Atypical MYC rearrangement pattern of 3’ deletion and 5’ amplification along with independent IGH rearrangement

A case study

Abstract

A spectrum of Cellular homolog of the v-myc oncogene (cMYC) alterations such as translocation, overexpression, mutation, and amplification plays an important role in lymphomagenesis, particularly in high-grade lymphomas, and are associated with prognostic significance. Accurate identification of cMYC gene alteration is important for diagnostic, prognostic, and therapeutic implications. With the application of different FISH (fluorescence in situ hybridization) probes that helped overcome the analytical diagnostic challenges as a result of variant patterns, we report rare, concomitant, and independent gene alterations in cMYC and Immunoglobulin heavy-chain gene (IGH) with detailed characterization of its variant rearrangement. Short-term follow-up post R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy seemed to be favorable. Accumulation of many more literature studies on such cases with their therapeutic implications would lead to the categorization of these cases as a separate subclass in large B-cell lymphomas followed by molecular targeted therapy.

Introduction

Among the various prognostic modulatory factors, such as clinical features, histology, tumor subtypes, and protein expressions, that impact lymphoma prognosis, genetic alterations of the MYC gene play a crucial role in modulating the biological and clinical effects of lymphomas.[¹]

MYC oncogene overexpression results from a spectrum of genomic alterations, such as translocations, mutations, increased gene copy number, and MYC rearrangement, which play a critical role in lymphomagenesis in aggressive B-cell lymphomas (BCL)such as Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), and B-cell lymphoma unclassifiable (BCLU) with features intermediate between DLBCL and BL.[¹]

The MYC proto-oncogene is located on chromosome 8q24 and encodes for a transcription factor with various roles in metabolism, protein synthesis, and cellular differentiation. MYC requires the contribution of other genetic events, that is, the expression of BCL2 and BCL6 and mutations in TP53 to promote proliferation and oncogenesis. MYC in large B-cell lymphoma can occur as a single hit referred to as single-hit lymphoma (SHL) or with concurrent BCL2 and/or BCL6 gene rearrangements referred to as double-hit/triple-hit lymphomas (DH/THL).[²]

Sole amplification of MYC without rearrangements as a rare event in BCL has also been described in literature studies with no significant association with survival. SHL is prevalent within cases of non-Burkitt MYC-rearranged B-cell lymphomas, representing 40% of cases of MYC-rearranged DLBCL and potentially up to 50% of cases of MYC-rearranged BCLU with unclear prognosis.[³]

It is apparent that certain clinicopathologic features, such as elevated R-IPI (revised International Prognostic Index) score and evidence of MYC translocation to an immunoglobulin (Ig) gene as a partner, may predict poor prognosis in SHL patients, even when treated with intensive induction therapy. However, some studies have not considered them to be associated with adverse outcomes and have treated them as DLBCL with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy. A subset of MYC-rearranged lymphomas known as double-hit lymphoma (DHL), which demonstrates rearrangement of BCL2 and/or BCL6 in addition to MYC, is associated with a particularly poor prognosis, although this may be improved for patients treated with frontline intensive immunochemotherapy.[⁴]

We describe a rare occurrence defined by the presence of an atypical MYC rearrangement pattern of 5’ deletion and 3’ amplification with non-Ig unknown partner fusion by fluorescence in situ hybridization (FISH).

Based on the histopathological classification of germinal center B-cell-like (GCB) subtype and the presence of cMYC overexpression, FISH was performed on formalin-fixed paraffin-embedded lymph node specimens using commercially available break-apart probes, that is, IGH, MYC, BCL2, and BCL6 and IGH/MYC fusion to rule out DH/TH lymphoma.

Case Study

We report an atypical, rare, complex pattern of MYC rearrangement on a biopsy of left tonsillar growth in a 43-year-old woman.

Histopathological findings showed fragments lined by stratified squamous epithelium with extensive ulceration, neutrophil-rich exudate, and granulation tissue with capillary proliferation and infiltrate sheets with vague clustering of atypical cells. The cells showed moderate to scant, pale clear to eosinophilic cytoplasm, round to ovoid nuclei with coarse chromatin, irregular nuclear contours, and conspicuous nucleoli. Patchy crushing of tumor cells was observed surrounding scant fibrotic stroma with mixed inflammatory cell infiltrate.

