IGH rod-like tracer: An AlphaFold2 structural similarity extraction-based predictive biomarker for minimal residual disease monitoring in children with precursor B-cell acute lymphoblastic leukemia

doi:10.21203/rs.3.rs-2071325/v1

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IGH rod-like tracer: An AlphaFold2 structural similarity extraction-based predictive biomarker for minimal residual disease monitoring in children with precursor B-cell acute lymphoblastic leukemia

https://doi.org/10.21203/rs.3.rs-2071325/v1

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Sequence variation resulting from the evolution of IGH clones and immunophenotypic drift makes it difficult to track abnormal B cells in children with precursor B-cell acute lymphoblastic leukemia (pre-B-ALL) by flow cytometry, quantitative polymerase chain reaction (qPCR), or next-generation sequencing (NGS). This study aimed to identify immunoglobulin heavy-chain genes (IGH) rod-like tracer as an effective biomarker for dynamic minimal residual disease (MRD) monitoring in children with pre-B-ALL. The V-(D)-J regions of immunoglobulin and T cell receptor of 47 pre-B-ALL samples were sequenced using the Illumina NovaSeq platform. The complementarity determining region 3 (CDR3) sequences (proportion > 5%) were identified, the IGH rod-like tracer consensus sequence was extracted based on its rod-like alpha-helices structural similarity predicted by AlphaFold2. Additional data from published 203 pre-B-ALL samples were applied for validation. NGS-IGH (+) patients with pre-B-ALL had a poor prognosis. Consistent CDR3 coded protein structures in NGS-IGH (+) samples could be extracted as a potential follow-up marker for children with pre-B-ALL during treatment. IGH rod-like tracer from quantitative immune repertoire sequencing may serve as a new class of biomarker with significant predictive values for the dynamic monitoring of MRD in children with pre-B-ALL.

Precursor B-cell acute lymphoblastic leukemia

Immunoglobulin and T-cell receptor gene rearrangements

Next-generation sequencing

Minimal residual disease

AlphaFold2

Pre-B-ALL is an aggressive immature B-cell neoplasm, and its outcomes have been substantially improved in pediatric and adult patients (1, 2). An important prognostic factor for B-ALL patients is the initial response to therapy, which is determined by the level of “measurable” (also referred to as “minimal”) residual disease (MRD) at the end of induction chemotherapy (2–4). Detection of MRD in pre-B-ALL patients helps to identify patients who may require treatment intensification (5). However, MRD assessment via flow cytometry can be challenging due to the difficulty in consistently interpreting complex multidimensional data, interfered with therapy-induced immunophenotypic drift and marrow regeneration. MRD assessment by quantitative polymerase chain reaction (qPCR) for tracking the patient-specific clonal rearrangements of the immunoglobulin B-cell receptor (BCR) and T-cell receptor (TCR) requires significant laboratory and institutional commitment to maintain the continuity and quality of testing. Recently, the next-generation sequencing (NGS) has been introduced to assess MRD in B-ALL patients (6). NGS universally amplifies antigen-receptor gene segments and identifies all clonal gene rearrangements at diagnosis, enabling the monitoring of disease progression and clonal evolution during therapy. Longitudinal BCR repertoire sequencing has also been used and revealed that the BCR underwent an unexpectedly high level of clonal diversification in B-ALL cells through both somatic hypermutation and secondary rearrangements (7). Furthermore, Gawad (8) and Bashford-Rogers (9) demonstrated massive clonal evolution in immunoglobulin heavy-chain genes (IGH). Clinical NGS has shown its superiority over flow cytometry and qPCR.

However, the immune repertoire of bone marrow samples from multiple recurrence However, the immune repertoire of bone marrow samples from multiple recurrence time points in children with pre-B-ALL has not yet been evaluated. Moreover, the state-of-the-art computational methods showed its potential to facilitate the evaluation of the clonal evolution of IGH. However, these methods are complicated and lack standardization (8, 9). Sequence variation resulting from the evolution of IGH clones and immunophenotypic drift make it more difficult to track abnormal B cells by flow cytometry, qPCR, or NGS as pre-B-ALL biomarkers. Consequently, there is an urgent need to identify valuable biomarkers from mass next-generation sequencing data for dynamic MRD monitoring in pre-B-ALL patients. In recent years, the predictive value of protein structure has been more accurate than the amino acid sequence in disease phenotyping (10). AlphaFold2 was a breakthrough in providing high-accuracy structural predictions, which facilitates unraveling previously unsolved questions from a brand-new structural perspective and as a next-generation biomarker. (11).

