NK cell activity and methylated HOXA9 ctDNA as prognostic biomarkers in patients with non-small cell lung cancer treated with PD-1/PD-L1 inhibitors.

BACKGROUND: PD-1/PD-L1 inhibitors have improved survival for patients with non-small cell lung cancer (NSCLC). We evaluated natural killer cell activity (NKA) and methylated HOXA9 circulating tumor DNA (ctDNA) as prognostic biomarkers in NSCLC patients treated with PD-1/PD-L1 inhibitors. METHODS: Plasma was prospectively collected from 71 NSCLC patients before treatment with PD-1/PD-L1 inhibitors and before cycles 2-4. We used the NK Vue® assay to measure the level of interferon gamma (IFNγ) as a surrogate for NKA. Methylated HOXA9 was measured by droplet digital PCR. RESULTS: A score combining NKA and ctDNA status measured after one treatment cycle had a strong prognostic impact. Group 1 had IFNγ < 250 pg/ml and detectable ctDNA (n = 27), group 2 consisted of patients with either low levels of IFNγ and undetectable ctDNA or high levels of IFNγ and detectable ctDNA (n = 29), group 3 had IFNγ ≥250 pg/ml and undetectable ctDNA (n = 15). Median OS was 221 days (95% CI 121-539 days), 419 days (95% CI 235-650 days), and 1158 days (95% CI 250 days-not reached), respectively (P = 0.002). Group 1 had a poor prognosis with a hazard ratio of 5.560 (95% CI 2.359-13.101, n = 71, P < 0.001) adjusting for PD-L1 status, histology, and performance status. CONCLUSIONS: Combining NKA and ctDNA status after one cycle of treatment was prognostic in patients with NSCLC treated with PD-1/PD-L1 inhibitors.

Antigen Expression Varies Significantly between Molecular Subgroups of Acute Myeloid Leukemia Patients: Clinical Applicability Is Hampered by Establishment of Relevant Cutoffs.

INTRODUCTION: In this single-center study of 268 acute myeloid leukemia (AML) patients, we have tested if a subset of 4 routinely employed immunophenotypic stem cell-associated markers correlated with the presence of recurrently mutated genes and if the markers were predictive for mutational status. METHODS: Immunophenotypic data from 268 diagnostic AML samples obtained in 2009-2018 were analyzed retrospectively for the antigens CD34, CD117, CD123, and CLEC12A. Correlation between immunophenotypes and mutations was analyzed by Fischer's exact test. Clinical applicability of the markers for predicting mutational status was evaluated by receiver operating characteristics analyses, where an area under the curve (AUC) of at least 0.85 was accepted as clinically relevant. RESULTS: For a number of genes, the antigen expression differed significantly between mutated and wild-type gene expression. Despite low AUCs, CD123 and CLEC12A correlated with FLT3+NPM1- and FLT3+NPM1+. Three subsets met the AUC requirements (CD34+, CD34+CD117+, and CD34-CD117+) for predicting FLT3-NPM1+ or FLT3+NPM1+. CONCLUSION: The value of immunophenotypes as surrogate markers for mutational status in AML seems limited when employing CD123 and CLEC12A in combination with CD34 and CD117. Defining relevant cutoffs for given markers is challenging and hampered by variation between laboratories and patient groups.

Blood natural killer cells during treatment in recurrent ovarian cancer.

