Subpar reporting of pre-analytical variables in RNA-focused blood plasma studies.

Extracellular RNA (cell-free RNA; exRNA) from blood-derived liquid biopsies is an appealing, minimally invasive source of disease biomarkers. As pre-analytical variables strongly influence exRNA measurements, their reporting is essential for meaningful interpretation and replication of results. The aim of this review was to chart to what extent pre-analytical variables are documented, to pinpoint shortcomings and to improve future reporting. In total, 200 blood plasma exRNA studies published in 2018 or 2023 were reviewed for annotation of 22 variables associated with blood collection, plasma preparation, and RNA purification. Our results show that pre-analytical variables are poorly documented, with only three out of 22 variables described in over half of the publications. The percentage of variables reported ranged from 4.6% to 54.6% (mean 24.84%) in 2023 and from 4.6% to 57.1% (mean 28.60%) in 2018. Recommendations and guidelines (i.e., BRISQ, ASCO-CAP, BloodPAC, PPMPT, and CEN standards) have currently not resulted in improved reporting. In conclusion, our results highlight the lack of reporting pre-analytical variables in exRNA studies and advocate for a consistent use of available standards, endorsed by funders and journals.

Detection of circulating tumor DNA in plasma of patients with primary CNS lymphoma by digital droplet PCR.

BACKGROUND: Primary central nervous system lymphoma (PCNSL) are rare mature B-cell lymphoproliferative diseases characterized by a high incidence of MYD88 L265P and CD79B Y196 hotspot mutations. Diagnosis of PCNSL can be challenging. The aim of the study was to analyze the detection rate of the MYD88 L265P and CD79B Y196 mutation in cell free DNA (cfDNA) in plasma of patients with PCNSL. METHODS: We analyzed by digital droplet PCR (ddPCR) to determine presence of the MYD88 L265P and CD79B Y196 hotspot mutations in cfDNA isolated from plasma of 24 PCNSL patients with active disease. Corresponding tumor samples were available for 14 cases. Based on the false positive rate observed in 8 healthy control samples, a stringent cut-off for the MYD88 L265P and CD79B Y196 mutation were set at 0.3% and 0.5%, respectively. RESULTS: MYD88 L265P and CD79B Y196 mutations were detected in 9/14 (64%) and 2/13 (15%) tumor biopsies, respectively. In cfDNA samples, the MYD88 L265P mutation was detected in 3/24 (12.5%), while the CD79B Y196 mutation was not detected in any of the 23 tested cfDNA samples. Overall, MYD88 L265P and/or CD79B Y196 were detected in cfDNA in 3/24 cases (12.5%). The detection rate of the combined analysis did not improve the single detection rate for either MYD88 L265P or CD79B Y196. CONCLUSION: The low detection rate of MYD88 L265P and CD79B Y196 mutations in cfDNA in the plasma of PCNSL patients argues against its use in routine diagnostics. However, detection of MYD88 L265P by ddPCR in cfDNA in the plasma could be considered in challenging cases.

Genomic profiling of post-transplant lymphoproliferative disorders using cell-free DNA.

Diagnosing post-transplant lymphoproliferative disorder (PTLD) is challenging and often requires invasive procedures. Analyses of cell-free DNA (cfDNA) isolated from plasma is minimally invasive and highly effective for genomic profiling of tumors. We studied the feasibility of using cfDNA to profile PTLD and explore its potential to serve as a screening tool. We included seventeen patients with monomorphic PTLD after solid organ transplantation in this multi-center observational cohort study. We used low-coverage whole genome sequencing (lcWGS) to detect copy number variations (CNVs) and targeted next-generation sequencing (NGS) to identify Epstein-Barr virus (EBV) DNA load and somatic single nucleotide variants (SNVs) in cfDNA from plasma. Seven out of seventeen (41%) patients had EBV-positive tumors, and 13/17 (76%) had stage IV disease. Nine out of seventeen (56%) patients showed CNVs in cfDNA, with more CNVs in EBV-negative cases. Recurrent gains were detected for 3q, 11q, and 18q. Recurrent losses were observed at 6q. The fraction of EBV reads in cfDNA from EBV-positive patients was 3-log higher compared to controls and EBV-negative patients. 289 SNVs were identified, with a median of 19 per sample. SNV burden correlated significantly with lactate dehydrogenase levels. Similar SNV burdens were observed in EBV-negative and EBV-positive PTLD. The most commonly mutated genes were TP53 and KMT2D (41%), followed by SPEN, TET2 (35%), and ARID1A, IGLL5, and PIM1 (29%), indicating DNA damage response, epigenetic regulation, and B-cell signaling/NFkB pathways as drivers of PTLD. Overall, CNVs were more prevalent in EBV-negative lymphoma, while no difference was observed in the number of SNVs. Our data indicated the potential of analyzing cfDNA as a tool for PTLD screening and response monitoring.

A population-based study of transformed marginal zone lymphoma: identifying outcome-related characteristics.

Histological transformation of marginal zone lymphoma (tMZL) into diffuse large B-cell lymphoma is associated with poor outcomes. Clinical characteristics associated with transformation risk and outcome after transformation are largely unknown due to scarcity of data. In this population-based study, competing risk analyses were performed to elucidate clinical characteristics associated with developing transformation among 1793 MZL patients using the Netherlands Cancer Registry. Cox regression analyses were performed to elucidate clinical characteristics associated with risk of relapse and mortality after transformation. Transformation occurred in 75 (4%) out of 1793 MZL patients. Elevated LDH and nodal MZL subtype at MZL diagnosis were associated with an increased risk, and radiotherapy with a reduced risk of developing tMZL. Most tMZL patients received R-(mini)CHOP (n = 53, 71%). Age >60 years and (immuno)chemotherapy before transformation were associated with an increased risk of relapse and mortality after transformation. Two-year progression-free survival (PFS) and overall survival (OS) were 66% (95% CI 52-77%) and 75% (95% CI 62-85%) for R-(mini)CHOP-treated tMZL patients, as compared to a PFS and OS both of 41% (95% CI 19-63%) for patients treated otherwise. Our study offers comprehensive insights into characteristics associated with transformation and survival after transformation, thereby optimizing guidelines and patient counseling.

Low Mutational Burden of Extranodal Marginal Zone Lymphoma of Mucosa-Associated Lymphoid Tissue in Patients with Primary Sjogren's Syndrome.

Patients with primary Sjogren's syndrome (pSS) are at risk of developing extranodal marginal zone lymphoma (ENMZL) of the mucosa-associated lymphoid tissue (MALT) in the parotid glands. Unlike recurrent genomic aberrations observed in MALT lymphoma, which were not associated with pSS (non-pSS), it is unknown which somatic aberrations underlie the development of pSS-associated MALT lymphomas. Whole-exome sequencing was performed on 17 pSS-associated MALT lymphomas. In total, 222 nonsynonymous somatic variants affecting 182 genes were identified across the 17 cases. The median number of variants was seven (range 2-78), including three cases with a relatively high mutational load (≥24/case). Out of 16 recurrently mutated genes, ID3, TBL1XR1, PAX5, IGLL5 and APC are known to be associated with lymphomagenesis. A total of 18 copy number alterations were detected in eight cases. MALT1 translocations were not detected. With respect to outcome, only two cases relapsed outside of the salivary glands. Both had a high mutational load, suggesting a more advanced stage of lymphoma. The low mutational load and lack of a clear lymphoma-related mutation profile suggests that localized pSS-associated MALT lymphomas are genomically more stable than non-pSS MALT lymphomas and most likely depend on a stimulatory micro-environment.