In this study, we used AlphaFold2 to generate protein structure predictions directly from NGS of bone marrow samples to characterize IGH, IGK, TRB, and TRG sequences during the treatment of children with pre-B-ALL. AlphaFold2 for the first was applied to extract the IGH rod-like tracer as a predictive biomarker of MRD in children with pre-B-ALL based on structural similarity analyses of IGH CDR3. We further investigated the dynamic MRD monitoring performance of IGH rod-like tracer in our sequencing data and published data, respectively.

Patient characteristics, treatment, and MRD sampling

The chemotherapy process was divided into the following phases: remission induction, early intensive treatment, consolidation treatment, delayed intensive treatment I, intermediate maintenance treatment, delayed intensive treatment II, and maintenance treatment. Patients were grouped according to the different risks. Multicolor flow cytometry (FCM)-MRD samples were obtained from bone marrow at different periods, and MRD (+) /MRD (-) change in bone marrow samples was also analyzed at each time point. All the sequenced 47 samples (13 patients) were from patients who received the "CCLG-2008" (12) chemotherapy regimen, including 13 pre-treatment, 13 post-treatment, and 21 samples with MRD (+) /MRD (-) change. Other IGH sequencing data from pre-B-ALL patients for validation were downloaded from the Sequence Read Archive (SRA) (SRA456729 and ERP115376), including 162 pre-treatment, 67 post-treatment, and 21 samples with MRD (+) /MRD (-) change during the chemotherapy process. The study was approved by the institutional ethics committee of our center, and written informed consent was obtained from the parents or guardians of each child (and assent from the patients as appropriate).

Detection of MRD with NGS

DNA from bone marrow samples was extracted using the QIAamp DNA Mini Kit (Qiagen) and quantified with the Qubit dsDNA HS analysis kit (Thermo Fisher Scientific) and Nanodrop 2000 (Thermo Fisher Scientific). According to the NGS of immunoglobulin (BCR) and T-cell receptor (TCR) gene recombination for MRD identification in acute lymphoblastic leukemia (13, 14), the IGH, IGK, TRB, and TRG VDJ regions were PCR amplified and sequenced on an Illumina NovaSeq system (PE250).

Detection of MRD using multicolor flow cytometry

MRD was detected using the 8-color FCM-MRD assay. Bone marrow samples were collected at different time points during the treatment. Approximately 106 nucleated cells were collected. BD FACSDivaTM software was used for analysis, FSC-A/FSC-H was used to set gates to remove adhesion cells, FSC-A/SSC-A was used to remove debris, SSC-A/CD45 was used to find lymphocytes at different stages, and CD45/CD19 was used to gate B lymphocytes. B cell markers (CD10/CD45, CD34/CD10, CD38/CD45, CD34/CD20 and CD20/CD10) were used to observe the differentiation and developmental trajectory of B cells. CD58/CD45 and CD13-33-15-117/CD45 were used to observe whether there were abnormal B cells (according to leukemia-associated immunophenotype [LAIP] and different from normal [DFN]).

Immune repertoire analysis

FLASH (15) was used to merge paired-end reads from NGS experiments. Seqkit (16) was used to convert FASTQ to FASTA. Gene quantification and BCR/TCR clonotype assignment were performed using MiXCR (17). All sequences we analyzed have excluded sequences with untranscribed/translated stop codons. The “immunarch” R package (https://github.com/immunomind/immunarch) was used for further analyses, including computing the number of clones or distributions of lengths and counts (repExplore), computing the clonality of repertoires (repClonality), computing the repertoire overlap (repOverlap), computing the distributions of V or J genes (geneUsage), and estimating the diversity of repertoires (repDiversity). Clonally related sequences were identified by sorting based on their complementarity determining region 3 (CDR3) nucleotide sequences. Group means were compared with the Wilcoxon rank-sum test if data were grouped. P-values were adjusted according to the Holm method.