OBJECTIVE: Recent research indicated favorable prognostic impact of intratumoral natural killer (NK) cells in ovarian carcinoma (OC). The role of NK cells during chemotherapy in OC is unknown. We investigated impact of NK cells in OC patients treated with palliative chemotherapy. METHODS: Participants receiving palliative chemotherapy for recurrent OC (N = 72) had prospectively blood samples at baseline and before cycle 2. NK cell counts were quantified by flow cytometry. NK cell activity was measured by the NK Vue® assay, estimating interferon-gamma production. Overall survival (OS) was the primary endpoint. Cutoffs were predefined, NK numbers (≥184 × 106 cells/L vs. <184 × 106 cells/L) and NK activity (<200 pg/mL vs. ≥200 pg/mL). RESULTS: Median OS in patients with low vs. high NK cell count at baseline was 7.1 months vs. 15.6 months (p = .028), respectively, and before cycle 2 was 5.7 vs. 17.3 months, p < .001, respectively. The difference in restricted mean survival (ΔRMST) was 5.7 months (95% CI: 3.3-8.0) at cycle 2 vs. 2.5 months (95% CI: -0.6 to 5.6) at baseline, showing a significant difference with no overlap of confidence intervals. In multivariate analyses, low NK cell count remained significant with a hazard ratio (HR)=2.83, 95% CI: 1.53-5.22, p = .001 (baseline) and HR = 3.34, 95% CI: 1.67-6.71, p = .001 (before cycle 2). Patients with both low NK count and NK activity at baseline (N = 20) had median OS 6.5 months vs. 11.5 months in patients with either high activity, high count or both (p = .007). In parallel, patients with both low NK activity and count at cycle 2 (N = 18) had a median survival of 4.0 months vs. 15.4 months (p < .001). CONCLUSIONS: A low blood NK cell count in recurrent metastatic ovarian cancer during chemotherapy is associated with unfavorable prognostic impact. Early increase in survival difference based on NK cell status suggests an association between NK cell count and treatment benefit.

Revisiting CLEC12A as leukaemic stem cell marker in AML: highlighting the necessity of precision diagnostics in patients eligible for targeted therapy.

Targeted therapy directed against rare disease-propagating leukaemic stem cells (LSCs) is a promising prospect for improving the outcome of acute myeloid leukaemia (AML) patients. Thus, distinguishing LSCs from normal haematopoietic stem and progenitor cells (HSPCs) is essential. The CLEC12A receptor has been proposed as a specific marker of LSCs, and consequently as an appealing treatment target. To explore the role of CLEC12A in further detail, we investigated whether a sorting strategy based on the activity of aldehyde dehydrogenase and CLEC12A expression could separate residual normal HSPCs from LSCs in bone marrow from 5 AML patients. We demonstrate that this distinction was possible in 2/5 cases, however with evidence of pre-leukaemic mutations in the CLEC12A- stem cells in one case. In contrast, cytogenetic and/or molecular aberrations were detected in both the CLEC12A+/- cell subsets in 3/5 AML cases studied. Furthermore, targeted next generation sequencing (NGS) of the sorted cell subsets revealed a pronounced clonal heterogeneity in the CLEC12A- cells suggestive of the leukaemia often originating in this immature cell subset. In conclusion, we provide proof-of-concept that precision diagnostics employing targeted cytogenetic/NGS-based analyses on highly purified cell subsets could be a powerful tool for selecting patients eligible for LSC-directed therapy.

Mapping the CLEC12A expression on myeloid progenitors in normal bone marrow; implications for understanding CLEC12A-related cancer stem cell biology.

The C-type lectin domain family 12, member A (CLEC12A) receptor has emerged as a leukaemia-associated and cancer stem cell marker in myeloid malignancies. However, a detailed delineation of its expression in normal haematopoiesis is lacking. Here, we have characterized the expression pattern of CLEC12A on the earliest stem- and myeloid progenitor subsets in normal bone marrow. We demonstrate distinct CLEC12A expression in the classically defined myeloid progenitors, where on average 39.1% (95% CI [32.5;45.7]) of the common myeloid progenitors (CMPs) expressed CLEC12A, while for granulocyte-macrophage progenitors and megakaryocyte-erythroid progenitors (MEPs), the average percentages were 81.0% (95% CI [76.0;85.9]) and 11.9% (95% CI [9.3;14.6]), respectively. In line with the reduced CLEC12A expression on MEPs, functional assessment of purified CLEC12A+/- CMPs and MEPs in the colony-forming unit assay demonstrated CLEC12A+ subsets to favour non-erythroid colony growth. In conclusion, we provide evidence that the earliest CLEC12A+ cell in the haematopoietic tree is the classically defined CMP. Furthermore, we show that CLEC12A-expressing CMPs and MEPs are functionally different than their negative counterparts. Importantly, these data can help determine which cells will be spared during CLEC12A-targeted therapy, and we propose CLEC12A to be included in future studies of myeloid cancer stem cell biology.

Unravelling the relevance of CLEC12A as a cancer stem cell marker in myelodysplastic syndrome.

