Diagnostic accuracy and prognostic relevance of immunoglobulin heavy chain rearrangement and 18 F-FDG- PET/CT compared with unilateral bone marrow trephination for detecting bone marrow involvement in patients with diffuse large B-cell lymphoma

This study aimed to determine whether molecular analysis of immunoglobulin heavy chain (IgH) genes and PET/CT could overcome the limitation of dening morphologic bone marrow involvement by trephination biopsy and could increase the diagnostic accuracy or prognostic prediction. 94 de novo patients with DLBCL underwent PET/CT, polymerase chain reaction (PCR) test for detection of IgH gene rearrangement, and unilateral BM trephination at diagnosis. Abbreviations: DLBCL, diffuse large B-cell lymphoma; IPI, international prognostic score; NCCN-IPI, National Comprehensive Cancer Network-IPI; LDH, lactate dehydrogenase; BMI, bone marrow involvement; PET, positron emission tomography-computed tomography; IgH, immunoglobulin heavy chain gene rearrangement; R-CHOP, rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone; IFRT, involved-eld radiation therapy; ICE, etoposide, carboplatin, and ifosfamide; DHAP, dexamethasone, cisplatin and cytarabine; IT MTX/AraC, intrathecal methotrexate and cytarabine; Auto PBSCT, autologous peripheral blood stem cell transplantation.


Background
In diffuse large B-cell lymphoma (DLBCL), bone marrow involvement (BMI) has the important clinical implication as a component of the Ann Arbor staging and clinical risk-strati cation index including the International Prognostic Index (IPI) [1]. For many years, unilateral bone marrow (BM) trephination has been regarded as the gold standard for the evaluation of BMI in patients with DLBCL [2]. The extent of lymphoma cell in ltration in the BM is a highly signi cant negative prognostic factor [3]. However, BM trephination biopsy has some limitations of, namely, low sensitivity to patchy or focal BM involvement, inter-observers' exibility, and technical problem such as inappropriately obtained specimens [4].
Recent studies demonstrated that 18 F-FDG positron emission tomography-computed tomography (PET/CT) possesses adequate sensitivity for the detection of BMI in patients with DLBCL [5,6]. The National Comprehensive Cancer Network (NCCN) guidelines also recommended that BM trephination biopsy is not necessary if the PET/CT scan demonstrates BMI and that the therapeutic modalities should not be changed [7,8]. However, the accuracy of PET/CT assessment on detecting BMI in patients with DLBCL remains unclear [9]. Our previous studies reported that high metabolic tumor volume, which indicates the extent of malignant lymphoid cell in ltration in the BM, had negative prognostic outcome compared with other clinical risk factors [10,11]. On the contrary, as 18 F-FDG is not a tumor-speci c contrast agent, it might accumulate in extra nodal sites in patients with other benign conditions [11]. These characteristics could lead to a false positive result. The pitfalls of PET/CT interpretation in BMI might be associated with the de nition of marrow involvement (focal vs diffuse in ltration) without taking into considerations the anatomic variations or in ammatory physiology in DLBCL [12].
Most of the patients with lymphoproliferative disorder can be diagnosed by histomorphology or cytomorphology [13,14]. However, the morphological features in 5-15% patients are not typical and can be di cult to diagnose [14]. In such cases, the immunoglobulin gene rearrangements could be useful for determining the clonality of lymphoproliferative tissues [14]. The detection of lymphoid clonality by immunoglobulin gene rearrangement is an important method in the diagnosis of and in predicting the prognosis of lymphoid malignancy [15]. Because the immunoglobulin heavy chain (IgH) gene rearranged when malignant B lymphoid cells were developing, IgH gene is considered the most valuable gene target for detecting B-cell clonality in previous studies [16,17]. Immunoglobulin gene rearrangement analysis performed by polymerase chain reaction (PCR) test such as the BIOMED-2 multi target PCR approach has been a helpful method for detecting the clonality of B-cell lymphoid malignancy, and the detection rates of PCR analysis increased with the combined use of immunoglobulin gene rearrangement [18,19]. However, only a few studies have evaluated the accuracy of PCR analysis for molecular staging and focused on adjusting the results based on the clinical outcome [20,21].
The aim of this prospective cohort study was to determine whether molecular analysis of immunoglobulin heavy chain (IgH) genes and PET/CT could increase the diagnostic accuracy or predict the survival outcome compared to conventional trephination biopsy in the rituximab-containing treatment of DLBCL.

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 con rmed diagnosis of DLBCL according to the Page 5/22 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-eld radiation therapy (IFRT).

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 in ltration and histology of the lymphoid in ltrates suggested an mBMI.

18 F-FDG PET/CT and image analysis
All patients underwent 18 F-FDG PET/CT with a PET/CT system Discovery ST scanner (GE Healthcare) at initial diagnosis. After fasting for 6 h, 18 F-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 18 F-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 eld 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 con rmed 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 de ned 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 in ammation or severe anemia was excluded.

