Donor Clonal Hematopoiesis and Recipient Outcomes After Transplantation

PURPOSE Clonal hematopoiesis (CH) can be transmitted from a donor to a recipient during allogeneic hematopoietic cell transplantation. Exclusion of candidate donors with CH is controversial since its impact on recipient outcomes and graft alloimmune function is uncertain. PATIENTS AND METHODS We performed targeted error-corrected sequencing on samples from 1,727 donors age 40 years or older and assessed the effect of donor CH on recipient clinical outcomes. We measured long-term engraftment of 102 donor clones and cytokine levels in 256 recipients at 3 and 12 months after transplant. RESULTS CH was present in 22.5% of donors, with DNMT3A (14.6%) and TET2 (5.2%) mutations being most common; 85% of donor clones showed long-term engraftment in recipients after transplantation, including clones with a variant allele fraction < 0.01. DNMT3A-CH with a variant allele fraction ≥ 0.01, but not smaller clones, was associated with improved recipient overall (hazard ratio [HR], 0.79; P = .042) and progression-free survival (HR, 0.72; P = .003) after adjustment for significant clinical variables. In patients who received calcineurin-based graft-versus-host disease prophylaxis, donor DNMT3A-CH was associated with reduced relapse (subdistribution HR, 0.59; P = .014), increased chronic graft-versus-host disease (subdistribution HR, 1.36; P = .042), and higher interleukin-12p70 levels in recipients. No recipient of sole DNMT3A or TET2-CH developed donor cell leukemia (DCL). In seven of eight cases, DCL evolved from donor CH with rare TP53 or splicing factor mutations or from donors carrying germline DDX41 mutations. CONCLUSION Donor CH is closely associated with clinical outcomes in transplant recipients, with differential impact on graft alloimmune function and potential for leukemic transformation related to mutated gene and somatic clonal abundance. Donor DNMT3A-CH is associated with improved recipient survival because of reduced relapse risk and with an augmented network of inflammatory cytokines in recipients. Risk of DCL in allogeneic hematopoietic cell transplantation is driven by somatic myelodysplastic syndrome–associated mutations or germline predisposition in donors.

Bockenheimer Disease is Associated With a TEK Variant

Bockenheimer disease is a venous malformation involving all tissues of an extremity. Patients have significant morbidity and treatment is palliative. The purpose of this study was to identify the cause of Bockenheimer disease to develop pharmacotherapy for the condition. Paraffin-embedded tissue from 9 individuals with Bockenheimer disease obtained during a clinically-indicated operation underwent DNA extraction. Droplet digital PCR (ddPCR) was used to screen for variants most commonly associated with sporadic venous malformations [TEK (NM_000459.5:c.2740C>T; p.Leu914Phe), PIK3CA (NM_006218.4:c.1624G>A; p.Glu542Lys and NM_006218.4:c.3140A>G; p.His1047Arg)]. ddPCR detected a TEK L914F variant in all 9 patients (variant allele fraction 2%-13%). PIK3CA E542K and H1047R variants were not identified in the specimens. Sanger sequencing and restriction enzyme digestion confirmed variants identified by ddPCR. A pathogenic variant in the endothelial cell tyrosine kinase receptor TEK is associated with Bockenheimer disease. Pharmacotherapy targeting the TEK signaling pathway might benefit patients with the condition.