CDR3 protein structure prediction and similarity comparison

AlphaFold2 (11) was used to predict the protein structure of CDR3 from the amino acid sequence of CDR3. Next, PyMOL (https://www.pymol.org) was used to calculate the structural similarity in NGS-IGH (+) and NGS-IGH (-) groups. The root mean squared deviations (RMSD) parameter was used to compare the similarity of protein tertiary structures. An RMSD of 0 between two structures suggested that they were the same and could be completely overlapped, and an RMSD (0–3) considered the two structures to be similar.

Statistical analyses

Progression-free survival (PFS) was defined as the time from the start of treatment until disease progression or death from any cause. Survival curves were calculated using Kaplan-Meier analysis. Receiver operating characteristic (ROC) curves were used to evaluate the performance of diagnostic tests and the accuracy of logistic regression that divided subjects into two categories. Continuous variables were described by the mean ± standard deviation (—x±s) (for normal distribution), or the median and interquartile range M (P25, P75) (for non-normal distribution). Moreover, the t-test or Mann-Whitney U test was used to identify statistically significant differences between two groups. The proportion of patients in each category was assessed using the chi-square test or Fisher’s exact test for categorical variables. All P values were 2-sided, with a significance level of 0.05. Statistical analyses were performed using SPSS version 22 (IBM Corp, Armonk, NY), GraphPad Prism 6 (La Jolla, CA), and R version 3.4.0 (Vienna, Austria).

IGH clonotypes were characteristically different from IGK, TRB, TRG

The principles of LAIP and DFN were used to distinguish abnormal tumor cells from normal differentiated cells by FCM (Fig. 1A). Multiplex PCR and NGS were used to amplify and sequence rearrangements within the IGH, IGK, TRB, and TRG V-(D)-J regions (Fig. 1B). Individual clonotypes with ≥ 5% frequency were designated disease clonotypes (8). Using this threshold, we identified 61 IGH disease clonotypes, 36 IGK disease clonotypes, 12 TRB disease clonotypes, and 2 TRG disease clonotypes in the bone marrow samples from children with pre-B-ALL (Supplemental Table 1). Compared to those of IGK, TRB, and TRG, IGH CDR3 may better reflect the abnormal immune cell status in children with pre-B-ALL. IGH rod-like tracer was defined as the class of disease IGH CDR3 coding domain with rod-like alpha-helices structure in NGS-IGH (+) samples. The consensus rod-like tracer was extracted from our IGH sequencing data, and alphafold2 predicted the structure. The length of most IGH clonotypes was greater than 20 amino acids (Fig. 1C). The length of clonotypes from other chains was mostly less than 20 amino acids long (medians: 10–15 amino acids) (Fig. 1D, 1E, 1F).

NGS-IGH (+) predicted a poor prognosis for patients with pre-B-ALL

We evaluated the prognostic value of NGS-IGH. Patients were dichotomized into two groups based on their NGS-IGH: NGS-IGH (+) group includes patients with the proportion of hyperexpanded clonotypes in the bone marrow samples greater than 50% at the primary diagnosis; and NGS-IGH (-) group includes those with the proportion of hyperexpanded clonotypes in the bone marrow samples less than 50% at the primary diagnosis. We found that NGS-IGH (-) patients had significantly improved survival rates than NGS-IGH (+) patients (P < 0.05) (Fig. 2A). We also used another dichotomization method (NGS-IGH > 5% or NGS-IGH < 5%). PFS showed no significant difference between patients with NGS-IGH > 5% and those with NGS-IGH < 5% (Fig. 2B).

