Genomic Mutation Landscape of Primary Breast Lymphoma: Next-Generation Sequencing Analysis

Primary breast lymphoma (PBL) is a rare subtype of non-Hodgkin's lymphoma (NHL) with rapid progression and high risk of central nervous system metastasis. We have investigated 40 PBL patients retrospectively, and 16 of them were sequenced by a target panel of 112 genes related with lymphoma. Next-generation sequencing (NGS) identified 203 mutations spanning 35 genes and revealed seven potential protein-changing genes (PIM1, MYD88, DTX1, CD79B, KMT2D, TNFAIP3, and ITPKB) with high frequency, referring crucial roles in lymphomagenesis. Our result suggested that PIM1 mutation is correlated with the age and pathological type of PBL patients. Gene TNFAIP3 and KMT2D mutation is only related to the pathological type and primary site, respectively. These high-mutant genes detected in PBL indicated a tendency to shorten overall survival (OS) and progression-free survival (PFS), which may lead to poor prognosis. Furthermore, the nuclear factor kappa-B (NF-κB) pathway and related regulatory factors are essential for the development of targeted therapy as well.


Introduction
Primary breast lymphoma (PBL) is defined as an extranodal lymphoma which presents as primary lesion within the breast without involvement of extramammary sites. It possesses an extremely rare incidence with rapid progression, poor prognosis, and high risk of central nervous system (CNS) relapse. The most common symptom is painless progressive breast mass with or without ipsilateral axillary fossa lymphadenectasis and usually involves the right breast for unknown reason. Histopathologic subtypes of PBL are variable including mucosa-associated lymphoid tissue (MALT) lymphoma, natural killer (NK)/T cell lymphoma, and T lymphocyte lymphoma, but diffuse large B-cell lymphoma (DLBCL) occurs in most cases [1][2][3][4][5]. At present, the most commonly applied chemotherapy regimen is rituximab combined with CHOP (cyclophosphamide, vincristine, doxorubicin, and prednisone) or CHOP-like treatment with-out any standard regimen. Prior studies suggest that the 5year overall survival (OS) rate of PBL may reach 87% in the rituximab era.
However, due to the rarity of PBL, its predictive mutational signature and genetic features remain poorly understood. Therefore, efforts are required to explore better prognosis prediction and improve the cure rate of this invasive disease in today's precision medicine era. The advancement of gene profiling technologies such as next-generation sequence (NGS) has significantly contributed to prognosis and therapy guidance for lymphomas. For example, CD47 has become a novel therapeutic strategy for DLBCL according to previous works [6]. Recently, the role of the signal transduction pathway in tumor received particular interest. It is discovered that no matter what the pathological type of lymphoma is, the genetic changes in the nuclear factor kappa-B (NF-κB) pathway are the most consequential and closely associate with prognosis by enrichment analysis of mutant genes. Here, we conducted the next-generation sequencer with high accuracy for screening genetic mutations through pathological tissues of 16 cases. Aimed to lay foundations for accurate diagnosis and applicable treatment methods, we have analyzed the correlation between gene mutation spectrum and clinical characteristics of PBL.