Highly selective Cooperative Primers for multiplex detection of rare mutations

We describe a real time PCR-based technique capable of detecting and quantifying rare somatic mutations in circulating tumor DNA reference materials. Our approach utilizes previously described Cooperative Primers, structurally modified to exhibit high allele-specificity and one-copy target sensitivity. Cooperative Primers are bi-functional molecules, consisting of a high affinity probe fragment that guarantees sensitivity, and a covalently attached lower affinity primer providing specificity. Additional optimization of Cooperative Primer structure generated molecules capable of reliable detection of allele changes as small as a single nucleotide. These highly selective Cooperative Primers maintain excellent discrimination properties in rare mutant allele scenarios, in both monoplex and multiplex assays. With synthetic DNA samples, Cooperative Primers can detect as little as 100 copies of mutant template amongst 1 000 000 copies of wild-type template (minor allele fraction of 0.01 %). Multiplex Cooperative Primer assay was validated with cell-free DNA reference materials and consistently detected the lowest minor allele fraction available (0.1 %) for EGFR L858R, G719S and V769-D770insASV mutations, while simultaneously providing qualitative and quantitative assessment of cell-free DNA with integrated β-Actin assay. Easy to design, rapid and inexpensive, Cooperative Primer - based real time PCR assays are a promising tool for evaluation of cancer therapy response, occurrence of resistance mutations and relapse monitoring.

Customisation of therapeutic strategy in metastatic colorectal cancer by use of liquid biopsies (detection of RAS mutations with digital PCR)

Εισαγωγή: Ο μεταστατατικός ορθοκολικός καρκίνος (μΟΚΚ) αφορά περίπου 60% των ασθενών με ορθοκολικό καρκίνο και σχετίζεται με υψηλή θνητότητα. Η κατάσταση των γονιδίων RAS είναι ένας σημαντικός βιοδείκτης που καθοδηγεί τη θεραπεία προβλέποντας την ανταπόκριση στα μονοκλωνικά αντισώματα έναντι του υποδοχέα του επιδερμιδικού αυξητικού παράγοντα (epidermal growth factor receptor, EGFR). Ωστόσο, η εξέταση των γονιδίων RAS πραγματοποιείται παραδοσιακά μόνο σε αρχειακό υλικό ιστού από τον όγκο, χωρίς να παρέχει ανανεωμένη πληροφορία για την κατάσταση RAS στη διάρκεια της νόσου, καθώς στους περισσότερους ασθενείς η επαναβιόψηση δεν είναι δυνατή ή πρακτική. Οι υγρές βιοψίες είναι μια αναδυόμενη και υποσχόμενη εναλλακτική στη βιοψία ιστού, καθώς παρέχουν με ακρίβεια μια εικόνα του μοριακού προφίλ του όγκου σε πραγματικό χρόνο.