Co-shared genomic alterations within tumors from patients with both myeloproliferative neoplasms and lymphoma

Myeloproliferative neoplasms (MPN) and lymphomas are generally considered to be distinct malignant diseases. MPN are clonal hematopoietic stem cell disorders characterized by proliferation of one or more of the myeloid-derived cell lineages. Lymphomas comprise a wide range of B- or T/ NK-cell-derived malignancies.^1,2 However, in patients diagnosed with both malignancies, some lymphoid entities are overrepresented.^3,4 In a nationwide cohort of patients presenting with both MPN and lymphoma, we observed that the occurrence of T-follicular helper cell lymphoma, angioimmunoblastic type (AITL), was five to seven times higher than in the general population.⁵ This, together with observations of similarities at genomic and proteomic level in patients with concurrent MPN and lymphoma,^6,7 has contributed to the hypothesis that MPN and some lymphoma subtypes may share common pathogenetic steps. To further explore this hypothesis, we investigated the mutational landscape in archival tumor samples from patients with both diagnoses.

We performed whole exome sequencing (WES) of paired bone marrow (BM) and lymphoma tissue samples from patients diagnosed, either simultaneously or metachronously, with both MPN and lymphoma, of either AITL or diffuse large B-cell lymphoma (DLBCL) type. To our knowledge, the present study is the first to report the occurrence of shared genomic alterations within the disease-specific tumor samples from patients diagnosed with MPN and either AITL or DLBCL.

A Danish cohort of patients diagnosed with both MPN and lymphoma between 1990-2015 was previously described.⁵ Diagnostic tumor samples from 14 patients with MPN, diagnosed prior or synchronous to either AITL (N=5) or DLBCL (N=9), were identified through the Danish National Pathology Registry (DNPR).⁸ We excluded patients with MPN diagnosed >6 months after lymphoma to reduce the probability of therapy-induced MPN. Clinicopathological characteristics are summarized in Table 1.

The study was approved by the Danish National Committee on Health Research Ethics (record no. 1609521) and the Danish Data Protection Agency (record no. 1-16-02-420-15) and was performed in compliance with the principles of the Helsinki Declaration. Before inclusion, written informed consent was obtained. Exception was made in cases where the patient had died at the time of the study, to which separate permission was granted in the ethics approval.

All patients were diagnosed with MPN based on BM samples, while the lymphoma diagnoses were based on either lymph node or extra-nodal tissue biopsies. Non-neoplastic tissue from archived specimens in the DNPR or saliva (in cases where no archived non-neoplastic tissue was available) were used as germline control. All specimens were reviewed by an experienced hematopathologist at a tertiary-care center. Immunohistochemical characterizations were performed to verify the adequacy of study specimens regarding the presence of MPN and lymphoma in relation to tumor content and to assess for any reciprocal infiltration of MPN into lymphoma or vice versa. A DNA library triad encompassing the 3-way paired MPN, lymphoma, and non-neoplastic tissue specimens was constructed for WES following standard protocols. Captured targets were paired-end sequenced according to standard protocols. Raw sequencing data was quality checked. We observed a high level of indels and C:G-T:A substitutions, probably induced by formalin-fixation of the specimens. To reduce the false-positive rate, the data was processed on two independent mutation-calling pipelines. Variants with allele frequencies (VAF) <10% were excluded from the analyses, and only somatic variants reported in COSMIC⁹ were retained for downstream evaluation. Variants within genes with a well-established relevance for the diseases addressed in the present study, were manually curated regardless of VAF. This involved variants in DNMT3A, TET2, IDH2, RHOA, and JAK2.^1,2 Alleles with depth coverage of ≥20 reads were evaluated. Variants present in the gnomAD database (version 2.1.1),¹⁰ European non-Finnish population, at a frequency above 0.01 were removed. Quality and read depth assessment were complemented with the inspection of focus variants in Integrative Genomics Viewer.¹¹

In patients diagnosed with MPN and AITL, the fraction of myelopoietic tissue in the MPN BM samples varied between 40-95% (Table 1). Tumor cell content in the AITL samples was estimated ranging between 60-90%. In patients diagnosed with MPN and DLBCL (patients #6-14), the fraction of myelopoietic tissue in the MPN BM samples varied between 80-95%. Tumor cell content in the DLBCL samples was estimated in the range 70-90%, except for patient #7 in which there was a low content of 15% due to adjacent salivary gland tissue.

Figure 1 shows an overview of the identified mutations with VAF ≥10% for both MPN/AITL and MPN/DLBCL patients. Specific mutations, well characterized in the literature as being associated with MPN and AITL are presented in Table 2 in more detail. Shared mutations were defined by an identical position and nucleotide change within the given gene sequence. In four of five AITL patients, mutations in either of the epigenetic modifier genes DNMT3A, TET2, or IDH2 were identified. Notably, a mutation in IDH2 leading to amino acid change at position R172 was found in two of five patients. A RHOA mutation resulting in the G17V change was found in the AITL sample of patient #1. In patient #5, MPN and AITL were simultaneously diagnosed. This patient had discrete lymphoma infiltration in the BM, where a RHOA mutation was identified. The same mutation could not be detected in the lymph node biopsy.

Table 1.Demographic and clinicopathological characteristics.

