Liquid biopsy testing in metastatic or advanced breast cancer patients during the COVID-19 pandemic

Objective: During the COVID-19 pandemic, cancer patients had restricted access to standard of care tissue biopsy. Liquid biopsy assays using next generation sequencing technology provides a less invasive method for determining circulating tumour mutations (ctDNA) associated with targeted treatments or prognosis. As part of deploying technology to help cancer patients obtain molecular testing, a clinical program was initiated to offer liquid biopsy testing for Canadian patients with advanced or metastatic breast cancer. Method(s): Blood was drawn in two 10 mL StreckTM DNA BCTs and sent to the CAP/CLIA/DAP accredited Imagia Canexia Health laboratory for testing using the clinically validated Follow ItTM liquid biopsy assay. Plasma was isolated using a double spin protocol and plasma cell-free DNA (cfDNA) extracted using an optimized Promega Maxwell RSC method. Extracted cfDNA was amplified using the multiplex amplicon-based hotspot 30 or 38 gene panel and sequenced. An inhouse developed bioinformatics pipeline and reporting platform were used to identify pathogenic single nucleotide variants (SNVs), indels (insertions and deletions), and gene amplification. Included in the panel are genes associated with metastatic breast cancer: AKT1, BRAF, ERBB2, ESR1, KRAS, PIK3CA, TP53. Result(s): To identify biomarkers, 1214 metastatic or advanced breast cancer patient cfDNA samples were tested. There were 15 cases sent for repeat testing. We reported 48% of samples harboring pathogenic ctDNA mutations in TP53 (22%), PIK3CA (19%), ESR1 (18%), AKT1 (2%), ERBB2 (1.5%). Co-occurring variants were identified in samples with ESR1/PIK3CA as well as TP53/PIK3CA (both p-values <0.001). Interestingly, 29% of samples with mutated ESR1 harbored >= 2 ESR1 ctDNA mutations. In 56% of cases, previous molecular testing indicated the cancer subtype as hormone receptor (ER, PR) positive with/without HER2 negative status. In this specific subgroup, 49% harbored ctDNA mutations with 63% of those being PIK3CA and/or ESR1 mutations. Conclusion(s): A population of Canadian women with metastatic breast cancer were tested using a liquid biopsy gene panel during the COVID-19 pandemic for identification of biomarkers for targeted therapeutic options. Over 50% of the samples were identified as hormone positive, with greater than 60% harboring PIK3CA and ESR1 ctDNA mutations. Studies have shown that metastatic PIK3CA mutated ER-positive/HER2-negative tumors are predictive to respond to alpelisib therapy and have FDA and Health Canada approval. Additionally, ESR1 mutations are associated with acquired resistance to antiestrogen therapies, and interestingly we identified 29% of ESR1 mutated samples with multiple mutations possibly indicating resistance subclones. In future studies, longitudinal monitoring for presence of multiple targetable and resistance mutations could be utilized to predict or improve clinical management.

Circulating tumor HPV DNA as an alternative method to determine HPV status in oropharyngeal squamous cell carcinoma.

Human Papilloma Virus (HPV) testing is mandatory for all newly diagnosed oropharyngeal squamous cell carcinoma (OPSCC) due to its importance for prognostication and aiding in treatment decision making. Fine needle aspiration (FNA) is a widely used and accepted diagnostic tool for OPSCC. Although FNA can accurately determine histological diagnosis, results are often indeterminate or lack insufficient samples for HPV testing. For samples with an indeterminant FNA, we propose an alternate method for determining HPV status using circulating tumor tissue modified HPV DNA (ctHPVDNA). We report three cases that confirmed HPV status using ctHPVDNA following an indeterminate FNA. If validated, this non-invasive assay could prevent the need for repeat FNAs or operative biopsies for the sole purpose of determining HPV status.

A case of refractory pelvic squamous cell carcinoma of unknown primary that responded to nivolumab.

Cancer of unknown primary (CUP) is a heterogeneous group of metastatic tumors with a usually unfavorable prognosis. A 33-year-old female was diagnosed with pelvic squamous cell carcinoma of unknown primary. The tumor was p16-positive, suggesting that it was human papillomavirus (HPV)-related. The tumor progressed for 4 months after concurrent chemoradiotherapy (initial treatment) and was refractory to paclitaxel plus carboplatin (second-line therapy). Liquid-based cancer genomic profiling identified five pathogenic variants, including Neurofibromin1 (NF1) (p.T1690Mfs*5); however, due to the lack of domestic clinical trials, the patient could not receive genome-based molecular-target therapies. Simultaneously, nivolumab was administered to the patient post its approval in Japan for CUP. The tumor responded to nivolumab, accompanied by decreased levels of tumor markers. NF1 mutations and HPV-related carcinogenesis may be associated with a favorable response to nivolumab treatment. It may therefore serve as a potential treatment against cancers of unknown primaries.