Next, we investigated the diagnostic value of IGH diversity, the proportion of hyperexpanded IGH for disease risk classification, and the number of relapses. IGH diversity had great diagnostic value in disease risk classification (Fig. 2C) (AUC = 0.88, P < 0.05) but had no significant value in the number of relapses (Fig. 2D) (ROC area = 0.67, P > 0.05) of patients. The proportion of hyperexpanded IGH also had a great diagnostic value in disease risk classification (Fig. 2E) (ROC area = 0.86, P < 0.05) and the number of relapses (Fig. 2F) (ROC area = 0.70, P < 0.05) of patients. Moreover, the ratio of IGH/TRB clonotypes was associated with disease risk classification (P < 0.001) (Fig. 2G) and also had a great diagnostic value in disease risk classification (Fig. 2H) (ROC area = 0.80, P < 0.05). These results showed that NGS-IGH (+) patients with pre-B-ALL had a poor prognosis.

The CDR3 protein structures were consistent across NGS-IGH (+) samples

AlphaFold2 was used to predict the protein structure of CDR3. Figure 3A showed that the CDR3 structure with alpha helices in the NGS-IGH (+) group had 23 corresponding atoms between protein chains, with the value of RMSD being 1.181. The CDR3 structure with alpha helices in the NGS-IGH (-) sample only had three corresponding atoms between protein chains (Fig. 3B). In both NGS-IGH (+) and NGS-IGH (-) groups, the CDR3 with beta folds all showed dissimilar structures (RMSD = 4.179, 3.292) (Fig. 3C, Fig. 3D). In both NGS-IGH (+) and NGS-IGH (-) groups, the CDR3 with irregular curls only had three corresponding atoms between protein chains (Fig. 3E, Fig. 3F). The CDR3 structures in the NGS-IGH (+) group were more similar, while the CDR3 structures in the NGS-IGH (-) group were more diverse. On the other hand, multiple sequence alignment (MSA) showed that the different CDR3 sequences were not similar, while the protein structures of the NGS-IGH (+) group were highly similar (Fig. 3A). These results suggested that consistent CDR3 rod-like protein structures in NGS-IGH (+) samples could be used as a potential follow-up marker in pre-B-ALL patients during treatment.

The predictive value of IGH rod-like tracer identified from the structure of IGH CDR3 with alpha-helices

The CDR3 structure with alpha helices was similar across the NGS-IGH (+) group. According to the proportion of primary diagnosis samples in each patient, we attempted to evaluate the predictive value of molecular tracers identified from the structure of IGH CDR3 with alpha-helices. For example, in patients L and J, the top 10 CDR3s that dominated the primary diagnosis sample were not tracked in IGH (Fig. 4A, Fig. 4B,). The IGH CDR3 structures with alpha helices were similar at different time points (Fig. 4E, Fig. 4F) (RMSD = 0.036, 2.031), while the sequences at different time points were not similar (Fig. 4C, Fig. 4D). On the other hand, the top 10 CDR3s tracked their distribution at other time points in patients L and J (Fig. 4G, Fig. 4H). The IGH rod-like tracer can better reflect the dynamic changes of abnormal B cells in patients. These data suggested that IGH rod-like tracer provided excellent dynamic MRD monitoring performance in pre-B-ALL patients.

Evaluate the predictive value of IGH rod-like tracer in tracking and monitoring abnormal B cells in pre-B-ALL patients in publish IGH sequencing data

To further evaluate the predictive value of IGH rod-like tracer in tracking and monitoring abnormal B cells in pre-B-ALL patients, we downloaded the published IGH sequencing data of 152 pre-treatment and 57 post-treatment pre-B-ALL patients. In pre-treatment samples, 213 IGH CDR3 sequences were defined as disease clonotypes, in which 147 clonotypes were identified with alpha-helices structures. One hundred nineteen sequences (81%) had high similarity with the consensus IGH CDR3 structures. In post-treatment samples, only 33 IGH CDR3 sequences were defined as disease clonotypes, in which only 22 clonotypes were identified with alpha-helices structures, and 10 sequences (45%) had high similarity with the consensus IGH CDR3 structures. The consensus IGH rod-like tracer had great predictive value in tracking and monitoring abnormal B cells in pre-treatment pre-B-ALL patients. We constructed the IGH rod-like tracer website (https://ai-lab.bjrz.org.cn/IR) and uploaded IGH sequences and PDB files of the consensus IGH rod-like tracer identified from our sequencing data. This website can predict the three-dimensional structure of IGH CDR3 online and compare the similarity with the structure of the consensus IGH rod-like tracer. These results verified the dynamic MRD monitoring performance of IGH rod-like tracer in published data.

