1. Patients’ characteristics
Patients diagnosed with de novo DLBCL between January 2017 and May 2018 were enrolled from single institution. Patients (a) aged 19 years or older with a confirmed diagnosis of DLBCL according to the 2016 World Health Organization (WHO) criteria; (b) who underwent PET/CT, IgH gene arrangement PCR assessment, and unilateral trephination BM biopsy at diagnosis; and (c) with no malignancy other than lymphoma at the time of diagnosis, were included in the cohort.
Of note, patients with primary central nervous system (CNS) involvement or who refused to participate in the study after the diagnosis were excluded. Clinical parameters, including age at diagnosis, sex, histology, Ann Arbor staging, IPI, initial rituximab-containing treatment schedule, date of relapse, date of death, or documented date of last visit, were collected. This study was approved by the Institutional Review Board of Chonnam National University Hwasun Hospital in accordance with the Declaration of Helsinki.
Patients received six cycles of rituximab (R) with cyclophosphamide, vincristine, doxorubicin, and prednisolone (CHOP) chemotherapy in standard doses every 3 weeks. Those with stage I received three cycles of R-CHOP chemotherapy prior to the administration of involved-field radiation therapy (IFRT).
2. Morphologic BMI by BM trephination biopsy
BM trephination section biopsy and aspirate smears from DLBCL patients who had positive morphologic BMI (mBMI) were reviewed by experienced hematopathologist in accordance with the WHO criteria. Based on the results of the morphological examination and immunohistochemistry (ICH), the extent of lymphoma cell infiltration and histology of the lymphoid infiltrates suggested an mBMI.
3.18F-FDG PET/CT and image analysis
All patients underwent 18F-FDG PET/CT with a PET/CT system Discovery ST scanner (GE Healthcare) at initial diagnosis. After fasting for 6 h, 18F-FDG was injected intravenously (calculated dose: 7.4 MBq per kg), and the patients’ serum glucose level were evaluated. CT scan was performed from the skull base to the proximal thighs. The transmission data were obtained 60 min after the injection of 18F-FDG with a low-dose CT using the following imaging parameters: rotation time (0.8 s), slice thickness (3.75 mm), automated from 10 to 130 mA, 120 kV, and a 50-cm field of view (FOV) with a 512 × 512 matrix. PET emission acquisition was performed in the same anatomic locations immediately after the CT scan using the following parameters: axial FOV (15.7 cm) with a 128 × 128 matrix. The examinations were reconstructed according to the conventional iterative algorithm (OSEM). The CT data were applied for attenuation correction. PET/CT images were evaluated and confirmed visually with standardized uptake value (SUV) by consensus of two experienced nuclear medicine physicians. The normal FDG BM uptake was determined when it was lower than or corresponding to that in the liver. Focal FDG BM uptake was visually defined as one or several focal bone uptakes in PET images with or without bone lesion in CT images and when it was higher than that in the liver and lower than that in the brain. We subdivided Focal FDG BM uptake into cases with iliac crest bone uptake and without iliac crest bone uptake. Diffuse FDG uptake in the BM was visually categorized as diffuse heterogenous FDG uptake higher than that of normal liver without focal lesions. Diffuse homogenous FDG BM uptake with other benign condition such as inflammation or severe anemia was excluded.
4. Clonal gene rearrangements by PCR analysis
DNA was extracted from the mononuclear cells of BM samples (94 patients) obtained from the Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimen submitted for unilateral trephination biopsy, which was conducted at the time of DLBCL diagnosis. QIAamp® Mini Kit (QIAGEN, Valencia, LA, USA) was used to isolate the DNA from FFPE specimens in accordance with the manufacturer’s instructions. The quantity of the extracted DNA was assessed using a spectrophotometric system (NanoDropTM ND-1000, NanoDrop Technologies, Wilmington, DE, USA).
The clonality of B-cell neoplasms was examined by conducting a BIOMED-2 clonality assay, while the IgH clonal gene rearrangements were detected using the IdentiClone IGH Gene Clonality Assay (Invivoscribe Technologies, San Diego, CA, USA) following the manufacturer’s instructions. The PCR IGH multiplex PCR reactions, such as VH-JH gene rearrangement and DH-JH gene rearrangement, were used to evaluate the IGH clonality (V, variable; D, diversity; and J, joining gene segments, respectively). The product of PCR reaction was diluted with Hi-Di TM Formamide (Applied Biosystems, Foster city, CA, USA) and distilled water. The sample was analyzed by laser-induced fluorescence capillary electrophoresis using Genetic Analyzer 3000 (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. Monoclonality was defined as the occurrence of one distinct peak within the expected size ranges as per the BIOMED-2 protocol and the largest peak being at least three times higher than the third largest peak in the polyclonal background [14, 22].
5. Statistical analysis
Data analysis was performed using SPSS software version 26.0. and R software version 3.1.0. Clinical characteristics and diagnostic assessments were analyzed using the chi-square tests for categorical variables, and two-sided Student’s t-test was used for analyzing the quantitative variables. When analyzing diagnostic assessments, morphologic BMI was taken as the reference standard. The parallel test was used to determine sensitivity, specificity of IgH PCR or PET/CT, the serial test was used to determine sensitivity and specificity of combined IgH PCR and PET/CT for detecting BMI. The receivers operating characteristic (ROC) for detecting PET BMI was measured using the area under the curve (AUC), Youden indexes, and optimal cut-off value. Positive predictive value (PPV) and negative predictive value (NPV) for detecting BMI were assessed based on Bayes’ rule. Progression-free survival (PFS) was the primary endpoint of this study and was calculated from the date of diagnosis of DLBCL to the date of disease progression, relapse, death, or last follow-up. The secondary endpoint was overall survival (OS), which was calculated from the period of DLBCL diagnosis to the date of death or last follow-up. The Kaplan-Meier method was used to analyze the PFS and OS. Breslow test and log rank test were used to compare the survival outcomes. Cox regression models and Breslow test were used for the multivariate analysis of various independent prognostic factors. P-values of less than 0.05 were considered significant.