Plasma cell-free DNA promise monitoring and tissue injury assessment of COVID-19.

Coronavirus 2019 (COVID-19) is a complex disease that affects billions of people worldwide. Currently, effective etiological treatment of COVID-19 is still lacking; COVID-19 also causes damages to various organs that affects therapeutics and mortality of the patients. Surveillance of the treatment responses and organ injury assessment of COVID-19 patients are of high clinical value. In this study, we investigated the characteristic fragmentation patterns and explored the potential in tissue injury assessment of plasma cell-free DNA in COVID-19 patients. Through recruitment of 37 COVID-19 patients, 32 controls and analysis of 208 blood samples upon diagnosis and during treatment, we report gross abnormalities in cfDNA of COVID-19 patients, including elevated GC content, altered molecule size and end motif patterns. More importantly, such cfDNA fragmentation characteristics reflect patient-specific physiological changes during treatment. Further analysis on cfDNA tissue-of-origin tracing reveals frequent tissue injuries in COVID-19 patients, which is supported by clinical diagnoses. Hence, our work demonstrates and extends the translational merit of cfDNA fragmentation pattern as valuable analyte for effective treatment monitoring, as well as tissue injury assessment in COVID-19.

Metagenomic Next-Generation Sequencing of Plasma for Diagnosis of COVID-19-Associated Pulmonary Aspergillosis.

Timely diagnosis remains an unmet need in non-neutropenic patients at risk for aspergillosis, including those with COVID-19-associated pulmonary aspergillosis (CAPA), which in its early stages is characterized by tissue-invasive growth of the lungs with limited angioinvasion. Currently available mycological tests show limited sensitivity when testing blood specimens. Metagenomic next-generation sequencing (mNGS) to detect microbial cell-free DNA (mcfDNA) in plasma might overcome some of the limitations of conventional diagnostics. A two-center cohort study involving 114 COVID-19 intensive care unit patients evaluated the performance of plasma mcfDNA sequencing for the diagnosis of CAPA. Classification of CAPA was performed using the European Confederation for Medical Mycology (ECMM)/International Society for Human and Animal Mycoses (ISHAM) criteria. A total of 218 plasma samples were collected between April 2020 and June 2021 and tested for mcfDNA (Karius test). Only 6 patients were classified as probable CAPA, and 2 were classified as possible, while 106 patients did not fulfill CAPA criteria. The Karius test detected DNA of mold pathogens in 12 samples from 8 patients, including Aspergillus fumigatus in 10 samples from 6 patients. Mold pathogen DNA was detected in 5 of 6 (83% sensitivity) cases with probable CAPA (A. fumigatus in 8 samples from 4 patients and Rhizopus microsporus in 1 sample), while the test did not detect molds in 103 of 106 (97% specificity) cases without CAPA. The Karius test showed promising performance for diagnosis of CAPA when testing plasma, being highly specific. The test detected molds in all but one patient with probable CAPA, including cases where other mycological tests from blood resulted continuously negative, outlining the need for validation in larger studies.

Investigation of liquid biopsy analytes in peripheral blood of individuals after SARS-CoV-2 infection.

BACKGROUND: Post-acute COVID-19 syndrome (PACS) is linked to severe organ damage. The identification and stratification of at-risk SARS-CoV-2 infected individuals is vital to providing appropriate care. This exploratory study looks for a potential liquid biopsy signal for PACS using both manual and machine learning approaches. METHODS: Using a high definition single cell assay (HDSCA) workflow for liquid biopsy, we analysed 100 Post-COVID patients and 19 pre-pandemic normal donor (ND) controls. Within our patient cohort, 73 had received at least 1 dose of vaccination prior to SARS-CoV-2 infection. We stratified the COVID patients into 25 asymptomatic, 22 symptomatic COVID-19 but not suspected for PACS and 53 PACS suspected. All COVID-19 patients investigated in this study were diagnosed between April 2020 and January 2022 with a median 243 days (range 16-669) from diagnosis to their blood draw. We did a histopathological examination of rare events in the peripheral blood and used a machine learning model to evaluate predictors of PACS. FINDINGS: The manual classification found rare cellular and acellular events consistent with features of endothelial cells and platelet structures in the PACS-suspected cohort. The three categories encompassing the hypothesised events were observed at a significantly higher incidence in the PACS-suspected cohort compared to the ND (p-value < 0.05). The machine learning classifier performed well when separating the NDs from Post-COVID with an accuracy of 90.1%, but poorly when separating the patients suspected and not suspected of PACS with an accuracy of 58.7%. INTERPRETATION: Both the manual and the machine learning model found differences in the Post-COVID cohort and the NDs, suggesting the existence of a liquid biopsy signal after active SARS-CoV-2 infection. More research is needed to stratify PACS and its subsyndromes. FUNDING: This work was funded in whole or in part by Fulgent Genetics, Kathy and Richard Leventhal and Vassiliadis Research Fund. This work was also supported by the National Cancer InstituteU54CA260591.

