.6ng (13-66 ng) were used for cfDNA MRD monitoring. All somatic mutations were considered potential MRD markers (Figure 1B), however the degree of MRD in each follow-up sample was defined by the somatic mutation with higher VRF. MRD values were significantly lower in complete response (CR) cases compared to those with active disease (p=0.001, Figure 1C, left). Notably, MRD positivity in the interim or at the end of treatment resulted in significantly inferior PFS (median 12 months vs not reached, P = 0.09, Figure 1C, right). An extension of the cohort and clinical impact of LiqBio-MRD test will be presented at the meeting. Our results demonstrate for the first time that NGS based MRD quantification is feasible in Liquid Biopsies from FL. Despite the marked spatial genetic heterogeneity of FL, which is better identified in cfDNA, the dilution of the signal in these samples suggests the use of both; lymph node biopsies and cfDNA at diagnosis to identify all potential MRD markers. The lower degree of MRD in CR evaluations (according to the 2014 Lugano response assessment) and the existence of patients in CR with positive and negative MRD suggest the potential of our LiqBio-MRD test to prospectively identify patients with different outcome. Nevertheless, more patients and a longer follow-up are necessary to draw meaningful conclusions. Figure 1 Disclosures Heredia: Altum sequencing: Current Employment. Rufian:Altum sequencing: Current Employment. Carrillo:Altum sequencing: Current Employment. Wang:Hosea Precision Medical Technology Co., Ltd: Current Employment. Canales:Janssen: Honoraria; Karyopharm: Honoraria; Celgene: Honoraria; Takeda: Speakers Bureau; Sandoz: Honoraria; Janssen: Speakers Bureau; Novartis: Honoraria; Sandoz: Speakers Bureau; Janssen: Speakers Bureau; Takeda: Speakers Bureau; Roche: Honoraria; iQone: Honoraria; Janssen: Honoraria; Sandoz: Honoraria; Novartis: Honoraria; Roche: Honoraria; Roche: Speakers Bureau; Karyopharm: Honoraria; Gilead: Honoraria; Sandoz: Speakers Bureau; Roche: Speakers Bureau. Martinez-López:Janssen, BMS, Sanofi, Novartis, Incyte, F. Hoffmann-La Roche and Amgen: Honoraria, Other: Advisory boards; Janssen, Novartis, BMS, Incyte: Consultancy; Hosea and Altum: Membership on an entity's Board of Directors or advisory committees.

ABL1 Kinase Domain (ABL1-KD) mutations are a common resistance mechanism to tyrosine-kinase inhibitors (TKIs) in Chronic Myeloid Leukemia (CML) and Philadelphia Positive Acute Lymphoblastic Leukemia (ALL). Different ABL1-KD mutations induce different degrees of resistance to different TKIs. The early detection of these resistant mutations helps to adjust patient's treatment. Here we present an Ultra-Deep Sequencing approach to detect and quantify acquired ABL1-KD mutations in genomic DNA (gDNA), aiming to define a robust test to detect such alterations in TKIs exposed Philadelphia-Positive Leukemia Patients with a resolution below 1E-4. Firstly, we defined an ABL1 specific next-generation sequencing (NGS) panel designed to cover all coding regions of ABL1 exons 4-10. The 9 amplicons were designed to cover full exons where possible to detect co-occurring mutations (Figure 1A). A panel was then applied to 3 biological replicates of 3 Healthy control donors (9 NGS data points each with 220ng of gDNA). The average coverage per amplicon in all samples was at least 500,000x. The NGS data was then analyzed applying the NGS-MRD algorithm described elsewhere (Onecha, E et al. Haematologica 2019) to 25 known ABL1-KD hotspots. After applying our error correcting algorithm, we obtained an average of 135,000 (22,000-503,000) refined reads for the 25 hotspots. The limit of detection (LOD) was calculated for every position in the DNA as the mean noise (Variant Read Frequency; VRF) per position in the controls ± 3SD (standard deviation); the limit of quantification (LOQ) being defined as mean ± 10SD. For all the hotspots analyzed, the LOD was below 1E-4 and the LOQ below 3E-4 (Figure 1B), except for p.F311L (c.931T>C; LOD=2.7E-3). The high level of noise in this position, constant in the different control samples sequenced in different sequencing runs, is most likely related to the high number of homopolymers in the region. Ten