Figure 1.Overview of shared and private mutations in the study cohort. Shared and private mutations with variant allele frequencies (VAF) ≥10% identified by whole exome sequencing. Canonical myeloproliferative neoplasm (MPN) and lymphoma-entity associated mutations are also reported at lower VAF levels. The numbers in the individual boxes indicate VAF (%). (A) Mutations identified in patients with angioimmunoblastic T-cell lymphoma (AITL) and MPN. ^§In patient #5, MPN and AITL were diagnosed simultaneously. The bone marrow was morphologically and immunohistochemically found to be infiltrated by both neoplasms. The mutational findings in this patient should therefore be interpreted with caution. (B) Mutations identified in patients with diffuse large B-cell lymphoma (DLBCL) and MPN. ^#Identical variant found in non-neoplastic tissue (germline mutation). *According to International Cancer Genome Consortium terminology, www.dcc.icgc.org. AITL: angioimmunoblastic T-cell lymphoma; DLBCL: diffuse large B-cell lymphoma; M: myeloid sample; L: lymphoid sample.

Table 2.Detailed representation of selected mutations identified in the study cohort.

Both AITL patients with IDH2 mutations had a concurrent RHOA mutation. Mutations in the JAK2 gene were found in three of the MPN samples. Two cases harbored the JAK2 V617F amino acid change, while the third was JAK2 exon 12 mutation-positive.

In the nine DLBCL patients, mutations in genes commonly associated with lymphoid neoplasms (B2M, BCL2, CCND3, EZH2, TP53, NKFBIE, PAX5 and MYD88), were identified with high allelic burdens (Figure 1). JAK2 mutations resulting in V617F amino acid change were found in five of these patients’ MPN samples. One patient harbored a MPL mutation in the MPN sample.

Shared mutations were found in three of five (60%) MPN/ AITL patients and involved DNMT3A, JAK2 and TET2. IDH2 was found in both tissue samples of patient #5, but due to simultaneous diagnosis of MPN and AITL and BM infiltration of lymphoma, this mutation could not confidently be classified as shared. Shared mutations were found in two of nine (22%) MPN/DLBCL patients involving only JAK2 (Table 2).

DNMT3A and TET2 play essential roles in hematopoietic stem cell differentiation and abnormal function of these genes may lead to impaired hematopoietic differentiation capacity and to the accumulation of clonal hematopoiesis (CH).^12-14 Shared mutations of DNMT3A (patient #2) and TET2 (patients #1 and #3) were present with high allelic burdens (range 19-49%), supporting the hypothesis that these mutations may be involved in the early common pathogenetic steps of both MPN and AITL. The allelic burden of DNMT3A and TET2 mutations were higher than those of IDH2 and RHOA in the AITL samples, suggesting that the latter mutations could represent more downstream events.¹⁴

These observations support the possible parallel evolution of two distinct neoplastic proliferations, a myeloid and a lymphoid, from a common hematopoietic progenitor cell population that carry CH features such as TET2 and DNMT3A mutations. These findings extend previous reports of the development of metachronous AITL and myeloid neoplasms from a common TET2/DNMT3A-mutated stem cell population in patients with CH.¹⁵

Eight of the 14 patients harbored JAK2 mutations: three of five (60%) of the MPN/AITL patients and five of nine (56%) of the MPN/DLBCL patients. In patient #2 (AITL) and patients #6 and #8 (DLBCL), the JAK2 mutation was shared. The non-neoplastic tissue of these patients also carried the same JAK2 variant with a low allele frequency (Table 2). While this may represent in vivo tumor cell contamination of the samples, another possible interpretation is that the presence of a JAK2 germline mutation predisposed to the subsequent development of MPN and lymphoma.

The risk of cross-contamination between MPN and lymphoma cannot be excluded with certainty, as demonstrated by the findings in patient #5. However, this risk is mitigated by factors such as: (i) expert specimen review by a tertiary-center hematopathologist; (ii) the diagnosis of MPN dating several years before the lymphoma diagnosis in most patients (10/14), reducing the probability of lymphoma cells being present in the MPN samples. Of the five patients with shared mutations (3 MPN/AITL and 2 MPN/DLBCL), four had an interval between the diagnosis of MPN and lymphoma of ≥2.6 years. In the last of these five patients (patient #1), AITL was diagnosed simultaneously with MPN, but without evidence of lymphoma in the BM; (iii) a high tumor cell content in most specimens, increasing the probability that the DNA extracted and sequenced from MPN and lymphoma tissue is representative of the respective neoplasm.

For future investigations, the application of single-cell and spatial multi-OMICS will likely improve the level of precision of clonal recognition and development.

In conclusion, we identified shared and private mutations in patients with co-occurrent MPN and lymphoma. Some of these mutations, particularly in the setting of MPN/AITL, may reflect ancestral pathogenetic alterations related to CH (e.g., TET2, DNMT3A), while others (e.g., IDH2, RHOA) may facilitate downstream clonal divergence and expansion. These events seem to be less frequent in DLBCL than AITL. However, additional data from larger, independent studies are required to provide support for these hypotheses.

Footnotes

• Received June 21, 2023
• Accepted July 15, 2024

Correspondence

F. D’AMORE - frandamo@rm.dk

Disclosures

No conflicts of interest to disclose.

Funding

Acknowledgments

The authors would like to thank the collaborating pathology and hematology departments for the retrieval of tissue samples and collection of clinical data. Special thanks for technical assistance to Kristina Lystlund Lauridsen, Laboratory of Molecular Pathology at Aarhus University Hospital, Jenny Zhaoying Xiang, Genomics Resources Core Facility at Weill Cornell Medicine, and Joelle Racchumi, Department of Pathology and Laboratory Medicine at Weill Cornell Medicine.