Liquid Biopsy: Clinically Integrated Research in the Modern Pandemic Era

The COVID-19 pandemic forced a redesign of clinical research to adapt to an ever-changing situation while minimizing patient and provider risks and preserving scientific discovery. During the initial surge of COVID-19, elective healthcare services and non-critical research operations were halted. These changes inspired dispersed health care operations and streamlined clinical research. The first wave of COVID-19 hit Detroit, Michigan, in March 2020, consuming Henry Ford Health (HFH), in COVID-19 emergency care. HFH has a clinically integrated liquid biopsy (LB) program where enrolled patients provide an LB sample via venipuncture within 7 days of each MRI, typically in the clinic at the point-of-care. Prior to COVID-19, 183 neurooncology patients were actively providing LB samples in clinic with a mean of 29.9 specimens monthly. Institutional COVID-19 restrictions on noncritical interactions resulted in months were nearly all outpatient encounters utilized telemedicine and decentralized testing off-site from research operations. This halted LB procurement to 4.55 specimens monthly during early pandemic months. To reduce patient-provider exposure, LB specimens were then procured with the venipuncture for MRI which streamlined LB operations and improved the patient experience. After this change, LB specimen procurement returned to near pre-pandemic levels with a mean of 28.1 monthly specimens, despite a significant population utilizing video visits and imaging at satellite or non-HFH centers. The pandemic forced adaptations to patient encounters that have changed how healthcare is delivered, resulting in parallel changes in research that must be considered in the design of future studies.

Next-generation proteomics of serum extracellular vesicles combined with single-cell RNA sequencing identifies MACROH2A1 associated with refractory COVID-19.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is widespread; however, accurate predictors of refractory cases have not yet been established. Circulating extracellular vesicles, involved in many pathological processes, are ideal resources for biomarker exploration. METHODS: To identify potential serum biomarkers and examine the proteins associated with the pathogenesis of refractory COVID-19, we conducted high-coverage proteomics on serum extracellular vesicles collected from 12 patients with COVID-19 at different disease severity levels and 4 healthy controls. Furthermore, single-cell RNA sequencing of peripheral blood mononuclear cells collected from 10 patients with COVID-19 and 5 healthy controls was performed. RESULTS: Among the 3046 extracellular vesicle proteins that were identified, expression of MACROH2A1 was significantly elevated in refractory cases compared to non-refractory cases; moreover, its expression was increased according to disease severity. In single-cell RNA sequencing of peripheral blood mononuclear cells, the expression of MACROH2A1 was localized to monocytes and elevated in critical cases. Consistently, single-nucleus RNA sequencing of lung tissues revealed that MACROH2A1 was highly expressed in monocytes and macrophages and was significantly elevated in fatal COVID-19. Moreover, molecular network analysis showed that pathways such as "estrogen signaling pathway," "p160 steroid receptor coactivator (SRC) signaling pathway," and "transcriptional regulation by STAT" were enriched in the transcriptome of monocytes in the peripheral blood mononuclear cells and lungs, and they were also commonly enriched in extracellular vesicle proteomics. CONCLUSIONS: Our findings highlight that MACROH2A1 in extracellular vesicles is a potential biomarker of refractory COVID-19 and may reflect the pathogenesis of COVID-19 in monocytes.

Saliva as a matrix for measurement of cancer biomarkers

To minimize exposure to contagions such as seasonal influenza and COVID-19, we need noninvasive sampling and diagnostic methods, such as human saliva. Saliva is gaining attention as a suitable diagnostic medium because of its applicability in a field setting. Compared to blood, the collection, transport, and storage of saliva samples are easy, noninvasive, cost-effective and cause little discomfort for patients. Evidence shows that salivary biomarkers can be used to detect and monitor a plethora of oral and systemic conditions, including cancer. Human saliva contains many biomolecules, such as proteins, RNA, and DNA. In this chapter, we highlight the potential clinical utility of saliva as a liquid biopsy and explore recent discoveries in the applications of salivary biomarkers in cancer research. In addition, we reviewed the current state of salivary diagnostics and its associated technologies, and its potential as the preferred medium of cancer diagnosis, monitoring, and prognosis and future aspirations. © 2022 Elsevier Inc. All rights reserved.

Plasma-first: accelerating lung cancer diagnosis and molecular profiling through liquid biopsy.

