An Angioimmunoblastic T-cell Lymphoma Patient Presented as Persistent Hypereosinophilia With a Novel RHOA Mutation Encoding p. Ala161Glu

doi:10.21203/rs.3.rs-1444059/v1

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An Angioimmunoblastic T-cell Lymphoma Patient Presented as Persistent Hypereosinophilia With a Novel RHOA Mutation Encoding p. Ala161Glu

https://doi.org/10.21203/rs.3.rs-1444059/v1

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Angioimmunoblastic T cell lymphoma (AITL) is a unique subtype of peripheral T cell lymphoma (PTCL). Studies have shown that RHOA p. Gly17Val mutations contribute to the specific pathogenesis of AITL. Here we reported a case of AITL with a novel RHOA mutation encoding p. Ala161Glu. This case presented with persistent hypereosinophilia for more than 3 years prior to diagnosis. At the time of diagnosis, the patient manifested with B symptoms, multiple lymphadenopathies, liver and spleen involvement. A retrospective analysis of whole exome sequencing and digital PCR of previous pathological specimens at different periods showed that RHOA p. Ala161Glu mutation and TP53 mutation occurred simultaneously. RHOA p. Ala161Glu mutation was found to be less frequent or undetectable in early gastrointestinal tract and lymph nodes. In the intestine, lymph nodes and other involved tissues, it increased with the progression of the disease. Notably, it was significantly elevated at the time of diagnosis. We presumed that the RHOA p. Ala161Glu mutation might be related to AITL pathogenesis. However, it needs further study to identify the specificity of RHOA p. Ala161Glu mutation in AITL.

Angioimmunoblastic T cell lymphoma

RHOA mutation

p. Ala161Glu

Pathogenesis

Angioimmunoblastic T-cell lymphoma (AITL) is a unique form of peripheral T cell lymphoma (PTCL), accounting for 18.5% of all PTCLs[1]. According to the WHO classification revised in 2016, AITL belongs to the nodular PTCL with a T follicular helper (Tfh) cell phenotype[2]. AITL is characterized as B-symptoms (fevers, unintentional weight loss, and/or night sweats), generalized lymphadenopathy and autoimmune-like manifestations[3]. Most AITLs are in advanced stages (III/IV) at the time of diagnosis, plausibly leading to adverse prognosis due to atypical clinical and laboratory results [4]. Hypereosinophilia is seen in one-third to one-half of the AITL cases and causes great difficulty in making diagnosis[5, 6]. In the past few decades, AITL's diagnostic capabilities have been continuously improved with the development of next-generation sequencing technologies and the development of AITL molecular markers. AITL genomic abnormalities are highly disease-specific and the ras homology family member A (RHOA) mutation is one of the most frequent recurrent mutations[7]. RHOA mutations in AITL invariably encode p.Gly17Val[8]. In this study, we identified a novel RHOA mutation encoding P. Ala161Glu in a patient with AITL.

A 57-year-old female was admitted to hospital on 6 April 2020 with hypereosinophilia for 3 years and abdominal discomfort for 8 months. Three years earlier, she had been treated at another hospital with imatinib plus prednisone for idiopathic eosinophilic hyperplasia. The regimen was initially effective but the eosinophil counts rebounded as prednisone was stopped. Physical examination showed that the upper abdomen was slightly tender on palpation. No skin lesions were observed. Laboratory data showed that eosinophil count was 33.67×10⁹/L. Lactate dehydrogenase (LDH) was slightly elevated (356U/L, literature upper limit 250). Serum total immunoglobulin E (IgE) was elevated (315.149 KIU/L, reference upper limit 100). Hepatitis B Virus (HBV) testing showed that HBsAg, anti-HBe and anti-HBc were all positive but no HBV-DNA was detected. EBV-DNA was also undetectable. Repeated examinations of stool specimens for ova and parasites were negative. Serum allergens and parasites were negative. Lung radio-graphs was normal. Bone marrow examination showed significantly iincreased eosinophils (Fig. 1A, B, C, D). The immunoglobulin heavy chain (IGH) and the T-cell receptor (TCR) gene rearrangements were negative based on PCR analysis of the bone marrow. Gene detection by Next Generation Sequencing (NGS) in bone marrow indicated that JAK2-V617F, platelet derived growth factor receptor alpha (PDGFRα), platelet derived growth factor receptor alpha belta (PDGFRβ) and fibroblast growth factor receptor 1 (FGFR1) were all negative. Cytogenetics showed an abnormal karyotype with 45, XX, -13 (1) [Fig. 2A]/45, XX, -16 (1) [Fig. 2B] /46, XX [Fig. 2C]. Positron-emission tomography with ¹⁸F-fluorodeoxyglucose (FDG-PET) was performed. As shown in Fig. 3A, the FDG-PET on Apr 9, 2020 showed positive FDG uptake in multiple enlarged lymph nodes throughout abdominal cavity and in the duodenum, terminal ileum, and colon. Two gastrointestinal specimens on Apr 12, 2020 and May 3, 2020 showed eosinophils invasion (Fig. 4). We also performed laparoscopic lymphadenectomy on Apr 17, 2020 but it didn’t show any diagnostic significance. Ultimately, the diagnosis of idiopathic eosinophilia was made and imatinib combined with prednisone regimen was restarted.