Relapsed samples had higher relative abundance, lower diversity of IGH, and the different V-J gene usage and related motifs

The samples were grouped according to the number of relapses, FCM-MRD status, gene mutation status, and disease risk level. Groups were divided as hyperexpanded (0.01 < X < = 1), large (0.001 < X < = 0.01), medium (1e-04 < X < = 0.001), and small (0 < X < = 1e-04) according to the percentage of relative abundance of IGH clonotypes. The samples of the 3rd relapse had the highest relative abundance, and the samples of the 1st relapse had the lowest relative abundance (P = 0.03) in the hyperexpanded group (Fig. 5A), indicating that hyperexpanded IGH clonotypes (0.01 < X < = 1) could reveal the number of relapses. There were no significant differences across different clonotypes in terms of FCM-MRD status, gene mutation status, and disease risk level (Fig. 5B, 5C, 5D). This result may be biased by the number of our patients. The diversity of IGH clonotypes, the different V-J gene usage and related motifs were also evaluated among the samples with different number of relapses (Supplement Fig. 1, Supplement Fig. 2).

The prognostic value of MRD during treatment has been demonstrated in numerous studies performed in patients with newly-diagnosed childhood and adult ALL (3, 18) or relapsed ALL (19–21), and in patients undergoing hematopoietic stem cell transplantation (22, 23). Despite these encouraging findings, MRD monitoring showed limited utility after induction in most studies, probably due to the sequence variations from the evolution of IGH clones and the immunophenotypic drift. Consequently, there is an urgent need to identify valuable predictive biomarkers from mass next-generation sequencing data for MRD monitoring. In our current study, the IGH rod-like tracer consensus sequence was extracted based on its rod-like alpha-helices structure predicted by AlphaFold2. Our findings underscored the prediction value of the novel biomarker-IGH rod-like tracer in children with pre-B-ALL, showing great potential for improving MRD monitoring.

Cross-lineage TCR gene rearrangements frequently occurred in immature B-cell malignancies, especially in pre-B-ALL (> 90% of cases) (14, 24). IGH, IGK, TRB, and TRG rearrangements were all detected in the study, and a few disease clonotypes were identified in IGK, TRB, and TRG. Focusing on the IGH clonotypes, we found that the proportion of hyperexpanded IGH could better reflect the abnormal immune cell status in children with pre-B-ALL. Since the immune repertoire of pre-B-ALL patients with multiple relapses has not been evaluated before, we focused on analyzing clonotypes among multiple relapses samples.

To further analyze the diagnostic and prognostic value of NGS-IGH in children with pre-B-ALL, we found that NGS-IGH (-) patients had a better prognosis, as seen in patients with chronic lymphocytic leukemia (25). Hyperexpanded IGH clonotypes and diversity of IGH clonotypes showed good prediction for the number of relapses and disease risk levels in children with pre-B-ALL. The ratio of IGH/TRB clonotypes also showed good prediction for disease risk level. In addition, to further elucidate the pathogenic mechanism of IGH clonotypes in the NGS-IGH (+) group, we used AlphaFold2 to predict the tertiary structures of the disease IGH clonotypes and used RMSD to analyze the similarity between NGS-IGH (+) and NGS-IGH (-) groups. To our knowledge, only one article had evaluated the CDR3 structure in B-ALL. Zha et al. (26) found that the TRB CDR3 regions contained a conserved amino acid motif with different spatial conformations in B-ALL. We predicted the high accurate structure of IGH CDR3 protein based on AlphaFold2 for the first time and found that deep learning approaches are valuable for advancing MRD detection. The structure of IGH-CDR3 was more similar within the NGS-IGH (+) group than that within the NGS-IGH (-) group, which may play the same role in tumorigenesis of pre-B-ALL; in addition, the protein structures could better reflect the role of abnormal B cells in disease compared to the amino acid sequences. Therefore, IGH rod-like tracer was defined as the class of disease IGH CDR3 coding domain with rod-like alpha-helices structure in IGH-NGS (+) samples.