Introduction: Molecular profiling of tumor tissue is the gold standard for treatment decision-making in advanced non-small cell lung cancer (NSCLC). Results may be delayed or unavailable due to insufficient tissue, prolonged wait times for biopsy, pathology assessment and testing. We piloted the use of plasma testing in the initial diagnostic workup for patients with suspected advanced lung cancer. Methods: Patients with ⩽15 pack-year smoking history and suspected advanced lung cancer referred to the lung cancer rapid diagnostic program underwent plasma circulating-tumor DNA testing using a DNA-based mutation panel. Tissue testing was performed per standard of care, including comprehensive next-generation sequencing (NGS). The primary endpoint was time from diagnostic program referral to cancer treatment in stage IV NSCLC patients (Cohort A) compared to a contemporary cohort not enrolled in the study (Cohort B) and an historical pre-COVID cohort referred to the program between 2018 and 2019 (Cohort C). Results: From January to June 2021, 20 patients were enrolled in Cohort A; median age was 70.5 years (range 33-87), 70% were female, 55% Caucasian, 85% never smokers, and 75% were diagnosed with NSCLC. Seven had actionable alterations detected in plasma or tissue (4/7 concordant). Fusions, not tested in plasma, were identified by immunohistochemistry for three patients. Mean result turnaround time was 17.8 days for plasma NGS and 23.6 days for tissue (p = 0.10). Mean time from referral to treatment initiation was significantly shorter in cohort A at 32.6 days (SD 13.1) versus 62.2 days (SD 31.2) in cohort B and 61.5 days (SD 29.1) in cohort C, both p < 0.0001. Conclusion: Liquid biopsy in the initial diagnostic workup of patients with suspected advanced NSCLC can lead to faster molecular results and shorten time to treatment even with smaller DNA panels. An expansion study using comprehensive NGS plasma testing with faster turnaround time is ongoing (NCT04862924).

Mechanisms of primary and secondary resistance to RET inhibitors in patients with RET-positive advanced NSCLC

Background: RET fusions are found in 1-2% of patients (pts) with advanced non-small cell lung cancer (aNSCLC). Targeted therapy with RET inhibitors (RETi) significantly improved prognosis. Molecular mechanisms of resistance are still incompletely characterized. Methods: This multicentric retrospective study included 24 centres. Eligible pts had a RET+ aNSCLC, were treated with a RETi and had at least one molecular profile by next-generation sequencing (NGS), performed before and/or after RETi, on tissue and/or plasma samples. Primary resistance under RETi was defined as disease progression (PD) within 6 months of therapy. Results: 95 patients were included with 112 biopsies: 93 at baseline, 19 at PD. 17 patients had paired NGS (baseline and PD). Median age was 65 years (range 56-72);62% were female, 54% were never smokers, 17% had brain metastasis (BM) at diagnosis. 55 patients received pralsetinib, 36 selpercatinib, 4 other RETi. Overall, median PFS under RETi was 17.1 months (95%CI 12.6-28). Primary resistance to RETi occurred in 22 (23%) patients. Primary resistant versus durable responders to RETi had non-adenocarcinoma histology in 9% vs 46% (p=0.61), smoking history in 57% vs 40% (p=0.21), BM in 5% vs 21% (p=0.1), TP53 mutations in 37% vs 22% (p=0.23). KRAS G12V mutation and SMARCA4 alterations were found only in poor responders (4.5% vs 0%, p=0.2;and 25% vs 0%, p=0.04, respectively). Among biopsies at PD (N=19, 13 liquid and 6 tissue biopsies), 7/13 (54%) liquid biopsies failed due to insufficient