KRAS mutation in secondary malignant histiocytosis arising from low grade follicular lymphoma

Follicular lymphoma (FL) is an indolent B-cell lymphoma composed of follicle center B cells [1]. While generally regarded as a manageable disease with a long survival rate, it can also progress to more aggressive forms of lymphoma, such as diffuse large B-cell lymphoma. Another rare and distinct form of disease progression is transdifferentiation from a B cell neoplasm to a neoplasm of another cell lineage [2‐9]. In these instances, the neoplastic cells demonstrate lineage plasticity, manifested as an apparent shift in cellular phenotype from one lineage to another while still carrying shared underlying clonal genetic abnormalities. Most reported cases have involved transdifferentiation of chronic lymphocytic leukemia to histiocytic neoplasms [2‐4, 6, 8, 9]. Transdifferentiation is an elusive process, and the exact pathogenic mechanisms remain unknown. Comprehensive genetic analysis could provide a useful tool by which to identify genetic alterations that occur during transdifferentiation and may provide clues to the underlying pathogenesis of this process. Furthermore, as this form of transformation heralds a very poor prognosis, identification of targetable genetic mutations could offer therapeutic options in cases where there are few alternatives. Here, we described a rare occurrence of a low grade FL transdifferentiating into a Langerhans cell sarcoma (LCS) with acquisition of KRAS mutation. As comparative whole exome sequencing of pre- and post-transdifferentiation lymphoma samples has not yet been reported in the literature, this report will contribute to the presently limited understanding of this rare phenomenon.

Our patient had a long history of follicular lymphoma which initially presented as a localized neck mass. A biopsy of the mass showed low grade follicular lymphoma. The patient was treated with radiation therapy, but 3 years later, was diagnosed with diffuse large B cell lymphoma involving lung with discordant low grade follicular lymphoma in the marrow. After eight cycles of R-CHOP therapy (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), the patient went into remission. Unfortunately, 6 years later, the patient presented with skin and breast lesions as well as increased adenopathy, and was treated with rituximab, with some improvement of adenopathy. The skin nodules on the left arm were noted enlarging, a subsequent biopsy revealed low-grade follicular lymphoma, which responded to rituximab and bendamustine therapy. A few months later, the patient noted an enlarged inguinal lymph node, a biopsy of which showed Langerhans cell sarcoma. Despite 4 cycles of ICE chemotherapy and 8 weeks of ibrutinib, a PET/CT showed evidence of disease progression with diffuse intensely hypermetabolic soft tissue nodules and lymph nodes. Biopsy of one of the lymph nodes was consistent with diffuse large B cell lymphoma. The patient received palliative radiation therapy and passed away in a hospice facility. The patient’s clinical history and treatment was summarized in Table 1.

Classification of hematopoietic neoplasms is predicated on the assumption that the phenotype of a neoplastic cell is intrinsically linked to a predefined lineage and that this lineage determines the type and nature of the neoplasm. However, as demonstrated in cases of transdifferentiation, these associations are not hard and fast rules, as cells, both physiologic and neoplastic, can demonstrate lineage plasticity [3‐9, 19‐21]. Our patient’s pathologic studies include a wide range of morphologic variation in the neoplastic clone, ranging from low grade FL to DLBCL to later on, LCS.

The mechanisms by which transdifferentiation occurs are largely unknown and previously described cases have relied upon molecular clonality or other genetic markers to establish a clonal relationship between what otherwise would appear as two phenotypically distinct neoplasms. Clonal IgH rearrangements have been reported in 39% of sporadic cases of histiocytic/dendritic cell sarcomas [22] which may reflect the close relationship and possible common precursor between neoplasms of histiocytic differentiation and lymphoid differentiation [4, 6, 7, 9, 19, 23‐25]. In our case, while a clonal IgH rearrangement was detected in the FL from the fresh bone marrow sample, PCR analysis failed to identify a clonal IgH rearrangement in LCS. The reason for the absence of clonal IgH rearrangement in the LCS sample is not entirely clear, but could be due to additional genetic alterations affecting the specific binding of the PCR primers or decreased assay sensitivity in formalin fixed tissue. However, the clonal relationship between FL and LCS was definitively confirmed by FISH analysis, which able to readily identify the t(14;18), IgH/BCL2 within morphologically-apparent sarcoma cells. The shared t(14;18), IGH/BCL2 translocation in both the follicular lymphoma and LCS by FISH provided strong evidence of their clonal relationship, as this translocation is considered very specific for follicular lymphoma and has not been reported in any de novo sarcoma.