Philadelphia-Positive Leukemia patients were then screened after TKI treatment (8 CML and 2 ALL). The median BCR-ABL1 defined by quantitative PCR (ratio BCR-ABL1 vs ABL1) in these follow-up samples was 0.6% (0.034% - 95%). All patients were screened in triplicates (220ng gDNA each) and the data-points ± 1SD from the mean were considered outliers (NGS false positives) and excluded from further analysis. Five patients presented a signal above the LOD for p.T315I (c.944C>T). This position is covered by 2 different amplicons in our panel. By bioinformatically demultiplexing the signal, the detection of those five mutations in both amplicons was confirmed (Amp_4; LOD=3E-5, Amp_5; LOD=4E-5). Moreover, aiming to validate this new approach, we applied to paired RNA samples an in-house BCR-ABL1/ABL1 nested PCR + NGS approach designed to quantify those alterations in cDNA. This approach confirmed the presence of 4 out of 5 gDNA detected mutations, with a Pearson correlation of 0.92 (Pval<0.001) (Figure 1C). The only mutation not confirmed by nested PCR presented the lowest BCR-ABL1 quantification (0.034%). Here we show an Ultra-Deep NGS based test which allows the early detection of TKI resistant emerging clones in genomic DNA samples with a resolution of 1E-4. Despite the facts that in Phi-positive leukemia patients' other techniques such as the nested PCR are available, for most of heme- dyscrasias it is not easy to detect acquired mutations below 1% VRF. Our test can reduce this limit by at least 2 logarithms. The clinical impact of this approach is illustrated by the two LLA patients included, both under dasatinib therapy when the p.T315I mutations were detected. Those 2 patients were changed to ponatinib, reducing BCR-ABL1 levels. An extension of the cohort and the validation of our test at clinical level will be presented at the meeting. Figure Disclosures Heredia: Altum sequencing: Current Employment. Carrillo:Altum sequencing: Current Employment. Rufian:Altum sequencing: Current Employment. Wang:Hosea Precision Medical Technology Co., Ltd: Current Employment. Ribera:Pfizer, Amgen: Research Funding; Pfizer, Amgen, Ariad, Novartis: Consultancy, Speakers Bureau. Martinez-López:Janssen, BMS, Sanofi, Novartis, Incyte, F. Hoffmann-La Roche and Amgen: Honoraria, Other: Advisory boards; Hosea and Altum: Membership on an entity's Board of Directors or advisory committees; Janssen, Novartis, BMS, Incyte: Consultancy.

BackgroundTelomeres are nucleoprotein structures present at the terminal region of the chromosomes. Mutations in genes coding for proteins involved in telomere maintenance are causative of a number of disorders known as telomeropathies. The genetic origin of these diseases is heterogeneous and has not been determined for a significant proportion of patients.MethodsThis article describes the genetic characterization of a cohort of patients. Telomere length was determined by Southern blot and quantitative PCR. Nucleotide variants were analyzed either by high-resolution melting analysis and Sanger sequencing of selected exons or by massive sequencing of a panel of genes.ResultsForty-seven patients with telomere length below the 10% of normal population, affected with three telomeropathies: dyskeratosis congenita (4), aplastic anemia (22) or pulmonary fibrosis (21) were analyzed. Eighteen of these patients presented known pathogenic or novel possibly pathogenic variants in the telomere-related genes TERT, TERC, RTEL1, CTC1 and ACD. In addition, the analyses of a panel of 188 genes related to haematological disorders indicated that a relevant proportion of the patients (up to 35%) presented rare variants in genes related to DNA repair or in genes coding for proteins involved in the resolution of complex DNA structures, that participate in telomere replication. Mutations in some of these genes are causative of several syndromes previously associated to telomere shortening.ConclusionNovel variants in telomere, DNA repair and replication genes are described that might indicate the contribution of variants in these genes to the development of telomeropathies. Patients carrying variants in telomere-related genes presented worse evolution after diagnosis than the rest of patients analyzed.