ctDNA. In 12 evaluable pts, 3 (25%) acquired secondary RET mutations (2 G810S and 1 S904F), 3 (25%) had novel RET rearrangements (2 in intron 11, 1 RET-DOCK1, 1 RET-CSGALNACT2) and 3 (25%) pts had off-target alterations (2 MET and 1 MYC amplification). Three pts (25%) developed novel TP53 mutations, while 3 (25%) had no novel identifiable alterations at PD. Conclusions: SMARCA4 and KRAS co-mutations may have a role in primary resistance to RETi. Secondary RET mutations, novel RET rearrangements and MET/MYC amplifications were identified after treatment with RETi. More than half of pts had insufficient ctDNA at PD, making tissue biopsy essential to identify resistance mechanisms. Legal entity responsible for the study: Institut Gustave Roussy. Funding: Has not received any funding. Disclosure: V. Fallet: Financial Interests, Personal, Advisory Board: AstraZeneca, BMS, Takeda, Roche, Pfizer, Sanofi, Sandoz, Jansen;Financial Interests, Personal, Invited Speaker: AstraZeneca, BMS, Takeda, Pfizer, MSD;Financial Interests, Personal, Expert Testimony: GSK, Boehringer. C. Audigier-Valette: Financial Interests, Personal, Advisory Role: AbbVie, AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Ipsen, Eli Lilly, Novartis, Pfizer, and Roche. A. Russo: Financial Interests, Personal, Advisory Board: Pfizer, AstraZeneca, MSD, Novartis;Financial Interests, Personal, Writing Engagements: AstraZeneca, Novartis. A. Calles Blanco: Financial Interests, Personal, Advisory Board: AstraZeneca, Boehringer Ingelheim, Pfizer, Roche, Lilly, Merck Sharp & Dohme, Novartis, Bristol-Myers Squibb, Takeda, Sanofi;Financial Interests, Personal, Other, Speaker honoraria: Bayer;Financial Interests, Institutional, Research Grant, Drug-only for Investigator-initiated trial: Merck Sharp & Dohme. P. Iranzo Gomez: Financial Interests, Personal, Advisory Role: Bristol-Myers Squibb Recipient, F. Hoffmann, La Roche AG, Merck Sharp & Dohme, Boehringer Ingelheim, MSD Oncology, Rovi, Yowa Kirin, Grunenthal Pharma S.A., Pfizer. M. Tagliamento: Financial Interests, Personal, Other, medical writer: Novartis, Amgen;Financial Interests, Personal, Invited Speaker, travel/accommodation: Roche, Bristol-Myers Squibb, AstraZeneca, Takeda. L. Mezquita: Financial Interests, Personal, Advisory Board: Takeda, AstraZeneca, Roche;Financial Interests, Personal, Invited Speaker: Roche, BMS, AstraZeneca, Takeda;Financial Interests, Personal, Research Grant, SEOM Beca Retorno 2019: BI;Financial Interests, Personal, Research Grant, ESMO TR Research Fellowship 2019: BMS;Financial Interests, Institutional, Research Grant, COVID research Grant: Amgen;Financial Interests, Institutional, Invited Speaker: Inivata, Stilla. C. Lindsay: Financial Interests, Institutional, Principal Investigator: Roche, Amgen, BI;Financial Interests, Personal, Advisory Role: CBPartners, Amgen. S. Ponce: Financial Interests, Institutional, Principal Investigator: Merck Sharp and Dohme, F. Hoffmann-La Roche, Foundation Medicine, PharmaMar. Personal fees: Merck Sharp and Dohme, Bristol-Myers Squibb, F. Hoffmann-La Roche, Foundation Medicine, AstraZeneca, Boehringer Ingelheim, Eli Lilly, Pfizer, Amgen, Celgene.;Financial Interests, Personal, Advisory Board: Merck Sharp and Dohme, Bristol-Myers Squibb, F. Hoffmann-La Roche, Foundation Medicine, AstraZeneca, Boehringer Ingelheim, Eli Lilly, Pfizer, Amgen, Celgene.;Non-Financial Interests, Personal, Other: Merck Sharp and Dohme, Bristol-Myers Squibb, F. Hoffmann-La Roche. M. Aldea: Financial Interests, Personal, Invited Speaker, travel/accommodation: Sandoz. All other authors have declared no conflicts of interest.