Evidence of distinct disease propagating stem cells in myelodysplastic syndrome (MDS) has emerged in recent years. However, immunophenotypic characterization of these cancer stem cells remains sparse. In acute myeloid leukaemia (AML), we have previously described aberrant expression of the C-type lectin domain family 12, member A (CLEC12A) as a stable and reliable marker of leukaemia blasts and as a tool for assessing minimal residual disease. Furthermore, CLEC12A has been proposed as a promising marker of leukaemic stem cells in AML. The role of CLEC12A in MDS, however, remains to be elucidated. In this study, we found CLEC12A aberrantly expressed on the CD34+ CD38- cell compartment in 71% (22/31) of MDS patients, distributed across all Revised International Prognostic Scoring System risk groups. We showed that the CD34+ CD38- CLEC12A+ cells were indeed malignant and possessed functional stem cell properties in the long-term colony-initiating cell assay. As opposed to reported findings in AML, we showed that cancer stem cells from MDS samples derived from both CLEC12A positive and negative CD34+ CD38- subpopulations. Due to the absence of CLEC12A on normal haematopoietic stem cells, CLEC12A stem cell immunophenotyping may contribute to diagnosing and monitoring MDS patients and could furthermore add knowledge about disease propagating cells in MDS.

Nature and nurture: a case of transcending haematological pre-malignancies in a pair of monozygotic twins adding possible clues on the pathogenesis of B-cell proliferations.

We describe a comprehensive molecular analysis of a pair of monozygotic twins, who came to our attention when one experienced amaurosis fugax and was diagnosed with JAK2+ polycythaemia vera. He (Twin A) was also found to have an asymptomatic B-cell chronic lymphocytic leukaemia (B-CLL). Although JAK2-, Twin B was subsequently shown to have a benign monoclonal B-cell lymphocytosis (MBL). Flow cytometric and molecular analyses of the B-cell compartments revealed different immunoglobulin light and heavy chain usage in each twin. We hypothesized that whole exome sequencing could help delineating the pattern of germline B-cell disorder susceptibility and reveal somatic mutations potentially contributing to the differential patterns of pre-malignancy. Comparing bone marrow cells and T cells and employing in-house engineered integrative analysis, we found aberrations in Twin A consistent with a myeloid neoplasm, i.e. in TET2, RUNX1, PLCB1 and ELF4. Employing the method for detecting high-ranking variants by extensive annotation and relevance scoring, we also identified shared germline variants in genes of proteins interacting with B-cell receptor signalling mediators and the WNT-pathway, including IRF8, PTPRO, BCL9L, SIT1 and SIRPB1, all with possible implications in B-cell proliferation. Similar patterns of IGHV-gene usage to those demonstrated here have been observed in inherited acute lymphoblastic leukaemia. Collectively, these findings may help in facilitating identification of putative master gene(s) involved in B-cell proliferations in general and MBL and B-CLL in particular.

Diagnosing and following adult patients with acute myeloid leukaemia in the genomic age.

The diagnosis and follow-up process of adult patients with acute myeloid leukaemia (AML) is challenging to clinicians and laboratory staff alike. While several sets of recommendations have been published over the years, the development of high throughput screening and characterization for both genetic and epigenetic events have evolved with astonishing speed. Here we attempt to provide a practical guide to diagnose and follow adult AML patients with a focus on how to balance the wealth of information on the one hand, with the restriction put on these processes in terms of time, feasibility and economy when caring for these patients, on the other.

Persistence of DNMT3A mutations at long-term remission in adult patients with AML.

Mutations in DNMT3A, the gene encoding DNA methyltransferase 3 alpha, have been identified as molecular drivers in acute myeloid leukaemia (AML) with possible implications for minimal residual disease monitoring and prognosis. To further explore the utility of DNMT3A mutations as biomarkers for AML, we developed assays for sensitive detection of recurrent mutations affecting residue R882. Analysis of DNA from 298 diagnostic AML samples revealed DNMT3A mutations in 45 cases (15%), which coincided with mutations in NPM1, FLT3 and IDH1. DNMT3A mutations were stable in 12 of 13 patients presenting with relapse or secondary myelodysplastic syndrome, but were also present in remission samples from 14 patients (at allele frequencies of <1-50%) up to 8 years after initial AML diagnosis, despite the loss of all other molecular AML markers. The mutant DNMT3A allele burden was not related to the clinical course of disease. Cell sorting demonstrated the presence of DNMT3A mutations in leukaemic blasts, but also at lower allele frequencies in T and B-cells from the same patients. Our data are consistent with the recent finding of preleukaemic stem cells in AML, which are resistant to chemotherapy. The persistence of DNMT3A mutations during remission may have important implications for the management of AML.