Immunohistochemistry studies were performed, which showed strong positivity for CD45, CD20, BCL6, multiple myeloma oncogene-1 (MUM-1), and moderate (2+) positivity for CD10 and BCL2, negativity for CD3, Pan CK, CD5 with strong Ki-67 of 80%, and MYC expression of 45%.

Based on the histomorphological and immunohistochemistry, the case was given an impression of large B-cell lymphoma, Germinal center B-cell (GCB) type.

FISH testing was done for cMYC, BCL2, and BCL6 rearrangement to rule out DH/TH lymphoma. IGH FISH testing was done for the designation of the fusion partner status.

FISH studies showed positivity for atypical MYC gene rearrangement and negative status for BCL2 and BCL6, classifying the case genetically as an SHL [Figures 1 and 2].

Figure 1: (a) MYC break-apart probe with one fusion and multiple orange signals indicating 3’green deletion and 5’orange amplification. (b) IGH/MYC dual-color fusion probe indicating negative for IGH/MYC fusion and three green signals indicating break in IGH and multiple orange signals indicating rearrangement in MYC. (c) Clone A with one fusion, one orange and one green using IGH break-apart probe indicating gene rearrangement. (d) Clone B with two fusion and two orange signals using IGH break-apart probe indicating duplication of IGH followed by break with 5’ deletion and 3’ duplication

Figure 2: (a) Two fusion (orange/green) signals indicative of negative pattern for BCL2 gene rearrangement. (b) Two fusion (orange/green) signals indicative of negative pattern for BCL6 gene rearrangement. (c) Hematoxylin and Eosin–stained slide showing tonsillar squamous mucosa with sheet-like infiltrate of atypical cells

MYC positivity showed a variant rearrangement pattern with concomitant 3’ deletion and 5’amplification [Figure 1a].

Independent application of IGH break-apart probe showed rearrangement for IGH. Rearrangement of IGH with break and multiple green signals probably indicates rearrangement with a variant pattern with an unknown fusion gene [Figure 1c, 1d]. IGH rearrangement was observed with the presence of two clones, one clone with one fusion, one orange and one green rearranged pattern; and another clone with two fusion (orange/green) and two orange signals indicating duplication of IGH followed by 3’ deletion and 5’ amplification. Considering the positivity for rearrangement of IGH, IGH/MYC fusion probe was applied to rule out IGH/MYC fusion, which showed negative status [Figure 1b].

The patient was treated on a standard R-CHOP therapy and is currently (till date) in 8 months remission post treatment.

Discussion

Extensive characterization using different FISH probes indicated a double independent rearrangement of MYC and IGH with partner gene for MYC being non-IG, thus classifying the case as an MYC-rearranged lymphoma with an atypical pattern.

MYC rearrangement with 5’ amplification seems to be a probable mechanism responsible for the overexpression of MYC protein.

Although both IG and non-IG partners fulfill the diagnosis of DHL or THL in the 2016 revised WHO (World Health Organization) classification, some studies showed a worse prognosis in cases with IGH/MYC translocation than non-IG-MYC translocation with R-CHOP treatment. The prognostic effect of lone MYC translocation or MYC overexpression is still in controversy, and the dominant view is that lone MYC translocation or MYC overexpression does not induce poorer clinical outcomes.[⁵]

Our case is the first in literature with concomitant cMYC and IGH, both as two independent-occurring rearrangements with variant patterns.

Our case study emphasizes that analytical challenges faced during FISH interpretation can be overcome by assessing the patterns of complex atypical rearrangements by the application of different combinations of probes to refine and understand the mechanism of leukemogenesis and to ascertain prognostic value.

Sun et al.[⁶] have reported a similar finding in two cases. Our case showed a lower level of 5’ amplification, whereas Pophali et al.[⁷] have observed high uncountable 5’ level of amplification.

The patient was put on a standard R-CHOP therapy and is in 8 months of remission; however, a long-term follow-up study is still required to access the prognosis.

A combination of complementary testing algorithms for conclusive diagnosis should be adopted to identify MYC-rearranged cohort so that risk-adjusted therapeutic approaches can be altered and refined with a personalized approach.

Reporting of such rare findings is essential to build up data with variant patterns and eventually help subclassify them as a distinct entity. Follow-up of such cases would then help establish the prognosis and improve disease risk stratification and genomic-based therapy selection.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.