Experimental Section
2.1. Patients. Our investigation cohort collected 40 patients who were diagnosed with primary breast lymphoma and treated in the Fourth Hospital of Hebei Medical University from June 2009 to October 2019. Their diagnosis and histopathologic subtypes were determined on tumor tissues through standard immunohistochemistry procedures. Meanwhile, pathology results were observed and analyzed independently by more than 2 experienced pathologists. The study was approved by the ethics committee of the fourth hospital of Hebei Medical University. Because of the rare incidence of this disease, we are trying to evaluate as much patients that we selected as possible. The baseline clinical characteristics of 40 patients were summarized in Table 1. Detailed description will focus on 16 patients who underwent NGS, including treatment, surgery approaches, and outcomes ( Table 2). The basis of screening for sequencing was mainly dependent on patients' will. Collectively, 16 patients were female with a median age of 59 years (range: 28-71 years). There were 7 patients aged ≥60 years (43.75%) and 9 patients aged <60 years (56.25%). Regarding the international prognostic index (IPI), 9 patients (56.25%) had a low-risk (0-1) score, 4 patients (25%) had a mediumrisk (2-3) IPI score, and 3 patients (18.75%) had a high-risk (4-5) score. Only 2 of them (12.5%) presented with B symptoms, 4 patients (25%) showed increasing lactate dehydrogenase (LDH) (>250 μ/l), and 3 patients (18.75%) had an elevated β 2 microglobulin level (>2.7 μg/ml). The pathological types were DLBCL in 14 cases (87.5%) and MALT lymphoma in 2 cases (12.5%) and 8 patients with Ki-67 index ≥ 70% among them (54.69%). All patients were receiving surgical treatment in the breast surgery department before chemotherapy. As for surgical approaches, modified radical mastectomy was conducted in 3 (18.75%) patients, total breast resection in 3 (18.75%) patients, and partial breast resection in 4 (25%) patients. Tumor resection was performed in 4 (25%) patients, and in 2 patients (12.5%), only tumor biopsy was performed. PBL is an aggressive subtype of non-Hodgkin lymphoma (NHL) with high risk of CNS metastasis. There were only 3 patients that presented with CNS metastasis and 10 patients with rapid progression.
A total of 15 patients underwent treatment, 12 (75%) only accepted chemotherapy or immunotherapy, 3 (21.43%) received radiotherapy combined with chemotherapy, and 3 (21.43%) combined intrathecal injection for CNS prevention. Just one patient received a novel option involving autologous stem cell transplantation (ASCT) and chimeric antigen receptor T cell therapy (CART) with radiotherapy plus chemotherapy.
2.2. Tissue DNA and NGS Library Preparation. The lymphoma plasma panel (Burning Rock Biotech, Guangzhou, People's Republic of China) is targeting 112 genes and spanning 314 K of human genomic regions, which strongly associated with the pathogenesis of lymphoma as well as    No difference was observed in both OS and PFS among the following clinical variables when analyzed individually: age, presence of B symptoms, IPI risk group, elevated LDH, and β 2 microglobulin level (P > 0:05). There was no significant difference in statistics but inferred a trend of shorter OS and PFS, which possibly led to inferior prognosis. Patients received different treatment regimens like chemotherapy combined with radiotherapy, applying rituximab with CHOP or CHOP like immune-chemotherapy, or accepting intrathecal injection to prevent central infiltration, etc. Different regimen presents no significant impact on further prognosis and survival.(P > 0:05).

Gene
Numbers of patients Numbers of mutation    Disease Markers variable shear mutation in 4 patients, which is only associated with the pathological type of PBL (P = 0:006). The median OS of TNFAIP3 mutant and nonmutant patients were 21 and 23 months, respectively. Five patients presented KMT2D gene mutation with 2 nonsense mutations, 2 frameshift mutations, and 1 shear region mutation. It showed the relationship with the primary site (P = 0:004). Additionally, we pay more attention to MYD88 and CD79B mutation in PBL. In our group, there are 9 patients with MYD88 mutation and 5 patients with CD79B mutation and 5 of them have double mutation. These mutations did not make any significant difference on the survival and other clinical features (including the primary site, Ann Arbor stage, IPI score, LDH and β 2 micro-globulin levels, bone marrow infiltration, B symptoms, and Ki-67); there was no significant correlation (P > 0:05).