Noncoding RNome as Enabling Biomarkers for Precision Health.

Noncoding RNAs (ncRNAs), in the form of structural, catalytic or regulatory RNAs, have emerged to be critical effectors of many biological processes. With the advent of new technologies, we have begun to appreciate how intracellular and circulatory ncRNAs elegantly choreograph the regulation of gene expression and protein function(s) in the cell. Armed with this knowledge, the clinical utility of ncRNAs as biomarkers has been recently tested in a wide range of human diseases. In this review, we examine how critical factors govern the success of interrogating ncRNA biomarker expression in liquid biopsies and tissues to enhance our current clinical management of human diseases, particularly in the context of cancer. We also discuss strategies to overcome key challenges that preclude ncRNAs from becoming standard-of-care clinical biomarkers, including sample pre-analytics standardization, data cross-validation with closer attention to discordant findings, as well as correlation with clinical outcomes. Although harnessing multi-modal information from disease-associated noncoding RNome (ncRNome) in biofluids or in tissues using artificial intelligence or machine learning is at the nascent stage, it will undoubtedly fuel the community adoption of precision population health.

Fatal Interstitial lung disease induced by Osimertinib: a case report

Introduction: Osimertinib is a selective third-generation EGFR-TKI inhibitor with an inhibitory effect on the T790M mutation. Interstitial lung disease (ILD) occurred in 3.9% of the Osimertinib-treated patients (with 0.4% fatal cases). Methods: Case report of fatal ILD induced by Osimertinib in a patient with metastatic lung adenocarcinoma. Results: We present the case of an 81-year-old female patient diagnosed with stage IVB lung adenocarcinoma (May 2020) with pulmonary, adrenal, and brain metastasis. Genetic sequencing showed an exon 19 deletion. She started erlotinib until documentation of disease progression in January 2021. In this context, she performed a liquid biopsy with the detection of a T790M resistance mutation. She started Osimertinib in February 2021. Her past medical history showed diabetes and dyslipidemia. Two months after starting Osimertinib, she went to the emergency department (ER) with a one-week evolution with progressive dyspnea, cough, and fever. Upon admission to the ER, she was conscious and cooperative, with respiratory distress signs, normal blood pressure, and hypoxemia. She had decreased breath sounds, and coarse crackles were audible bilaterally. In the blood sampling, Haemoglobin was 7.7 mmol/L, creatinine 0.08 mmol/L, platelets 257000x10ˆ9/L, C-reactive protein 28.6 nmol/L, and NT-proBNP 98 pmol/L. Rt-PCR for sars-CoV-2 detection was negative. X-ray showed bilateral diffuse infiltrates. She started oxygen therapy via nasal cannula at 3l/min and IV antibiotics. ABG values were pH 7.44, pCO2 37 mmHg, pO2 69 mmHg, HCO3 26 mEq/L, sO2 94%. On reassessment after 3 hours, she presented worsening dyspnea and dizziness, with higher oxygen needs (venturi mask, 60%). Chest CT angiography showed extensive bilateral diffuse ground-glass densification with crazy-paving areas. It also showed no signs of pulmonary embolism. We admitted her to a level 2 ICU unit for surveillance. Due to suspected drug toxicity, she started Methylprednisolone pulses (1000mg/3days). Six hours after admission, due to hypoxemia worsening, non-invasive ventilation was started with the need to escalate oxygen therapy to 100% FiO2. At 24h, she showed clinical and blood analysis improvement. Nonetheless, she still needed 100% fiO2 to maintain >92% oxygen saturation. On the 4th day of hospitalization, she was hypotensive, prostrated, and with little reaction to painful stimulation. She started palliative treatment and died on the same day. Conclusions: ILD is a rare adverse effect of the treatment with Osimertinib, and fatal ILD is even rarer. The time from starting Osimertinib to this side effect is variable between patients. Awareness is necessary for a rapid diagnosis and early treatment. [Formula presented] Keywords: Osimertinib, Intersticial Lung Disease, Adverse effect

Diagnostic Approach and Treatment of Lung Cancer Patients in Portugal: Portuguese Lung Cancer Study Group Survey

Introduction: In Portugal, in 2020, 5415 new lung cancer patients were diagnosed and 4797 deaths were caused by lung cancer. Lung cancer ranks third in terms of cancer incidence and is the leading cause of cancer mortality. Early diagnosis, complete and fast patient assessment and staging, multidisciplinary approach, access to personalized medicine, new treatment options and research are essential to improve survival and quality of life. Access to clinical trials is critical for this improvement.The aim of this study is to assess the techniques available to the diagnostic work-up, treatments, the waiting time and the needs perceived by physicians. Methods: The Portuguese Lung Cancer Study Group launched a survey in order to study the diagnostic approach and treatment of lung cancer patients in Portugal. An online survey with 47 questions was sent to all Portuguese hospitals that treat lung cancer, referring to the pre-COVID-19 pandemic experience. Results: Responses from 31 Portuguese hospitals were collected, between May and September 2020. Availability to bronchoscopy, image-guided transthoracic needle biopsy (TNB), endobronchial ultrasound- transbronchial needle aspiration (EBUS-TNBA), PET/CT, molecular biology testing is presented in table I. In 58% (n=18) the molecular biology test was performed as a "reflex test". About 68% (n=21) of hospitals used next generation sequencing. Two hospitals (7%) reported not having access to liquid biopsies.Video-assisted thoracoscopic surgery was the main surgical technique (61%;n=19). The waiting time for the first radiation oncology consultation was less than 15 days in 71% (n=26). About 61% (n=19) of hospitals had clinical trials. A wide majority of doctors (77%) would like to have more clinical trials. In 71% (n=22) of the hospitals, it was possible to refer patients to Palliative Care receiving systemic anticancer therapy. [Formula presented] Conclusions: Despite the limitations of the methods, this study allowed us to deepen our knowledge about the work-up technologies and treatments available for lung cancer patients in Portugal. It has also identified future opportunities, such as increasing accessibility to some diagnostic tools and clinical trials. Keywords: Diagnosis and treatment approach, Lung Cancer in Portugal, Health Services Research - Portugal

