FEASIBILITY AND PRELIMINARY RESULTS OF A PROSPECTIVE EVALUATION OF RESIDUAL DISEASE IN DIFFUSE LARGE B-CELL LYMPHOMAS USING DEEP SEQUENCING OF CELL FREE DNA AND DNA FROM FORMALIN FIXED BIOPSIES
Author(s): ,
Cristiana Carniti
Affiliations:
Medical Oncology and Hematology Department,Fondazione IRCCS Istituto Nazionale Tumori,Milano,Italy
,
Giulia Biancon
Affiliations:
Medical Oncology and Hematology Department,Fondazione IRCCS Istituto Nazionale Tumori,Milano,Italy
,
Stefania Banfi
Affiliations:
Medical Oncology and Hematology Department,Fondazione IRCCS Istituto Nazionale Tumori,Milano,Italy;Department of Oncology and Hemato-Oncology,Università degli Studi di Milano,Milano,Italy
,
Cristina Vella
Affiliations:
Medical Oncology and Hematology Department,Fondazione IRCCS Istituto Nazionale Tumori,Milano,Italy
,
Martina Magni
Affiliations:
Medical Oncology and Hematology Department,Fondazione IRCCS Istituto Nazionale Tumori,Milano,Italy;Department of Oncology and Hemato-Oncology,Università degli Studi di Milano,Milano,Italy
,
Martina Pennisi
Affiliations:
Medical Oncology and Hematology Department,Fondazione IRCCS Istituto Nazionale Tumori,Milano,Italy
,
Dodero Anna
Affiliations:
Medical Oncology and Hematology Department,Fondazione IRCCS Istituto Nazionale Tumori,Milano,Italy
,
Anna Guidetti
Affiliations:
Medical Oncology and Hematology Department,Fondazione IRCCS Istituto Nazionale Tumori,Milano,Italy;Department of Oncology and Hemato-Oncology,Università degli Studi di Milano,Milano,Italy
Paolo Corradini
Affiliations:
Medical Oncology and Hematology Department,Fondazione IRCCS Istituto Nazionale Tumori,Milano,Italy;Department of Oncology and Hemato-Oncology,Università degli Studi di Milano,Milano,Italy
EHA Library. Carniti C. Jun 15, 2019; 266930; PS1313
Cristiana Carniti
Cristiana Carniti
Contributions
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Abstract

Abstract: PS1313

Type: Poster Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 17:30 - 19:00

Location: Poster area

Background

The clinical utility of the deep sequencing of clonal immunoglobulin heavy chain (IGH) gene rearrangements in cell free DNA (cfDNA) has been documented in diffuse large B cell lymphoma (DLBCL)  patients, although results are generated retrospectively using commercial NGS platforms that limit the routine clinical application due to elevated costs and turn-around time. 

Aims
As we have recently developed an Ion Torrent-based sequencing method to analyze the IGH hypervariable region in genomic DNA (gDNA) and cfDNA of multiple myeloma patients (Biancon et al, 2018), we have designed a prospective study to: i) profile the IGH repertoire from formalin-fixed paraffin embedded (FFPE) lymph node biopsies of newly diagnosed DLBCL patients; ii) track the disease during therapy using cfDNA; iii) assess if our liquid biopsy approach can be used for the non-invasive disease monitoring in DLBCL complementing standard imaging techniques.

Methods

So far, we profiled 30 DLBCL patients. Genomic DNA was isolated from pre-treatment archival FFPE samples and cell free DNA extracted from plasma collected at diagnosis and at different time points during R-CHOP based therapy. IGH gene rearrangements were amplified and NGS data were analyzed as described (Biancon et al, 2018). 

Results

A malignant IGH clonotype was successfully identified from baseline FFPE samples at frequencies ranging from 6.3% to 69% (median: 10.9%) in 60% (18/30) of DLBCL patients. Seven out of 30 (23%) turned out to be polyclonal with different clones present at similar low frequencies (< 5%). Five patients (17%) had off-target results suggestive of poor quality DNA.

For patients with a lymph-node derived IGH rearrangement successfully identified, we analyzed paired baseline plasma samples. The same tumor clonotype was identified in 15 out of 18 patients (83%) in cfDNA  (frequencies from 1.4% to 74.8%, median: 5.6%). Pearson’s correlation test demonstrated a strong positive correlation between frequencies of clonal IGH rearrangements in FFPE gDNA and cfDNA samples (r=0.9754, P<0.001). The longitudinal analysis revealed that levels of circulating tumor-associated IGH gene rearrangements reflected changes in tumor burden as detected by imaging modalities, including escalating levels at progressive disease and declining or undetectable levels at complete response. Of note, in patient #9, the analysis of cfDNA after 4 cycles of R-CHOP therapy, suggested a possible disease progression,  preceding its documentation by imaging at the end of cycle 6. In patient #5, cfDNA analysis helped clarifying uncertain imaging results: the levels of the clonotipic IGH rearrangement in cfDNA became undetectable after cycle 2 and remained negative to the EOT. Despite this, interim and end of treatment (EOT) PET-CT scans, reported the presence of possible residual disease that was subsequently proven to be a false-positive result when subjected to biopsy and histological examination.

Conclusion
These data confirm that: i) our custom sequencing workflow can be successfully applied to the analysis IGH clonal rearrangements form archival FFPE samples and paired cfDNA in DLBCL; ii) IGH cfDNA levels reflect DLBCL tumor burden at diagnosis and can be used for the noninvasive disease monitoring with results complementing imaging data. The combination with the tumor genotyping on cfDNA (Rossi et al, 2018) that is ongoing, will allow to track clonal evolution and the emergence of treatment resistant clones and increase the number of patients receiving residual disease monitoring using the liquid biopsy. 

Session topic: 20. Lymphoma Biology & Translational Research

Keyword(s): Diffuse large B cell lymphoma, Molecular markers

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