Discussion
In recent years, a lot work focusing on the signal transduction pathway and gene alternation in tumor researches captured much attention. With the advancements of NGS technology, the molecular landscape of PBL could be profiled in order to explore the standard treatment scheme and realize individualized precision treatment for our patients. Due to the low incidence rate, its clinical characteristic and genetic features required further investigation. We detected biopsy specimens of 16 PBL patients by NGS technology, and biological effects of related genes, pathogenesis, and prognosis were also analyzed. There were 7 high-mutation frequent genes among 112 gene panels including PIM1, MYD88, DTX1, CD79B, KMT2D, TNFAIP3, and ITPKB.
PIM1 is a serine/threonine kinase acting as the most common mutant gene in PBL involving a series of biological functions like survival, proliferation, and differentiation. It has universally acknowledged the critical role of PIM1 in the occurrence and development of hematological malignancies and identified as the target of abnormal somatic hypermutation in DLBCL [7][8][9]. PIM family kinases were activated by JAK-STAT signaling pathways and downstream of NF-κB transcription factors, inducing and regulating protein activity to promote tumorigenesis. Plenty of studies claimed that the PIM kinase inhibitor has become a promising candidate of highly specific and selective drugs with superior toxicity characteristics [10,11].
Genes MYD88 L265P and CD79B were essential in PBL lymphomagenesis and were frequently detected in PBL. There were totally 9 patients that harbored MYD88 L265P mutation; it was functional gain-driven mutation and located in the MYD88 Toll-like/interleukin-(IL-) 1 receptor domain, which is related with unfavourable prognosis among aggressive lymphoma. L265P mutants promote cell survival by spontaneously assembling protein complexes containing interleukin-1 receptor-associated kinase 1   M   PIMI  IGHJ  ITPKB  MYD88  TNFAIP3  CD79B  IRF4  KIR3DL 2  DTX1  CD58  KMT2D  CREBBP  DNMT3A  CDKN2A  TET2  BCL6  TP53  PIK3CA  PRDMI  EP300  PTEN  DD3X3  CIITA  BCOR  ALK  SF3B1  BCORLI  ARIDIB  ID3  CCND3  CARD11  KLHL6  TSC2  MYC  IGHD   0 6 Disease Markers (IRAK1) and interleukin-1 receptor-associated kinase 4 (IRAK4) [12]. It may lead the activation of IRAK4, NF-κB signaling, JAK kinase in signal transducer and activator of transcription 3 (STAT3), IRAK phosphorylation, and secretion of IL-6, IL-10, and interferon β (IFNβ). Zhang et al. hold the view that downregulation of IRAK4 and NF-κB may cause tumor matrix disorganization through investigation. Consequently, the signal complex coordinated by MYD88 and L265P is an appealing target and the IRAK4 kinase inhibitor is also expected to become a therapeutic approach for malignancies with MYD88 mutation. The CD79B gene, encoding a subunit of the B-cell antigen receptor, contains the domain of immune receptor tyrosine activation motif (ITAM) which plays a critical role in signal transduction from the B-cell receptor (BCR). The presence of CD79B mutations has always been recognized in the first tyrosine (Y196) of ITAM [13,14]. Previous findings described the upregulation of BCR on the cell surface resulting in NF-κB signaling activation via tyrosine protein kinase (LYN), tyrosine kinase in the spleen (SYK), and the tyrosine kinase of Bruton (BTK), which inhibits B cell apoptosis and promotes proliferation in lymphoma cells [15,16]. In B cells, NF-κB signaling was initially transferred by CD79A (Ig-α) and CD79B (Ig-β). Meanwhile, the phosphorylated ITAM mediated with SYK activation triggered signal cascade reaction involving BTK, PLCγ, and PKCβ. Then, the "CBM complex" was formed and activated direct phosphorylation of Iκ-B kinase (IKK) to start transduction [14]. Genes MYD88, PIM1, and CD79B were connected and interacted with each other according to the NF-κB pathway as the primary target signal node. TNFAIP3, also known as A20, represents a tumor suppressor gene in lymphomas that synergistically attenuates NF-κB signaling induced by tumor necrosis factor (TNF) and TLR signal transduction. Alternations including deletion, promoter methylation, point mutation, and frameshift mutation lead to inactivation of RIP protein ubiquitination mediated by TNF [17,18]. Meanwhile, epigenetic gene like KMT2D has stimulation on the methylation of H3K4 and modulates genes related with B cell differentiation containing CD40, JAK-STAT, and TLR. Previously published studies pointed out that the deficiency and mutations in KMT2D could hinder B cell   PIM1  IGHJ  MYD88  ITPKB  TNFAIP3  KMT2D  CD79B  CD58  DTX1  IRF4  TP53  EP300  BCL6  CDKN2A  CREBBP  PIK3CA  IGHD  IGHD  TSC2  KLHL6  CARD11  CCND3  ID3  ARID1B  BCORL1  SF3B1  ALK  BCOR  CIITA  DD3X3  PTEN  PRDM1  TET2  DNMT3A  KIR3DL2 [19,20]. Numerous works confirmed that calcium reaction was a key factor during cancer invasion and metastasis. ITPKB becomes a target during the process by encoding isoenzymes of inositol 1,4,5-triphosphate 3kinase (Ins (1,4,5) p3) in B cells.
The NF-κB pathway modulated cell proliferation and angiogenesis by transcriptional regulation and inhibiting apoptotic genes resulting in tumorigenesis. The signaling could be stimulated according to activation mutations in CD79B and MYD88 and inactivation mutations in TNFAIP3. Shorter survival was observed in patients mutated with PIM1, MYD88, CD79B, TNFAIP3, DTX1, KMT2D, and ITPKB, consisting with other literatures, but there was no statistically significant correlation between alternations and prognosis. However, the statistical results of such small cohort may not be representative. Considering the rare incidence of PBL and limited sample size, further study could be verified in a large cohort trail.

Conclusion
In summary, the NGS analysis for 16 PBL patients among 35 hotspot genes provides an implication that PIM1, MYD88, DTX1, CD79B, KMT2D, TNFAIP, and ITPKB are possibly consistent with the trend for shorter survival and poor prognosis as high-frequency mutant genes. Nevertheless, we found that patients diagnosed with marrow infiltration and stage IV are correlated with inferior overall survival. Other clinical characteristics and therapy approaches make no significant differences on survival status.

Data Availability
Our NGS results were processed by Burning Rock Biotech. We appreciated all the patients, families, and investigators that participated in our research.