Ataxia telangiectasia (AT) is a genetic disease caused by mutations in the ATM gene but the mechanisms underlying AT are not completely understood. Key functions of the ATM protein are to sense and regulate cellular redox status and to transduce DNA double-strand break signals to downstream effectors. ATM-deficient cells show increased ROS accumulation, activation of p38 protein kinase, and increased levels of DNA damage. GSE24.2 peptide and a short derivative GSE4 peptide corresponding to an internal domain of Dyskerin have proved to induce telomerase activity, decrease oxidative stress, and protect from DNA damage in dyskeratosis congenita (DC) cells. We have found that expression of GSE24.2 and GSE4 in human AT fibroblast is able to decrease DNA damage, detected by γ-H2A.X and 53BP1 foci. However, GSE24.2/GSE4 expression does not improve double-strand break signaling and repair caused by the lack of ATM activity. In contrast, they cause a decrease in 8-oxoguanine and OGG1-derived lesions, particularly at telomeres and mitochondrial DNA, as well as in reactive oxygen species, in parallel with increased expression of SOD1. These cells also showed lower levels of IL6 and decreased p38 phosphorylation, decreased senescence and increased ability to divide for longer times. Additionally, these cells are more resistant to treatment with H202 and the radiomimetic-drug bleomycin. Finally, we found shorter telomere length (TL) in AT cells, lower levels of TERT expression, and telomerase activity that were also partially reverted by GSE4. These observations suggest that GSE4 may be considered as a new therapy for the treatment of AT that counteracts the cellular effects of high ROS levels generated in AT cells and in addition increases telomerase activity contributing to increased cell proliferation.

&NA; NHEJ1‐patients develop severe progressive lymphocytopenia and premature aging of hematopoietic stem cells (HSCs) at a young age. Here we show a patient with a homozygous‐NHEJ1 mutation identified by whole exome‐sequencing that developed severe pancytopenia and bone marrow aplasia correlating with the presence of short telomeres. The mutation resulted in a truncated protein. In an attempt to identify the mechanism behind the short telomere phenotype found in the NHEJ1‐patient we downregulated NHEJ1 expression in 293T and CD34+cells. This downregulation resulted in reduced telomerase activity and decreased expression of several telomerase/shelterin genes. Interestingly, cell lines derived from two other NHEJ1‐deficient patients with different mutations also showed increased p21 expression, inhibition in expression of several telomerase complex genes and shortened telomeres. Decrease in expression of telomerase/shelterin genes did not occur when we inhibited expression of other NHEJ genes mutated in SCID patients: DNA‐PK, Artemis or LigaseIV. Because premature aging of HSCs is observed only in NHEJ1 patients, we propose that is the result of senescence induced by decreased expression of telomerase/shelterin genes that lead to an inhibition of telomerase activity. Previous reports failed to find this connection because of the use of patient's cells immortalized by TERT expression or recombined telomeres by ALT pathway. In summary, defective regulation of telomere biology together with defective V(D)J recombination can negatively impact on the evolution of the disease in these patients. Identification of telomere shortening is important since it may open new therapeutic interventions for these patients by treatments aimed to recover the expression of telomerase genes.