The patient was readmitted 3 months later with obvious abdominal pain and abdominal distension. At the same time, she complained of night sweats and apparent weight loss (5kg). Laboratory data revealed that the leukocyte count was 1.9×10⁹/L and an eosinophil of 1.8%. LDH was 546U/L and total serum IgE was 134.06 KIU/L. EBV-DNA was 8.41×10³IU/ml (upper limit of reference 1×10³). Liver function showed Albumin (ALB), 28.9g/L; Alanine aminotransferase (ALT), 254 (upper limit 50) U/L; Asparagus aminotransferase (AST), 122 (upper limit 40) U/L; γ-glutamyl transferase (GGT), 310 (reference upper limit 60) U/L; Alkaline phosphatase (ALP), 156 (upper limit of reference 125) U/Land Direct bilirubin (D-Bil), 11(upper limit 5)µmol/L. The Ultrasound and CT both demonstrated diffuse changes of the liver and multiple enlarged lymph nodes in the retroperitoneum, mesenteric, pelvic cavity and groin. The FDG-PET on July 30, 2020[Fig. 3B] showed multiple enlarged lymph nodes in the body with increased metabolism, enlarged spleen with increased metabolism and segmental thickening of the intestinal wall of the gastric antrum, duodenum, colon and rectum with increased FDG metabolism. Compared with previous CT images, lymph node and spleen lesions progressed, with newly emerged pathological changes of gastric antrum. Retroperitoneal lymph node puncture guided by ultrasound was performed on Aug 6, 2020. The sample of the retroperitoneal lymph node showed that the neoplastic T cells with intermediate-sized nuclei, copious pale/clear cytoplasm and abundant clear cytoplasm [Fig. 5A]. Immunohistochemistry showed that the atypical largel lyphoid cells were CD3+[Fig. 5B], CD4+ [Fig. 5C], CD5+, CD8-, ICOS+ [Fig. 5D], Bcl-6+ [Fig. 5E], PD-1+ [Fig. 5F], Bcl-2+, Ki-67+ (70~80%), CD56-, TIA-1-, CD20-, CD79a-, CD10-; immunoblasts CD30+; follicular dendritic cells CD21+ [Fig. 5G] and scattered lymphocytes EBER+ [Fig 5H]. TCR gene rearrangements were positive [Table 1]. The patient was eventually diagnosed as AITL. After diagnosis, whole exome sequencing was conducted on the lymphoma specimens, followed by Digital PCR analysis on all the specimens before diagnosis involving the bone marrow tissues, two gastrointestinal and abdominal lymph node ones. Two mutations were identified, RHOA (c.482C>A; p. Ala161Glu) and TP53 (c.298C>T; p.Q100X). As shown in Fig.6A and Fig.6B, RHOA and TP53 gene mutations were 0.015% [Fig. 6A(a)] and 4.127% [Fig. 6A(b)] in the gastric specimens on Apr 12, 2020[Fig.6B]. They were equally <0.001 [Fig. 6A(c,d)] in the intestinal specimens on Apr 12, 2020 and increased at a frequency of 0.023% [Fig. 6A(e)] and 1.198% [Fig. 6A(f)] on May 3, 2020[Fig.6B]. They were 1.299% [Fig. 5A(g)] and 3.303% [Fig. 6A(h)] in the abdominal lymph node specimens on Apr 17, 2020 [Fig. 6B] and were elevated by 16.00% and 20.60% respectively on Aug 6, 2020 [Fig. 6B]. Unfortunately, due to tissue problems, we were unable to detect these two mutations in the bone marrow regimens and the gastric ones on May 3, 2020.

As for treatment, anti-CD30 monoclonal antibody (Brentuximab vedotin) combined with CHP chemotherapy (cyclophosphamide 750 mg/m², pegylated liposomal doxorubicin 40mg/m², and prednisone 100 mg) was given for seven cycles, followed by autologous stem cell transplantation (ASCT). However, relapse occurred two months later. Then, targeted therapy including Chidamide, Selinexor and Decitabine was tried, but all failed. Now she is undergoing CD30-directed Chimeric Antigen Receptor T(CAR-T) cells therapy at another hospital.