We further evaluated the predictive value of the IGH rod-like tracer in the NGS-IGH (+) group. In patients for whom clonal evolution cannot be dynamically monitored by flow cytometry, qPCR, or NGS, IGH rod-like tracer in samples collected at different time points helped to distinguish abnormal B cells from the same source and was validated in published IGH sequencing data in pre-treatment pre-B-ALL patients. Thus, this study provided a new method to track pre-B-ALL patients at the molecular level. IGH CDR3 structures can be predicted and identified as IGH rod tracers by visiting the IGH rod-like tracer website (https://ai-lab.bjrz.org.cn/IR).

Furthermore, we found that the 3rd relapse sample had the highest relative abundance and the lowest diversity. The V-J gene usage and related motifs in the 3rd relapse samples differed from the 1st and 2nd relapse samples. The high-frequency use of IGHJ6 and IGHV4-34 genes was considered self-reactive (27–32), and the 9G4 antibody encoded by IGHV4-34 could bind to self-antigens and cause damage (31). GMDVW had similar motifs to ZNF24, TBX20, DBD1, and TBX20. YGMDV had similar motifs to ZNF410, ZNF410 DBD, and GMEB2 DBD3, indicating the motifs of IGH may promote tumorigenesis(33, 34). In our pre-B-ALL samples, the biased V-J gene usage and the presence of a shared antigen-binding motif in the CDR3 region of tumor B cells demonstrated a significant antigen-driven process. Precursor B-cell receptor (pre-BCR) signaling and spleen tyrosine kinase (SYK) have recently been introduced as therapeutic targets for pre-B-ALL (35). These results suggested that the influence of antigen-stimulated self or foreign antigens on BCR may play a key role in disease progression and the initiation of B cell malignant transformation.

In summary, we for the first time performed immune repertoire sequencing on bone marrow samples from children with pre-B-ALL with multiple recurrences, followed by AlphaFold2 structural similarity analyses of IGH CDR3 coding region. The IGH rod-like tracer was then extracted from mass quantitative immune repertoire sequencing data with great predictive values as a novel biomarker for the dynamic monitoring of MRD in children with pre-B-ALL. The high-accuracy protein structural predictions by AlphaFold2 will greatly facilitate further clinical interpretations of the mass NGS data, and AlphaFold2 itself will also be a powerful tool to identify robust biomarkers in clinical diagnosis and monitoring in future studies.

Funding

This work was supported by CAMS Innovation Fund for Medical Sciences (2021-I2M-1-050), the National Key Research and Development Program of China (2020YFC2009006), National Natural Science Foundation of China (Grants 81870048 & 81902618), Beijing Hospital Nova Project (No.BJ-2020-083) and Beijing Hospital Project (NO. BJ-2019-153), Fundamental Research Funds for the Central Universities (Grant 3332020023), Beijing Hope Run Special Fund of Cancer Foundation of China (LC2019A04).

Data availability

The sequencing data has been submitted to the Genome Sequence Archive and is available under the accession number HRA003517.

Author contributions

ZZL and WQC analyzed the data and wrote the main text of the manuscript. LC, ZLL, ZLX, YR, GY, HY analyzed the data. MLZ, BJF, ZCL, FR, CM and SF prepared figures. QCX and FX supervised this work. All authors reviewed and approved the submitted manuscript.

Competing interests

The authors declare no competing interests.

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IGH rod-like tracer: An AlphaFold2 structural similarity extraction-based predictive biomarker for minimal residual disease monitoring in children with precursor B-cell acute lymphoblastic leukemia

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Abstract

Figures

Introduction

Materials And Methods

Patient characteristics, treatment, and MRD sampling

Detection of MRD with NGS

Detection of MRD using multicolor flow cytometry

Immune repertoire analysis

CDR3 protein structure prediction and similarity comparison

Statistical analyses

Results

IGH clonotypes were characteristically different from IGK, TRB, TRG

NGS-IGH (+) predicted a poor prognosis for patients with pre-B-ALL

The CDR3 protein structures were consistent across NGS-IGH (+) samples

Discussion

Declarations

Funding

Data availability

Author contributions

Competing interests

References

Additional Declarations

Supplementary Files

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