Cell sorting enables interphase fluorescence in situ hybridization detection of low BCR-ABL1 producing stem cells in chronic myeloid leukaemia patients beyond deep molecular remission.

The exact disease state of chronic myeloid leukaemia (CML) patients in deep molecular remission is unknown, because even the most sensitive quantitative reverse transcription polymerase chain reaction (qPCR) methods cannot identify patients prone to relapse after treatment withdrawal. To elucidate this, CD34(+) stem cell and progenitor cell subpopulations were isolated by fluorescence-activated cell sorting (FACS), and their content of residual Philadelphia positive (Ph(+) ) cells was evaluated in 17 CML patients (major molecular response, n = 6; 4-log reduction in BCR-ABL1 expression (MR(4) ), n = 11) using both sensitive qPCR and interphase fluorescence in situ hybridization (iFISH). Despite evaluating fewer cells, iFISH proved superior to mRNA-based qPCR in detecting residual Ph(+) stem cells (P = 0·005), and detected Ph(+) stem- and progenitor cells in 9/10 patients at frequencies of 2-14%. Moreover, while all qPCR(+) samples also were iFISH(+) , 9/33 samples were qPCR-/iFISH(+) , including all positive samples from MR(4) patients. Our findings show that residual Ph(+) cells are low BCR-ABL1 producers, and that DNA-based methods are required to assess the content of persisting Ph(+) stem cells in these patients. This approach demonstrates a clinically applicable manner of assessing residual disease at the stem cell level in CML patients in MR(4) , and may enable early and safe identification of candidates for tyrosine kinase inhibitor withdrawal.

hMICL and CD123 in combination with a CD45/CD34/CD117 backbone - a universal marker combination for the detection of minimal residual disease in acute myeloid leukaemia.

Real-time quantitative polymerase chain reaction (qPCR) has been extensively validated for the detection of minimal residual disease (MRD) in acute myeloid leukaemia (AML). Meanwhile, multicolour flow cytometry (MFC) has received less attention because the so-called leukaemia-associated immunophenotypes (LAIPs) are generally of lower sensitivity and specificity, and prone to change during therapy. To improve MRD assessment by MFC, we here evaluate the combination of human Myeloid Inhibitory C-type Lectin (hMICL, also termed C-type lectin domain family 12, member A, CLEC12A) and CD 123 (also termed interleukin-3 receptor alpha, IL3RA) in combination with CD34 and CD117 (KIT), as an MRD assay in pre-clinical and clinical testing in 69 AML patients. Spiking experiments revealed that the assay could detect MRD down to 10(-4) in normal bone marrow with sensitivities equalling those of validated qPCR assays. Moreover, it provided at least one MFC MRD marker in 62/69 patients (90%). High levels of hMICL/CD123 LAIPs at the post-induction time-point were a strong prognostic marker for relapse in patients in haematological complete remission (P < 0·001). Finally, in post induction samples, hMICL/CD123 LAIPs were strongly correlated (r = 0·676, P = 0·0008) to applied qPCR targets. We conclude the hMICL/CD123-based MFC assay is a promising MRD tool in AML.

Unraveling stem cell and progenitor subsets in autologous grafts according to methods of mobilization: implications for prediction of hematopoietic recovery.