The shared CREBBP mutation (NM_001079846:exon29:c.4920_4922del:p.1640_1641del) is an interesting finding. CREBBP mutations, particularly mutations of the acetyltransferase domain, has been implicated as an early driver event in follicular lymphoma [26, 27]. Although the specific CREBBP mutation that we have detected in this case has not been previously reported in COSMIC database, it does occur within this functional acetyltransferase domain (p.1342-p.1649) [28, 29]. Thus, it could potentially represent a shared acquired somatic driver mutation and further support a model of direct transdifferentiation of LCS from antecedent follicular lymphoma rather than, for example, evolution from a common precursor neoplastic cell harboring an early translocation t(14;18) event.

The additional SNP microarray results also suggest that the LCS likely originated from an earlier follicular lymphoma with t(14;18). As the LCS does not share the other complex genetic alterations with the exception of the shared CNV gain involving 22q11.23. However, it is worth pointing out that the LCS sample contains relatively low tumor burden (20–30% by visual estimation) with a dense background of inflammatory cells, whereas the follicular lymphoma contains almost 100% tumor cells. Therefore, other more subtle genetic alterations in the sarcoma cells may not be identified by the OncoScan SNP microarray which requires at least 25% tumor content.

The most intriguing finding in this case is the detection of KRAS p. G13D, a known pathogenic mutation, detected solely in the LCS sample. RAS mutations have been implicated in a number of hematologic malignancies, with NRAS mutations being more prevalent than KRAS mutations, particularly in myeloid neoplasms [30]. RAS-MAPK alterations have also been frequently described in hairy cell leukemia and multiple myeloma, but are more rarely seen in other lymphoproliferative disorders, such as diffuse large B-cell lymphoma [31, 32]. In studies analyzing mutational profiles of follicular lymphoma, a pathogenic role for RAS mutation in the development of follicular lymphoma has not been described [33, 34], although it has been reported as a rare event in cases of histologic transformation [35]. In a study of 55 primary DLBCL samples, KRAS p.G13D mutation was identified in 2 cases, thus is it possible that RAS mutations could drive the pathogenesis of a relatively rare subset of primary and secondary DLBCL [32]. A separate study described differential gene expression of genes in the MAPK signaling pathway, including NRAS, in cases of follicular lymphoma compared with transformed follicular lymphoma, suggesting activation of this pathway could be important in disease transformation [36]. In our case, the KRAS p.G13D may have arisen during the transformation process. Further studies to completely delineate clonal derivation were desired but precluded by the absence of material from original FL and subsequent DLBCL for additional testing. Nonetheless, the high prevalence of KRAS p.G13D mutation in the sarcoma cells, inferred from a variant allele frequency similar to the sarcoma tumor burden, suggests that it is a predominant mutation within the sarcoma cells.

Our finding raises a couple of important questions. What is the significance of KRAS mutation in histiocytic neoplasms? Could this mutation potentially drive transdifferentiation process itself? A recent abstract presentation [37] retrospectively reviewed cases of histiocytic sarcomas and discovered RAS/MAPK mutations in 44% (n = 8) of cases. KRAS p.G13D was present in 3 of the cases. Some of the cases of histiocytic sarcoma also possessed mutations that are common in B-cell lymphoma (such as MYD88, SOCS1, KMT2D, ARID1A) and furthermore, a subset of patients with histiocytic sarcoma had concurrent or prior history of B-cell lymphoma, including follicular lymphoma, which raised the possibility of transdifferentiation. This suggests that RAS/MAPK mutations are actually quite common in histiocytic neoplasms, even some that may be secondary to B-cell lymphoma, and that given their prevalence, may actively participate in driving this disease process.