Plasma first: Accelerating lung cancer diagnosis through liquid biopsy

Background: Molecular profiling of tumor tissue is the gold standard for treatment decision making in advanced non-small cell lung cancer. Results may be delayed or unavailable due to insufficient tissue samples or prolonged wait times for biopsy, pathology assessment and testing. We piloted the use of plasma molecular testing as part of the initial diagnostic work-up for patients with suspected advanced lung cancer (NCT04863924). Methods: Patients with radiologic evidence of advanced lung cancer referred to the lung rapid diagnostic program underwent plasma circulating tumor DNA (ctDNA) testing using InVisionFirst-Lung, a next-generation sequencing (NGS) assay targeting 37 genes. Standard tissue testing was performed with comprehensive NGS (Oncomine). The primary endpoint was time to treatment in stage IV NSCLC patients compared to an historical pre-COVID-19 cohort (2018-9). Secondary endpoints included actionable targets identified in plasma, % of patients starting targeted therapy based on liquid biopsy and result turnaround time (TAT). Results: Between July 1 to December 31, 2021, 60 patients were enrolled. Median age was 70 years (range 33-91), 52% were female, 57% Caucasian, 48% never smokers. Of these, 73% had NSCLC, 12% small cell, 10% non-lung pathology and 5% declined tissue biopsy. Of 44 NSCLC patients, 5 (11%) had early-stage disease and underwent curative therapy. Most stage IV patients (79%) had systemic treatment. Median time to treatment initiation in the study cohort was 34 days (n = 31, range 10-90) versus 62 days (n = 101, range 13- 159) in the historical cohort (p<0.0001). Two thirds (N = 23) of stage IV NSCLC patients had actionable alterations identified, (30% in current/ex-smokers);18 started targeted therapy including 10 based on plasma results before tissue results were available. Median TAT was 7 days for plasma from blood draw to reporting (range 4-14) and 26 days for tissue molecular testing (range 11-42), p<0.0001. Concordance was high between plasma and tissue testing (70%). Liquid biopsy identified actionable alterations for 3 patients not identified by tissue NGS. In 4 cases, plasma testing failed to identify actionable alterations detected in tissue, due to undetectable plasma ctDNA. Conclusions: Liquid biopsy in the initial diagnostic workup of patients with suspected advanced NSCLC leads to faster molecular results and shortens time to treatment compared to tissue testing alone. Supplementing the current standard of tissue molecular testing with a plasma-first approach during the diagnostic work up of patients with suspected advanced lung cancer may increase access to precision medicine and improve patient outcomes. (Table Presented).

DESC-1, a prospective study on epidemiologic factors influencing general population to engage with liquid biopsy based cancer screening

Background: Liquid biopsy technology has delivered promising results for therapy monitoring and disease relapse detection. The simplicity of the procedure makes it attractive for early cancer detection despite its clinical value in this setting being as yet unknown. The objective of the study is to evaluate the acceptance of this new screening technology by the general population. Methods: Participants in a digital health study originally selected randomly from the general population of the canton of Geneva (Switzerland) were invited to participate in a survey investigating (1) their motivation and (2) what factors might affect their decision regarding whether to proceed to a liquid biopsy test for cancer screening. Results: 2'898 participants responded to the survey (participation rate: 35%), 1'568 are > 50yo. Two thirds are female, 7.6% have a history of cancer, 80.8% have a family relative with a history of cancer. In the whole population: 97.7% are ready to use liquid biopsy as a cancer screening test. In the > 50yo, 97.3% of women and 98.5% of men would do the test. In the < 50yo, 97.2% of women and 98.6% of men would take it with X2 = 0.018 (p = 0.89) for independence with sex. Age category, education level, income band, professional status, self-rated health, tobacco status, chronic disease, personal or familial history of cancer and COVID pandemic have no statistical impact on the incentive to do a liquid biopsy test. In the whole population, 94.6% would take the test knowing that there is a risk of a false positive. The median accepted false positive percentage error rate is 10% with interquartile rate (IQR) Q1: 5% and Q3: 15%. Ninety-six percent of respondents would take the test knowing that there is a risk of a false negative, accepting a median false negative percentage error rate of 10% with IQR Q1: 5% and Q3 : 20%. Most respondents (97.6%) will do the test knowing that additional tests or procedures could be required in case of positivity and 58.8% would take it yearly, while 24.6% only every two years. Fifty-nine percent believe that taking the test could influence their lifestyle, regardless of the result. Conclusions: This study shows that the surveyed population is ready to engage with liquid biopsy as a cancer screening tool despite its limitations and drawbacks. Willingness to take the test might depend on sex only in the < 50yo population. Knowing the lack of demonstrated benefit, it is urgent to conduct trials assessing the clinical value, psychological impact and financial burden of liquid biopsy tests before incorporating this technology into cancer screening programs. Due to the very high level of interest demonstrated in our study, the possibility of direct-to-consumer availability should prompt health authorities and accreditation bodies to carefully weigh the impact of authorizing market access to liquid biopsy technologies for this purpose.

Liquid biopsy as a tool for predicts of severe COVID-19 and hospitalised community-acquired pneumonia.