Clonal gene rearrangements by PCR analysis
DNA was extracted from the mononuclear cells of BM samples (94 patients) obtained from the Formalin-Fixed Para n-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 uorescence capillary electrophoresis using Genetic Analyzer 3000 (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. Monoclonality was de ned 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].

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 [23]. The parallel test was used to determine sensitivity, speci city of IgH PCR or PET/CT, the serial test was used to determine sensitivity and speci city 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 signi cant. ) as the high-risk group, respectively. Positive mBMI using trephination section biopsy was detected in 9 patients (9.6%; concordant mBMI = 6 and disconcordant mBMI = 3), while IgH clonality (IgH BMI) was detected in 21 patients (22.3%). On the other hands, positive bone marrow 18 F-FDG PET uptake (PET BMI) was observed in 16 patients (17.0%). Among those with positive PET BMI, 11 patients had a focal type (68.8%, 11/16; focal with iliac crest lesion = 1 and focal without iliac crest lesion = 10) and 5 (31.2%) had a diffuse type. In addition, 5 patients with positive PET BMI were concordant with conventional mBMI and 6 patients were with positive IgH clonality. All patients were basically treated with six cycles of R-CHOP chemotherapy, except 8 (8.5%) patients with stage I who treated with involved-eld radiation therapy (IFRT) after three cycles of R-CHOP. Other details of the clinical characteristics were summarized in Table 1. The distribution classi cation of patients with BMI is shown in Table 2.    Table 2 were used for calculations as standard formulas for sensitivity, speci city, PPV, NPV. § Parallel test was used to determine sensitivity, speci city of IgH PCR or PET/CT. Serial test was used to determine sensitivity and speci city of combined IgH PCR and PET/CT for detecting BMI.

Clinical correlation according to the results of the combined detection of PET/CT and IgH rearrangement
A signi cant difference of positivity was observed in PET BMI status depending on clinical stages (p < 0.001). Moreover, a clinical correlation was also found between positive PET BMI and IPI risk groups (p = 0.004). A signi cant difference of the IgH BMI status was observed between patients with Stage I-II (5 of 47 patients; 10.6%) and those with stage III-IV (16 of 47 patients; 30.0%, p = 0.017). The detection of IgH BMI was signi cantly increased in high-risk patients, depending on the IPI risk classi cation (IPI non-high risk vs high risk, p = 0.039) in Table 4.  Fig. 1a). The survival of patients (n = 15) with negative mBMI and positive IgH BMI were similar to patients (n = 9) with positive mBMI (p > .05; Fig. 1b). Patients (n = 11) with PET BMI (+) and mBMI (-) were associated with shorter survival outcome than those (n = 74) with mBMI (-) and PET BMI (-) (p > .05; Fig. 1c).  Fig. 2b).  (Table 5).

Discussion
Bone marrow in ltration of lymphoma cells is one of the important prognostic factors of DLBCL [1].
Unilateral BM trephination biopsy has been conducted to con rm BMI [2]. If the additional less invasive tools for determining BMI can use for detecting focal or diffuse BM involvement, it will be helpful to diagnosis or prognosis in patients with DLBCL. Many approaches have been used for determining BMI, such as PCR ampli cation of immunoglobulin gene rearrangement or FDG/PET-CT [7,19,20,[24][25][26]. To our knowledge, this was the rst trial to evaluate the prognostic value of a combination of FDG PET/CT and PCR-based clonality for the treatment of DLBCL. This prospective cohort study from a single institution aimed to evaluate the diagnostic and prognostic signi cance of BM assessment in DLBCL according to the IgH gene rearrangement as well as FDG PET/CT compared with conventional BM biopsy.
In the study, 22 patients (22 out of 85 patients) with negative mBMI were detected with positive PET BMI or IgH BMI. The characteristics of positive mBMI were not concordant with those of focal PET BMI without iliac crest lesion. The discordance of three detecting methods mainly occurred due to anatomic sites. Focal FDG uptake of bone marrow without diffuse iliac crest may not be detected as morphologic BM biopsy, resulting in inconsistency between positive PET CT and morphologic BMI. This nding may suggest that focal PET BMI without iliac crest could indicate BMI without mBMI. The assessment of diffuse FDG uptake remained controversial in several studies [9,24]; however, some patients with diffuse BM uptake showed positive mBMI [25]. Our study showed that three patients with diffuse PET BMI were matched with positive conventional mBMI. Berthet  negative mBMI and positive IgH BMI were signi cantly worse than negative mBMI and negative IgH BMI [19]. In our study, patients with combined positive IgH BMI and PET BMI without detecting mBMI showed poor survival outcome than those with negative conventional mBMI. It could give an additional information to patients with negative morphologic BMI.

Conclusions
In conclusion, the study suggested that the combined assessment with IgH gene rearrangement PCR and PET/CT could give additional information of detecting the BMI in patients with DLBCL. The assessment of BMI based on PET/CT and IgH gene rearrangement PCR could be indicator to predict the survival outcomes of DLBCL, particularly in patients with negative conventional mBMI.