When and how to test for C-MYC in aggressive B cell lymphomas
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C-MYC (MYC) is a regulator gene that plays an important role in cell cycle progression, apoptosis, and cellular transformation. It is believed to regulate expression of up to 15 % of all human genes. In recent years, C-MYC has been extensively studied in blastic B cell lymphomas, and currently C-MYC is regarded not only as a diagnostic tool, but also a promising prognostic biomarker. C-MYC testing is therefore no longer simply a confirmational analysis for Burkitt’s lymphoma, but also provides important information on prognosis and risk stratification of diffuse large B cell lymphoma patients. This review outlines our approach to integrate this new role of C-MYC in a convenient way in routine diagnostics and discusses the application of different techniques such as immunohistochemistry and FISH for C-MYC testing.
Keywordsc-myc C-MYC DLBCL Burkitt lymphoma Immunohistochemistry FISH Prognosis Double-hit score
The C-MYC gene was identified more than 20 years ago as the cellular homolog of the retroviral v-myc oncogene [1, 2, 3]. Since then, its protein function, expression, and the epidemiology of its gene alterations have been intensively studied. C-MYC has been shown to be a regulator gene that codes for a transcription factor, a multifunctional nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis, and cellular transformation, and is believed to regulate expression of up to 15 % of all human genes . Recent work suggests that c-myc is a TATA-Box-binding factor that regulates all genes transcribed by RNA-polymerase 2, meaning that c-myc is acting as a transcriptional amplifier rather than as a transcription factor per se . Importantly, C-MYC plays a major role in the pathogenesis of malignant neoplasms, particularly lymphomas, and its gene is recurrently deregulated either by rearrangements, amplifications, or mutations . Over 90 % of Burkitt’s lymphomas (BL) carry a t(8;14) or, less commonly, a t(2;8), t(8;22), or t(3;8) [6, 7], juxtaposing the C-MYC gene to the immunoglobulin heavy chain, or the light chain gene. However, 5–14 % of diffuse large B cell lymphomas (DLBCLs) also display translocations involving C-MYC [8, 9, 10], occasionally leading to a diagnostic challenge since aggressive lymphomas with C-MYC rearrangement can be either (i) BL, (ii) B cell lymphomas, or unclassifiable, with features intermediate between DLBCL and BL (BCL-U, iBL/DLBCL), or (iii) DLBCL not otherwise specified. The exact classification relies on the interpretation of clinical data, morphology, immunohistochemical profile, and molecular genetics.
Should any blastic B cell lymphoma be analyzed for C-MYC aberrations?
When is it appropriate to use immunohistochemistry for c-myc, and when is it more appropriate to perform C-MYC FISH?
If the above analyses are performed, how should the results be interpreted?
The Basel algorithm in diffusely growing blastic lymphomas
If morphology, immunohistochemistry (CD20+/CD5−/CD10+/CyclinD1−/BCL2−/BCL6+/CD44−/CD38+/Ki67 > 95 %), and clinical context (age and topography) are in line with BL, and c-myc is immunohistochemically detectable in >95 % of tumor cells, diagnosis of BL can be made without FISH confirmation. This phenotype is highly characteristic and allows also the exclusion of blastic mantle cell lymphoma. Importantly, CD10− and bcl2 + BL do not exist.
All cases expressing c-myc in lower percentages should be subjected to FISH analysis for C-MYC (first line break-apart probe and, if negative, double-fusion probe in a second run). If both FISH examinations suggest a non-rearranged C-MYC gene, then aggressive “Burkitt’s-like” lymphomas (BCL-U) with recurrent 11q aberrations  or with c-myc activation due to miRNA-34 downregulation  should be considered.
If everything is in line with BL but c-myc immunohistochemistry is completely negative, C-MYC point mutations, which truncate the protein, should be considered, and C-MYC FISH should be performed. If C-MYC FISH is also negative in such a case, it should be classified as BCL-U according to the suggestion of Salaverria et al. .
Diffuse large B cell lymphoma, not otherwise specified
In DLBCL, the situation is more complex. In unselected DLBCL series, rearrangements of the C-MYC gene were discovered in approximately 10 % of cases [9, 10, 36, 37, 38, 39]. Of these, 20 to 30 % may have an additional break in the BCL2- and/or BCL6 genes [40, 41, 42], fulfilling the criteria of so-called genotypic double-hit lymphomas.