BACKGROUND AIMS: In the autologous setting, granulocyte colony-stimulating factor (G-CSF) (G), or, when failing, G plus plerixafor (G+P), are common regimens for mobilization of stem cells into peripheral blood. To delineate mobilization effects on graft composition and hematopoietic recovery, we compared contents of stem cells and progenitor cells in products of G+P- and G patients. Paired samples of G+P patients and prior insufficient G mobilization were available for analyses. METHODS: Subset analyses of grafts were performed by flow cytometry and myeloid colony-forming assay. In search of new markers to ascertain graft quality, we determined the fractions of aldehyde dehydrogenase bright (ALDH(br)) cells. RESULTS: G grafts contained higher percentages of CD34+ cells, CD34+CD38- cells, and committed progenitors (CD34+CD38+) compared with G+P grafts. A detailed characterization of the mobilized CD34+ cell subset showed higher percentages of CD38- among the CD34+ cells of the G+P group (P = 0.032). In contrast, the CD34+ cell subset in G grafts was characterized by a higher percentage of ALDH(br) cells (P < 0.0001). Studying engraftment and day +100 graft function the G and G+P transplanted patients were comparable with respect to neutrophils, whereas in platelets they differed. In the prediction of engraftment and hematopoietic recovery, the dose of infused ALDH(br) cells correlated best to both platelet (r = 0.565, P = 0.002) and neutrophil reconstitution (r = 0.366, P = 0.06). CONCLUSIONS: Besides showing dissimilar distributions of CD34+CD38- cells and progenitors in G and G+P grafts, this study further designated ALDH(br) as a promising marker in determination and prediction of graft quality and hematopoietic recovery.

Soluble programmed death ligand 1 as prognostic biomarker in non-small cell lung cancer patients receiving nivolumab, pembrolizumab or atezolizumab therapy.

Many studies have focused on the prognostic role of soluble programmed death ligand 1 (sPD-L1) in non-small cell lung cancer (NSCLC), but outcomes are ambiguous and further investigations are needed. We addressed the matter by studying sPD-L1 in baseline samples and in longitudinal samples taken prior to three subsequent cycles of anti-PD-1/anti-PD-L1 treatments. Eighty patients with NSCLC were enrolled. Median sPD-L1 level at baseline was 52 pg/mL [95% confidence interval (CI) 49-57]. In patients treated with pembrolizumab and nivolumab, the concentration of sPD-L1 remained rather stable throughout treatment. In contrast, sPD-L1 rose by 50-fold following the first cycle of atezolizumab therapy. We found the baseline level of sPD-L1 to be related to overall survival (OS) after two years of follow-up in simple Cox analysis (p = 0.006) and multiple Cox Regression, hazard ratio 1.02 (95% CI 1.00-1.03) (p = 0.033). There was no association between sPD-L1 and tissue PD-L1 expression, overall response rate, or progression free survival. In conclusion, sPD-L1 measured in baseline serum samples may be associated with OS in NSCLC patients receiving anti-PD1/anti-PD-L1 treatment. Importantly, the results signify that further research is warranted to explore the clinical utility of sPD-L1 in patients treated with anti-PD-L1.

Description of an activity-based enzyme biosensor for lung cancer detection.

BACKGROUND: Lung cancer is associated with the greatest cancer mortality as it typically presents with incurable distributed disease. Biomarkers relevant to risk assessment for the detection of lung cancer continue to be a challenge because they are often not detectable during the asymptomatic curable stage of the disease. A solution to population-scale testing for lung cancer will require a combination of performance, scalability, cost-effectiveness, and simplicity. METHODS: One solution is to measure the activity of serum available enzymes that contribute to the transformation process rather than counting biomarkers. Protease enzymes modify the environment during tumor growth and present an attractive target for detection. An activity based sensor platform sensitive to active protease enzymes is presented. A panel of 18 sensors was used to measure 750 sera samples from participants at increased risk for lung cancer with or without the disease. RESULTS: A machine learning approach is applied to generate algorithms that detect 90% of cancer patients overall with a specificity of 82% including 90% sensitivity in Stage I when disease intervention is most effective and detection more challenging. CONCLUSION: This approach is promising as a scalable, clinically useful platform to help detect patients who have lung cancer using a simple blood sample. The performance and cost profile is being pursued in studies as a platform for population wide screening.

Lung cancer is responsible for more deaths worldwide than all other cancers. It is often detected with the appearance of symptoms when treatment is limited and outcomes for the patient are much worse. While imaging chest scans can detect disease, they are poorly used even in the United States where it is an approved screening method. When cancer is present, protease enzymes are responsible for making space and modifying the lung tissue for the growing tumor. This report describes a panel of 18 sensors that release a fluorescent signal when these enzymes are present in a blood sample. The signal acts like a fingerprint of activity that can be used to identify people with lung cancer. This sensor platform can detect patients with curable lung cancer and could provide a platform for screening very large populations of at-risk individuals.

Assessment of circulating biomarkers for detection of lung cancer in a high-risk cohort.