Cell-free DNA (cfDNA) is released into the circulation after apoptosis, necrosis, and active secretion from cells. In a healthy individual, cfDNA is present in small amounts, has a short half-life, and is predominantly derived from circulating hematopoietic cells. The composition and quantity of cfDNA dramatically changes during pathological conditions. Indeed, several studies reported elevated cfDNA concentration as a potential noninvasive biomarker in many diseases. AIM: The aim of the study was evaluation of the circulating cell-free DNA in patients with severe Covid-19 in comparison with patients with hospitalised community-acquired pneumonia (with and without hyperglycemia and type 2 diabetes mellitus) to determine the specificity, sensitivity and cutoff value of cfDNA for each nosology. MATERIALS AND METHODS: The studies were carried out on the basis of city and regional hospitals in the Luhansk region between 2015 to 2021. Were examined in the study 28 patients with a positive diagnosis of COVID-19 according to PCR analysis (14 women and 14 men), 60 patients with community- acquired pneumonia (CAP) (30 women and 30 men), 101 patients with community-acquired pneumonia and hyperglicemia (CAP+HH) (44 women and 57 men), 70 patients with type 2 diabetes mellitus (T2DM) (37 women and 33 men), 42 patients with community-acquired pneumonia in combination with type 2 diabetes mellitus (CAP+T2DM) (27 women and 15 men). The control group consisted of 81 healthy volunteer donor (46 women and 35 men). DNA fragmentation was measured with the diphenylamine assay. Statistical and graphical analyses were done using Statistica 7.0 StatSoft software and using GraphPad Prism version 9.0 (GraphPad Software, La Jolla, CA, USA) software. RESULTS: We found 3-4-fold higher concentration of serum cfDNA levels in COVID-19 patients (womens and mens) compared with healthy controls. Similarly, the levels of cfDNA were 1,5- to 2-fold higher in pneumoniawomens and pneumonia-mens, pneumonia+hyperglycemia-womens and pneumonia+hyperglycemia-mens pneumonia+Type2 Diabetes-womens and pneumonia+Type2 Diabetes-mens, compared with healthy controls. Our results indicate cfDNA profiles on admission can discriminate between patients with COVID-19 and community-acquired pneumonia at risk of severe disease and death with better performance than previously reported inflammatory markers. CONCLUSIONS: Circulating cell-free nucleic acids (cfDNA) are novel potential biomarkers of COVID-19 and community-acquired pneumonia identified. Our study is one of the first to analyze cfDNA level (the cutoff value of cfDNA concentration) for prediction of COVID-19 and community-acquired pneumonia (with and without complications and comorbidity diseases).

17th Congress of the Italian Society of Experimental Hematology

The proceedings contain 170 papers. The topics discussed include: circular RNAs in pediatric t-cell acute lymphoblastic leukemia: new biomarkers;microRNAs in B cell precursor acute lymphoblastic leukemia: strict cooperation with oncogenes and tumor suppressors;metabolic strategies of myeloid blast survival;SARS-COV2 coagulopathy: an intricated puzzle;role of the microenvironment in the pathogenesis of myelodysplastic syndromes;the tumor microenvironment in Hodgkin lymphoma: clinical implications;liquid biopsy provides complementary information to tissue biopsies for the molecular classification of DLBCL patients;and prognostic value of immune cells in the multiple myeloma bone marrow microenvironment: a meta-analysis with in silico and in vitro validation.

Liquid Biopsy at Home: Delivering Precision Medicine for Patients with Cancer During the COVID-19 Pandemic.

CoronaVirus disease-2019 has changed the delivery of health care worldwide and the pandemic has challenged oncologists to reorganize cancer care. Recently, progress has been made in the field of precision medicine to provide to patients with cancer the best therapeutic choice for their individual needs. In this context, the Foundation Medicine (FMI)-Liquid@Home project has emerged as a key weapon to deal with the new pandemic situation. FoundationOne Liquid Assay (F1L) is a next-generation sequences-based liquid biopsy service, able to detect 324 molecular alterations and genomic signatures, from May 2020 available at patients' home (FMI-Liquid@Home). We analyzed time and costs saving for patients with cancer, their caregivers and National Healthcare System (NHS) with FMI-Liquid@Home versus F1L performed at our Department. Different variables have been evaluated. Between May 2020 and August 2021, 218 FMI-Liquid@Home were performed for patients with cancer in Italy. Among these, our Department performed 153 FMI-Liquid@Home with the success rate of 98% (vs. 95% for F1L in the hospital). Time saving for patients and their caregivers was 494.86 and 427.36 hours, respectively, and costs saving was 13 548.70€. Moreover, for working people these savings were 1084.71 hours and 31 239.65€, respectively. In addition, the total gain for the hospital was 163.5 hours and 6785€, whereas for NHS was 1084.71 hours and 51 573.60€, respectively. FMI-Liquid@Home service appears to be useful and convenient allowing time and costs saving for patients, caregivers, and NHS. Born during the COVID-19 pandemic, it could be integrated in oncological daily routine in the future. Therefore, additional studies are needed to better understand the overall gain and how to integrate this service in different countries.