Σκοπός: Η μελέτη στοχεύει στη χρήση και τον έλεγχο της υψηλής ευαισθησίας πλατφόρμας υγρής βιοψίας ψηφιακής PCR BEAMing για την ανίχνευση μεταλλάξεων RAS σε μια κόορτη ασθενών με μΟΚΚ σε ένα συνεργαζόμενο δίκτυο ογκολογικών κέντρων στην Ελλάδα. Επιπρόσθετα, στοχεύει στη μελέτη της χρησιμότητας των επαναλαμβανόμενων υγρών βιοψιών, παράλληλα με τον standard έλεγχο ιστού, για την εξατομίκευση της θεραπευτικής στρατηγικής στον μΟΚΚ.Μέθοδοι: Κλινικά δεδομένα και δεδομένα μοριακής ανάλυσης συλλέχθηκαν προοπτικά από τον φάκελο των ασθενών με μΟΚΚ. Δείγματα πλάσματος συλλέχθηκαν από ασθενείς με μΟΚΚ στο Πανεπιστημιακό Γενικό Νοσοκομείο Ιωαννίνων, το Πανεπιστημιακό Γενικό Νοσοκομείο Λάρισας και τη EUROMEDICA Γενική Κλινική Θεσσαλονίκης, στην Ελλάδα. Η ψηφιακή PCR BEAMing χρησιμοποιήθηκε για τον ορισμό της κατάστασης RAS στο κυκλοφορούν DNA (circulating tumour DNA, ctDNA) του όγκου σε τέσσερα στιγμιότυπα-«κλειδιά»: διάγνωση, μέσον της θεραπείας πρώτης γραμμής, πρώτη και δεύτερη επιδείνωση.Αποτελέσματα: Εξήντα οκτώ ασθενείς με μΟΚΚ εντάχθηκαν στη μελέτη μεταξύ Ιανουαρίου 2018 και Οκτωβρίου 2019 με διάμεσο χρόνο παρακολούθησης 13.3 μήνες. Μεταλλάξεις RAS ανιχνεύτηκαν στον ιστό και στο ctDNA σε 51.5% και 47.8% των ασθενών, αντίστοιχα, με ολική ποσοστιαία συμφωνία 72.3%. Η συμφωνία ιστού-ctDNA σχετιζόταν θετικά με την ύπαρξη ηπατικών μεταστάσεων (p=0.010) και αρνητικά με τη χαμηλή ευαισθησία της μεθόδου ελέγχου ιστού (p=0.092). Η κατάσταση RAS διέφερε μεταξύ της διάγνωσης και του μέσου της θεραπείας πρώτης γραμμής (p=0.046), καθώς το κλάσμα μεταλλαγμένων αλληλομόρφων RAS (RAS mutant allele fraction, RAS MAF) έγινε μη ανιχνεύσιμο σε 28.6% των ασθενών, και ο μέσος όρος του RAS μειώθηκε από 12.27% στη διάγνωση σε 5.63% στο μέσο της θεραπείας πρώτης γραμμής (p=0.147). Στην πρώτη επιδείνωση, νέες μεταλλάξεις RAS αναδύθηκαν σε σύγκριση με τη διάγνωση (p=0.014), καθώς 35.7% των ασθενών ανέπτυξαν νέα μετάλλαξη RAS, οδηγώντας σε μεταβολή της κατάστασης RAS από φυσιολογική σε μεταλλαγμένη στο 21.4% των ασθενών. Στη δεύτερη επιδείνωση, η κατάσταση RAS διατηρήθηκε στο 62.5% και στο 100% των ασθενών σε σύγκριση με τη διάγνωση και την πρώτη επιδείνωση, αντίστοιχα.Συμπεράσματα: Η ανάλυση του ctDNA πλάσματος με την ψηφιακή PCR BEAMing βρέθηκε να έχει ικανοποιητική συμφωνία με τον ιστό και να ανιχνεύει αποτελεσματικά ακόμα και μικρές διαφορές τόσο στην κατάσταση RAS όσο και στο RAS MAF στα στιγμιότυπα που ελέγχθηκαν. Συνεπώς, μπορεί να χρησιμοποιηθεί παράλληλα με τον έλεγχο του ιστό στη διάγνωση και, αποτυπώνοντας τις δυναμικές αλλαγές RAS, μπορεί να υποβοηθήσει τη λήψη εξατομικευμένων θεραπευτικών αποφάσεων για τους ασθενείς με μΟΚΚ και τον σχεδιασμό μελλοντικών κλινικών δοκιμών.