Studies have shown that RHOA mutations exhibit different patterns depending on tumor types. Arg5 and Tyr42 are predominant mutations in Burkitt lymphoma[9] and gastric adenocarcinoma[10–12], while adult T-cell leukemia/lymphoma (ATLL) has two types of RHOA mutations, loss-of-function (Gly17Val and Ala161Glu) and gain of function (Cys16Arg, Cys16Phe, Lys118Glu, Ala161Val, and Ala161Pro) mutations[13]. In AITL, the type of RHOA mutation has been reported to be Gly17Val[14, 15]. The Gly17Val mutant protein could enhance both TCR and inducible T-cell co-stimulator (ICOS) signaling pathways to specify Tfh lineage and maintain Tfh phenotype[8]. RHOA p. Gly17Val mutations were discovered in nearly 70% of AITLs[14, 16–18] and a smaller proportion of PTCL-NOS[14, 16]. Gly17Val mutants were found in Tfh-derived neoplastic cells rather than other cells[16, 19, 20]. Studies have shown that RHOA p. Gly17Val mutations contribute to AITL-specific pathogenesis[19]. Hence, the functional consequences of RHOA mutation in tumor development may depend on the tissue and cell from which it originates.

In this AITL patient, we identified a novel RHOA p. Ala161Glu mutation. As shown in Fig. 6, the frequencies of this mutation are low or undetectable in the earlier tissues including the gastrointestinal tract and lymph node. However, in the same tissues such as intestine and lymph nodes, they showed a tendency to increase as the disease progresses. Notably, the frequency of mutations at the time of diagnosis was significantly higher, approaching 20%. It is suggested that the mutation of RHOA p. Ala161Glu may be associated with the pathogenesis of AITL. This patient presented with persistent hypereosinophilia, causing a great difficulty in the diagnosis of AITL. In fact, hypereosinophilia is present in 1/3 to 1/2 of AITL cases[5, 8]. Therefore, the clinical characteristics of the specific RHOA mutation in this case do not appear to differ significantly from those in AITL with Gly17Val mutated RHOA or wild-type RHOA. The p. Ala161Glu mutation has been identified in 1 case of PTCL-NOS[16] and 3 cases of adult T-cell leukemia/lymphoma (ATLL)[13]. Up to date, few studies have been conducted on this kind of RHOA mutation. It is speculated that Ala161Glu mutants have no ability to bind GTP and inhibit the binding of wild-type RHOA proteins (e.g., Gly17Val) to GTP[13, 16]. The clear molecular mechanism for the the mutation of RHOA P. Ala161Glu, a new AITL tumor type, needs to be further studied.

In addition to RHOA mutations, the most commonly mutated genes in AITL involve epigenetic pathways [tet methylcytosine dioxygenase 2 (TET2), DNA methyltransferase 3A (DNMT3A) and isocitrate dehydrogenase 2 (IDH2), TCR and costimulatory signaling pathways[21–25]. Unlike RHOA mutations, this mutation may be present in both tumor and non-tumor cells of patients with AITL[19]. It has been reported that RHOA P. Gly17Val mutations in AITL are often co-occurring with TET2 mutations, and usually with DNMT3A mutations[16]. The coexistence of these mutations may play a synergistic role in the pathogenesis of AITL[16]. Nonetheless, the TP53 mutations in this case occurred simultaneously with RHOA mutations. TP53 mutations are considered to be a disadvantage in several cancer sub-types. A few studies suggest that it tends to be a risk factor for PTCL[26, 27]. The poor prognosis in our case may be related to TP53 mutation.

In summary, our findings introduce a new RHOA mutation in a patient with AITL. The role of the RHOA p. Ala161Glu mutation in AITL development needs to be further explored.

Ethical approval and consent to participate

The study was approved by the Ethics Committee of Shulan （Hangzhou）Hospital Affiliated to Zhejiang Shuren University Shulan International Medical College. Informed written consent was obtained from the patient for the studies conducted and for publication of any potentially identifiable images or data included in this article.

Consent for publication

All authors of the manuscript have read and agreed to its content for publication.

Availability of data and materials

The single institute data from this study is available from the corresponding author upon reasonable request.

Conflict of interest

All authors declare no conflict of interest.

Funding

This work was funded by the Program for the 2021 Zhejiang Provincial Traditional Chinese Medicine Scientific Research Fund Project (Grant number: 2021ZB248) .

Authors’ contributions

LHC and HYT conceived the study, analyzed data, and drafted the manuscript. XL, FY, ZMW and YQX prepared figures 1-6 and table 1. JJ reviewed the manuscript. The remaining authors participated in the providing clinical materials and some ideas.

Acknowledgments

Not applicable.

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Table. 1 TCR gene rearrangements showed positive.

Gene	Site	Result
TCRB gene rearrangements	Tube A(V 𝛃+J 𝛃 1/2)	（+）
	Tube B(V 𝛃+J 𝛃 2)	（-）
	Tube C(D 𝛃+J 𝛃 1/2)	（+）
TCRG gene rearrangements	Tube A（V 𝛄 1-8、V 𝛄 10, +multiple J 𝛄 regions）	（+）
TCRG gene rearrangements	Tube B（V 𝛄 9、V 𝛄 11, +multiple J 𝛄 regions）	（-）
TCRD gene rearrangements	V 𝛅+D 𝛅+J 𝛅	（-）

No competing interests reported.

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An Angioimmunoblastic T-cell Lymphoma Patient Presented as Persistent Hypereosinophilia With a Novel RHOA Mutation Encoding p. Ala161Glu

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