In terms of Langerhans cell neoplasms, the RAS/MAPK signaling pathway has been strongly implicated as a pathogenic driver. Recent mouse models have demonstrated that expression of KRAS p.G12D mutation in lung myeloid cells induce the development of pulmonary Langerhans cell histiocytosis (LCH) [38]. NRAS p.Q61K mutations have also been reported at higher frequency in cases of pulmonary LCH [39], but have not thus far been seen in non-pulmonary cases. A study of 61 cases of non-pulmonary LCH reported BRAF V600E mutations in half of cases, also detected several other point mutations including TP53 p.R175H, KRAS p.G13D and MET p.E168D [10]. BRAF is an essential component of the RAS-RAF-MEK-ERK signaling cascade, which is triggered by binding of extracellular growth factor or cytokines to surface tyrosine kinase receptors, eventually leading to the modulation of downstream gene expression [40]. The activation of this pathway in LCH is further supported by the fact that MEK and ERK phosphorylation could be detected by immunohistochemistry in all cases of LCH, regardless of BRAF mutational status [41]. Activating mutations in BRAF other than V600E have also been detected in LCH [42‐44]. Furthermore, cases without BRAF V600E mutation have demonstrated a high prevalence of somatic MAP2K1 mutation, which are mutually exclusive with BRAF mutations [43, 45]. Thus, there is a substantial amount of evidence to suggest that the RAS-RAF-MEK-ERK signaling pathways is important to the evolution of LCH.

LCS is an extremely rare malignancy thought to be derived from Langerhans histiocytes, and because of the scarcity of reported cases, the molecular pathogenesis of this entity is mostly unknown. However, it is not unreasonable to infer that some of the same pathways that drive LCH could also drive LCS. Interestingly, a recent report of histiocytic sarcoma transdifferentiation from B-lymphoblastic leukemia demonstrated NRAS p.G12D mutation, further suggesting RAS pathway alterations may be important in the transdifferentiation process [46]. It is also possible that other genetic or epigenetic changes which are as yet uncharacterized may contribute to lineage conversion and phenotypic switch seen during transdifferentiation.

Because of its prevalent role in neoplasms of multiple different organ systems, the RAS-RAF-MEK-ERK signaling pathway is also a target for novel therapeutics, including monoclonal antibodies and small molecule inhibitors, for example BRAF and MEK inhibitors in melanoma [47‐49]. KRAS is one integral component of the RAS-RAF-MEK-ERK signaling pathway that acts upstream of BRAF [50]. Detection of KRAS mutation is now recommended in colorectal carcinoma, since its presence indicates resistance to targeted therapy against upstream signaling proteins (i.e. EGFR) [51]. Recent studies have shown specific efficacy of cetuximab in colorectal cancer patients with KRAS p.G13D mutations (the same mutation detected in our patient) and not other KRAS mutations [52, 53]. Ongoing studies in a variety of neoplasms examining RAS proteins and other downstream mediators, such as BRAF and MEK, as druggable targets offer additional therapeutic possibilities in patients with applicable genetic alterations, such as the current case [54‐56].

In summary, this case describes transformation/transdifferentiation of a low grade FL into a LCS with detection of KRAS p.G13D mutation in the transformed sample. Our findings demonstrate characterize genetic events that may occur during transdifferentiation and further emphasizes the role of alterations of RAS-RAF-MEK-ERK signaling in the pathogenesis of Langerhans cell neoplasms. Importantly, the discovery of acquired KRAS mutation raises the possibility of targeted therapies (e.g. small molecule inhibitors) [50, 57] in the treatment of patients with these neoplasms who otherwise have limited therapeutic options.