Dyskeratosis congenita (DC) is a rare genetic disease of telomere biology and is characterized by the classic triad of reticular skin pigmentation, dysplastic nails, and oral leukoplakia (4, 6,16). Autosomal dominant, autosomal recessive and X-linked recessive forms of the disease have been defined, and it is associated with the genes encoding telomerase and components of telomere-related proteins (12,15,17). The risk for progressive bone marrow failure, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), solid tumors (especially squamous cell carcinomas of the head/neck or anogenital cancers) and pulmonary fibrosis is increased (1,3). To date, mutations in the DKC1 (X-linked), TERT (autosomal dominant or autosomal recessive), TERC and TINF2 (autosomal dominant), NHP2, CTC1, NOP 10 and WRAP53 (autosomal recessive) genes have been identified as causing DC (14). The most widely known genetic model is the X-linked recessive form, and it is associated with a missense mutation of the DKC1 gene. This gene encodes a nucleolar protein (dyskerin) that is essential for ribosome biogenesis and telomere maintenance (18). We present here the molecular and cl inical findings of a case of DC. In our case, a hemizygotic mutation in the DKC1 gene (c.227C>T, p.Ser76Leu) was detected.The 20-year-old male presented herein was born weighing 2100 gr as the second live birth of the second pregnancy of a 22-year-old mother. The boy did not have any hallmarks reported in his prenatal history. He had frequent infections. The boy, whose nails did not grow for approximately 10 years, had pigmentation anomalies in the skin for about the same period of time. The patient was diagnosed with splenomegaly 2 years ago. He had complaints of anemia, imperception and failure to gain weight for a long time. In his physical examination, he had blepharitis, tooth decay, microcephaly, sparse hair, moustache and beard, cachectic appearance and pes planus, in addition to a classical triad of symptoms: reticular, hyperpigmented skin lesions in the neck and shoulders, leukoplakia of the tongue, and dystrophy in the nails of the hands and feet (Fig. la-c). All of his hand fingerprints were absent. In the gastroscopic examination, he exhibited esophageal stenosis (web) at the cervical level and grade 3 esophageal varices in the distal esophagus. He exhibited pancytopenia in the complete blood count analysis and osteoporosis and osteopenia in the DEXA scan. Portal hypertension was observed in the portal vein Doppler US as well as periportal fibrosis in the liver, massive splenomegaly and ascites in the abdomen. His echocardiogram revealed normal cardiac function. His 42-year-old mother had prematurely gray hair and dystrophic toe nails. The case was assessed as DC due to these findings. The patient exhibited short telomeres and a hemizygotic mutation in the DKC1 gene (c.227C>T, p.Ser76Leu), and his mother had the same heterozygous mutation. No mutations were found in the father.Abnormal skin pigmentation, nail dystrophy, bone marrow failure and leukoplakia are among the major clinical characteristics of dyskeratosis congenita. Other well-defined somatic features include epiphora, learning disability/growth retardation/mental retardation, pulmonary disease, short stature, excessive tooth decay/tooth loss, esophageal stenosis, premature hair loss/prematurely gray hair/sparse eyebrow, malignancy, intrauterine growth retardation, liver disease/peptic ulcer/ enteropathy, ataxia/cerebellar hypoplasia, hypogonadism/undescended testes, microcephaly, urethral stenosis/phimosis, osteoporosis/aseptic necrosis/scoliosis and deafness. For a diagnosis of DC, the patient must have 2 or more of the 4 major features and 2 or more of the somatic features (7). Our case satisfied the diagnostic criteria of DC.The most commonly reported form of DC is the X-linked form. Mutations in the DKC1 gene result in DC cases of varying severity. While the children appear normal at birth, the classical triad of mucocutaneous lesions are observed in the first decade of life, bone marrow failure develops in the second decade of life, and death occurs due to bone marrow failure and complications of bleeding and infections in the third decade (2, 6). …

Recently, we observed that telomeres of BRCA1/2 mutation carriers were shorter than those of controls or sporadic breast cancer patients, suggesting that mutations in these genes might be responsible for this event. Given the contradictory results reported in the literature, we tested whether other parameters, such as chemotherapy, could be modifying telomere length (TL). We performed a cross-sectional study measuring leukocyte TL of 266 sporadic breasts cancer patients treated with first-line chemotherapy, with a median follow-up of 240 days. Additionally, we performed both cross-sectional and longitudinal studies in a series of 236 familial breast cancer patients that included affected and non-affected BRCA1/2 mutation carriers. We have measured in leukocytes from peripheral blood: the TL, percentage of short telomeres (<3 kb), telomerase activity levels and the annual telomere shortening speed. In sporadic cases we found that chemotherapy exerts a transient telomere shortening effect (around 2 years) that varies depending on the drug combination. In familial cases, only patients receiving treatment were associated with telomere shortening but they recovered normal TL after a period of 2 years. Chemotherapy affects TL and should be considered in the studies that correlate TL with disease susceptibility.