BACKGROUND: There is an urgent need for early detection of lung cancer. Screening with low-dose computed tomography (LDCT) is now implemented in the US. Supplementary use of a lung cancer biomarker with high specificity is desirable. OBJECTIVE: To assess the diagnostic properties of a biomarker panel consisting of cytokeratin 19 fragment (CYFRA 21-1), carcinoembryonic antigen (CEA) and cancer antigen 125 (CA125). METHODS: A cohort of 250 high-risk patients was investigated on suspicion of lung cancer. Ahead of diagnostic work-up, blood samples taken. Cross-validated prediction models were computed to assess lung cancer detection properties. RESULTS: In total 32% (79/250) of patients were diagnosed with lung cancer. Area under the curve (AUC) for the three biomarkers was of 0.795, with sensitivity/specificity of 57%/93% and negative predictive value of 83%. When combining the biomarkers with US screening criteria, the AUC was 0.809, while applying only US screening criteria on the cohort, yielded an AUC of 0.62. The ability of the biomarkers to detect stage I-II lung cancer was substantially lower; AUC 0.54. CONCLUSIONS: In a high-risk cohort, the detection properties of the three biomarkers were acceptable compared to current LDCT screening criteria. However, the ability to detect early stage lung cancer was low.

High Expression of the Costimulatory Checkpoint Factor DNAM-1 by CD4+ T-Cells from Multiple Myeloma Patients Refractory to Daratumumab-Containing Regimens.

Multiple myeloma is an incurable disease characterized by unregulated growth of malignant plasma cells in the bone marrow (BM). Tumor-induced dysfunction of T-cells may be responsible for immune evasion and failure of immunotherapy. Therefore, a better understanding of the phenotype of T-cells at the tumor site is needed. We assessed the expression of immune regulatory receptors on T-cell subsets from peripheral blood (PB) and BM using multicolor flow cytometry. Paired PB and BM samples were collected from newly diagnosed, treatment-naïve myeloma patients (n = 19) and patients progressing during treatment with the CD38 monoclonal antibody daratumumab alone or in combination with other anti-myeloma drugs (n = 39). We observed that CD4+ T-cells from both PB and BM of patients relapsing on daratumumab have a higher expression of the costimulatory checkpoint receptor DNAM-1. The potential role of DNAM-1+CD4+ T-cells in the development of resistance to daratumumab needs further exploration. We also observed that the inhibitory checkpoint receptor TIGIT is more frequently expressed by BM CD8+ T-cells from myeloma patients than PD-1 and CTLA-4, which supports the hypothesis that TIGIT may play a central role in the immune escape of the malignant plasma cells.

Natural killer cell activity as a biomarker for the diagnosis of lung cancer in high-risk patients.

OBJECTIVE: Natural killer (NK) cells play an essential role in the immune response against cancer. However, immune escape mechanisms may cause inferior NK cell activity (NKA) in patients with cancer. This prospective study examined the relationship between NKA and lung cancer in a high-risk cohort. METHODS: In a cohort study, 250 participants referred by their general practitioner for suspicion of lung cancer were included. Before clinical investigation, blood was collected into NK Vue tubes, and the level of interferon gamma after 24 hours served as a surrogate marker for NKA. RESULTS: Among 250 patients, 79 were diagnosed with lung cancer. No difference in NKA was found between patients with lung cancer and control participants in which lung cancer was ruled out (median 226 pg/mL vs. 450 pg/mL). However, there was a significant difference in NKA between patients with late-stage lung cancer and controls (median 161 pg/mL vs. 450 pg/mL). A linear regression model showed that NKA was not influenced by age, sex or smoking status. CONCLUSIONS: The significantly lower NKA in patients with late-stage lung cancer warrants further investigation combining NKA with other biomarkers and examining the potential role of NKA as a marker of disseminated disease.

Acute myeloid leukemia exhibiting clonal instability during treatment: Implications for measurable residual disease assessments.