Frequency and Prognostic Impact of ALK Amplifications and Mutations in the European Neuroblastoma Study Group (SIOPEN) High-Risk Neuroblastoma Trial (HR-NBL1)

PURPOSE In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through activating point mutations or genomic amplification. We studied ALK genetic alterations in high-risk (HR) patients on the HR-NBL1/SIOPEN trial to determine their frequency, correlation with clinical parameters, and prognostic impact. MATERIALS AND METHODS Diagnostic tumor samples were available from 1,092 HR-NBL1/SIOPEN patients to determine ALK amplification status (n = 330), ALK mutational profile (n = 191), or both (n = 571). RESULTS Genomic ALK amplification ( ALKa) was detected in 4.5% of cases (41 out of 901), all except one with MYCN amplification (MNA). ALKa was associated with a significantly poorer overall survival (OS) (5-year OS: ALKa [n = 41] 28% [95% CI, 15 to 42]; no- ALKa [n = 860] 51% [95% CI, 47 to 54], [ P < .001]), particularly in cases with metastatic disease. ALK mutations ( ALKm) were detected at a clonal level (> 20% mutated allele fraction) in 10% of cases (76 out of 762) and at a subclonal level (mutated allele fraction 0.1%-20%) in 3.9% of patients (30 out of 762), with a strong correlation between the presence of ALKm and MNA ( P < .001). Among 571 cases with known ALKa and ALKm status, a statistically significant difference in OS was observed between cases with ALKa or clonal ALKm versus subclonal ALKm or no ALK alterations (5-year OS: ALKa [n = 19], 26% [95% CI, 10 to 47], clonal ALKm [n = 65] 33% [95% CI, 21 to 44], subclonal ALKm (n = 22) 48% [95% CI, 26 to 67], and no alteration [n = 465], 51% [95% CI, 46 to 55], respectively, P = .001). Importantly, in a multivariate model, involvement of more than one metastatic compartment (hazard ratio [HR], 2.87; P < .001), ALKa (HR, 2.38; P = .004), and clonal ALKm (HR, 1.77; P = .001) were independent predictors of poor outcome. CONCLUSION Genetic alterations of ALK (clonal mutations and amplifications) in HR-NB are independent predictors of poorer survival. These data provide a rationale for integration of ALK inhibitors in upfront treatment of HR-NB with ALK alterations.

Dynamics of circulating tumor DNA in patients with advanced solid tumors treated with cediranib and olaparib.

3035 Background: Circulating tumor DNA (ctDNA) has emerged as a potential biomarker to monitor treatment response in solid tumors. Our group previously showed that changes in ctDNA levels were predictive of radiographic response and survival in NSCLC patients receiving immunotherapy. Here we evaluated whether ctDNA dynamics could similarly be used to assess response in a PARP inhibitor-based therapy. Methods: A total of 122 patients with NSCLC, TNBC, PDAC or SCLC received cediranib (C) 30mg daily and Olaparib (O) 200mg twice daily in a phase II study NCI9881. Using a multigene NGS assay, ctDNA was measured longitudinally at baseline (T0), after 3 to 7 days of C monotherapy (T1), after 1 week of C+O combination (T2), after 4 weeks of C+O (T3), and every 4 weeks (T4+) thereafter. The first radiographic assessment was done after 8 weeks of C+O and every 8-12 weeks thereafter. CtDNA was quantified by determining the allele fraction of cancer-associated somatic mutations in plasma. We defined an early ctDNA response (e-ctDNA-R) as a >10% decrease in mutant allele fraction from T0 to T2, and an early ctDNA progression (e-ctDNA-P) as a > 10% increase; otherwise, it was stable ctDNA (e-ctDNA-S). Results: In total, 493 samples were analyzed from 94 patients, and 40 unique patients had both T0 and T2 ctDNA measurements, as well as corresponding radiographic assessments. These included 10 NSCLC, 17 TNBC, 3 SCLC and 10 PDAC. Of these patients, 4, 21, and 15 patients had PR, SD, and PD as best overall radiographic response respectively. Twenty-three (57.5%) patients had either e-ctDNA-R (17, 42.5%) or e-ctDNA-S (6, 15.0%), 18 (78.3%) of whom subsequently had either radiographic partial response (PR) or stable disease (SD). Seventeen (42.5%) patients had e-ctDNA-P, 10 (58.8%) of whom then had PD. A fair agreement was observed between e-ctDNA-R/S or e-ctDNA-P and radiographic PR/SD or PD with Cohen’s k 0.38 (70% agreement). The correlation between early ctDNA changes and PFS/OS are summarized in the table. All 25 patients with PR/SD eventually progressed. Of these, 17 (68%) had >10% increase in ctDNA from the nadir prior to disease progression (median 94.6%, 95%CI 38.2%-389.1%). The time between ctDNA progression and ctDNA nadir was significantly shorter (median 21 days, 95%CI 21-28) than the time between radiographic/clinical progression and initial PR/SD (median 107 days, 95%CI 56-204, P=0.0015). Conclusions: Longitudinal ctDNA measurements could enable early assessment of treatment response, resistance, and disease progression in patients treated with PARP inhibitor-based therapy. However, in this study, tumor responses and ctDNA changes were generally not as robust as have been observed with other classes of therapy.[Table: see text]