Next-generation sequencing (NGS) is an excellent methodology for measuring residual disease in acute myeloid leukemia and surveying several subclones simultaneously. There is little experience with interpretation of differential clonal responses to therapy. We hypothesized that differential clonal response could best be studied in patients with residual disease at the time of response evaluation. We performed targeted panel sequencing of paired diagnostic and first treatment evaluation samples in 69 patients with residual disease by morphology or measurable residual disease (MRD) level >0.02. Five patients had a rising clone at the time of evaluation. In a representative case, the rising clone was present only in the putative healthy stem cells (CD45lowCD34+CD38-CD123-CD7-) and not in the putative leukemic stem cells (CD34+CD38-CD123+CD7+) cells, thus indicating nonmalignant clonal hematopoiesis. In contrast, 17 of 43 evaluable patients exhibited a differential response in genes related to the leukemic clone. Twenty-six of 43 patients exhibited a clonal response that followed the overall treatment response. Patients with a differential response had better event-free survival (EFS) and overall survival (OS) than those in whom the clonal response followed the overall response (log-rank test, EFS: p = 0.045, OS: p = 0.050). This indicates that when following multiple leukemia-related clones, the less chemotherapy-responsive clone could, in some cases, have lower relapse potential, contrary to what is known when using standard mutation or fusion transcript-based disease surveillance. In conclusion, our results confirm the potential of refining MRD assessments by following multiple clones and warrants further studies on the precise interpretations of multiclone NGS-MRD assays.

Performance of the EarlyCDT® Lung test in detection of lung cancer and pulmonary metastases in a high-risk cohort.

OBJECTIVES: Early detection of lung cancer is pivotal for an optimal prognosis. CT screening is currently implemented in USA. To decrease the amount of CT scans, the application of a blood-based biomarker as part of screening criteria is desirable. MATERIALS AND METHODS: The EarlyCDT® Lung test was performed in a high-risk cohort composed 246 patients referred from their GP on suspicion of lung cancer. Blood samples were taken at first visit and patients underwent diagnostic workup on suspicion of lung cancer resulting in either a malignant diagnosis or ruled out cancer. Sensitivity and specificity of the EarlyCDT® Lung were calculated in the cohort and subgroups based on age, smoking history, sex and lung cancer stage. RESULTS: Overall sensitivity in the cohort was 33 % for lung cancer and 31 % for primary lung cancer and lung metastases combined. Sensitivity in age groups was 11 % (60 years or below), 31 % (61-75 years) and 55 % (>75 years). In patients with at least 10 tobacco pack years, sensitivity was 33 % while the sensitivity in patients with at least 50 tobacco pack years was 44 %. The assay sensitivity in stage I-II lung cancer patients was 21 %, while this was 40 % in stage III-IV lung cancer patients. In a subgroup of patients that met current CT screening criteria (age 55-80 years and minimum 30 tobacco pack years) the sensitivity was 37 %. CONCLUSION: The rationale of screening for lung cancer is to find patients in an early and resectable stage. However, the EarlyCDT® Lung test performed best in elderly, late stage lung cancer patients with a heavy smoking history. Based on these results, the current study finds insufficient sensitivity of the EarlyCDT® Lung test to be used as part of inclusion criteria in a low-dose CT program for detection of lung cancer.

Validating Methylated HOXA9 in Bronchial Lavage as a Diagnostic Tool in Patients Suspected of Lung Cancer.

Diagnosing lung cancer requires invasive procedures with high risk of complications. Methylated tumor DNA in bronchial lavage has previously shown potential as a diagnostic biomarker. We aimed to develop and validate methylated HOXA9 in bronchial lavage as a diagnostic biomarker of lung cancer. Participants were referred on suspicion of lung cancer. Ten mL lavage fluid was collected at bronchoscopy for analysis of methylated HOXA9 based on droplet digital PCR according to our previously published method. HOXA9 status was compared with the final diagnosis. The Discovery and Validation cohorts consisted of 101 and 95 consecutively enrolled participants, respectively. In the discovery cohort, the sensitivity and specificity were 73.1% (95% CI 60.9-83.2%) and 85.3% (95% CI 68.9-95.0%), respectively. In the validation cohort, the values were 80.0% (95% CI 66.3-90.0%) and 75.6% (95% CI 60.5-87.1%), respectively. A multiple logistic regression model including age, smoking status, and methylated HOXA9 status resulted in an AUC of 84.9% (95% CI 77.3-92.4%) and 85.9% (95% CI 78.4-93.4%) for the Discovery and Validation cohorts, respectively. Methylated HOXA9 in bronchial lavage holds potential as a supplementary tool in the diagnosis of lung cancer with a clinically relevant sensitivity and specificity. It remained significant when adjusting for age and smoking status.