Serial Monitoring of Circulating Tumor DNA by Next-Generation Gene Sequencing as a Biomarker of Response and Survival in Patients With Advanced NSCLC Receiving Pembrolizumab-Based Therapy

PURPOSE Although the majority of patients with metastatic non–small-cell lung cancer (mNSCLC) lacking a detectable targetable mutation will receive pembrolizumab-based therapy in the frontline setting, predicting which patients will experience a durable clinical benefit (DCB) remains challenging. MATERIALS AND METHODS Patients with mNSCLC receiving pembrolizumab monotherapy or in combination with chemotherapy underwent a 74-gene next-generation sequencing panel on blood samples obtained at baseline and at 9 weeks. The change in circulating tumor DNA levels on-therapy (molecular response) was quantified using a ratio calculation with response defined by a > 50% decrease in mean variant allele fraction. Patient response was assessed using RECIST 1.1; DCB was defined as complete or partial response or stable disease that lasted > 6 months. Progression-free survival and overall survival were recorded. RESULTS Among 67 patients, 51 (76.1%) had > 1 variant detected at a variant allele fraction > 0.3% and thus were eligible for calculation of molecular response from paired baseline and 9-week samples. Molecular response values were significantly lower in patients with an objective radiologic response (log mean 1.25% v 27.7%, P < .001). Patients achieving a DCB had significantly lower molecular response values compared to patients with no durable benefit (log mean 3.5% v 49.4%, P < .001). Molecular responders had significantly longer progression-free survival (hazard ratio, 0.25; 95% CI, 0.13 to 0.50) and overall survival (hazard ratio, 0.27; 95% CI, 0.12 to 0.64) compared with molecular nonresponders. CONCLUSION Molecular response assessment using circulating tumor DNA may serve as a noninvasive, on-therapy predictor of response to pembrolizumab-based therapy in addition to standard of care imaging in mNSCLC. This strategy requires validation in independent prospective studies.

Quality threshold evaluation of Sanger confirmation for results of whole exome sequencing in clinically diagnostic setting

BackgroundWith the ability to simultaneously sequence more than 5,000 disease-associated genes, next-generation sequencing (NGS) has replaced Sanger sequencing as the preferred method in the diagnostic field at the laboratory level. However, Sanger sequencing has been used routinely to confirm identified variants prior to reporting results. This validation process causes a turnaround time delay and cost increase. Thus, this study aimed to set a quality threshold that does not require Sanger confirmation by analyzing the characteristics of identified variants from whole exome sequencing (WES).MethodsOur study analyzed data on a total of 694 disease-causing variants from 578 WES samples that had been diagnosed with suspected genetic disease. These samples were sequenced by Novaseq6000 and Exome Research Panel v2. All 694 variants (513 single-nucleotide variants (SNVs) and 181 indels) were validated by Sanger sequencing.ResultsA total of 693 variants included 512 SNVs and 181 indels from 578 patients and 367 genes. Five hundred seven heterozygous SNVs with at > 250 quality score and > 0.3 allele fraction were 100% confirmed by Sanger sequencing. Five heterozygous variants and one homozygous variant were not confirmed by Sanger sequencing, which showed 98.8% accuracy. There were 146 heterozygous variants and 35 homozygous variants among 181 indels, of which 11 heterozygous variants were not confirmed by Sanger sequencing (93.9% accuracy). Five non-confirmed variants with high quality were not identified on the ram .bam file.ConclusionOur results indicate that Sanger confirmation is not necessary for exome-derived SNVs with > 250 quality score and 0.3 > allele fraction set to an appropriate quality threshold. Indels or SNVs that do not meet the quality threshold should be reviewed by raw .bam file and Sanger confirmation should be performed to ensure accurate reporting.