Recent data point to the additive prognostic information obtained from FISH for individual risk estimation, since all types of C-MYC rearrangements, “IG and non-IG,” were associated with poorer disease-specific survival in DLBCL . Several studies demonstrated that immunohistochemical staining for c-myc is a good screening tool, since almost 50 % of cases expressing c-myc in over 90 % of tumor cells are also C-MYC rearranged. There are also cases (approximately 10 %) that present without (or without relevant) immunohistochemically stainable c-myc despite bearing a rearrangements [26, 27, 43]. The significance of the latter set of cases (C-MYC FISH-positive, but immunohistochemically negative) is unclear; additional studies, including identification of the C-MYC translocation partners and sequencing for epitope-abrogating point mutations, will be required to further clarify the biological and clinical significance of these subsets . Importantly, recent evidence shows that rearrangements with low c-myc protein expression might be associated with a better prognosis than the presence of rearrangements or high c-myc expression , pointing towards the relevance of c-myc protein overexpression. From the technical point of view, it should be stressed that, analogously to BL, it is essential not to use only the break-apart probe or the double-fusion probe in an isolated manner for the detection of C-MYC rearrangements, since a subset of translocations may be missed by either probe [8, 45]. The best practical way is to use the double-fusion probe, if in a first attempt the break-apart probe is negative (see below).
The puzzling role of c-myc overexpression in DLBCL can be sought in the fact that, not only the C-MYC translocation itself, but particularly the deregulation of c-myc by other mechanisms, can give rise to a BL-like GEP pattern ; in the group of lymphomas showing typical gene expression profiles of BL, there were cases without any detectable translocation of C-MYC (4 cases of 38). These findings were confirmed by recent studies showing significantly elevated c-myc protein expression in tumors lacking translocations of the C-MYC gene [24, 43, 46]. Alternative mechanisms equally presaging upregulation of C-MYC have been suggested. miRNAs regulating c-myc expression have been described and, accordingly, miRNA profiles show differences in C-MYC-rearranged and non-rearranged BL [35, 47], and also in DLBCL . Amplifications of C-MYC (encountered in <1 % of DLBCL) have also been shown to be associated with c-myc overexpression and poorer prognosis [49, 50]. Therefore, given the important role of the c-myc protein, which regulates a substantial number of genes, and possible alternative causes (other than C-MYC gene rearrangements) of c-myc overexpression, it is not surprising that protein overexpression of c-myc, regardless of gene translocations, plays an important prognostic role in DLBCL. Moreover, DLBCL with immunohistochemical c-myc overexpression, particularly in combination with bcl2 positivity (referred as phenotypic double-hit score (DHS) positive cases), follows an aggressive clinical course with adverse prognosis [13, 24, 26, 28, 43]. The adverse effect of c-myc deregulation (overexpression on protein level, mRNA level, or rearrangement) was recently confirmed by a meta-analysis of 24 eligible studies comprised of 4,662 patients who did or did not have Rituximab as part of their chemotherapy regimen .
Is it recommended that all DLBCL be stained for c-myc (and bcl2)?
What are the best prognostic cut-off values for c-myc (and bcl2)?
Which are the most relevant immunohistochemical cut-off values predicting C-MYC breaks in DLBCL?
When should C-MYC FISH be performed?
Considering recent data from a large, independent patient cohort [24, 25, 28, 43], we recommend staining of all DLBCL for both bcl2 and c-myc at initial diagnosis (Table 1) . As is true for all immunohistochemically assessed biomarkers, specific attention should be paid to proper fixation, robust staining protocols, and precise counting. The information gained from these stainings can help physicians to stratify the cohort of DLBCL patients into patients with superior and inferior prognosis even though we acknowledge that a subgroup of high-risk patients such as elderly patients or patients with significant comorbidities cannot be treated more aggressively (see discussion below).
In the literature, different prognostic cut-off scores for c-myc and bcl2 were reported, varying from ≥10 % , ≥40 % [24, 28, 43, 44] and >75 %  for c-myc, and ≥30 % , ≥50 % [26, 43], and ≥70 % [24, 28] for bcl2. The cut-offs most often used (and reproduced) for prognostication are ≥40 % for c-myc and ≥50 or ≥70 % for bcl2.
The question of the most relevant immunohistochemical values for c-myc is difficult to answer, since there are both immunohistochemically negative/FISH positive cases and immunohistochemically positive/FISH negative cases. The optimal immunohistochemical cut-off for c-myc to predict FISH-positive cases is unclear, and data in the literature vary widely between a minimum of ≥30 % , ≥50 % , ≥80 % , and >95 % , although lower percentages also exist, e.g., 5 and 19 % . This difference is obviously due to the different weighting of the specificity and sensitivity of the respective cut-off scores (Fig. 1).
In our series of 432 cases, a positive predictive value of c-myc protein overexpression to detect C-MYC rearranged cases was 60 % using a cut-off of >95 % . This value was established by analysis of the area under the receiver operating characteristic curves, and had a maximum specificity (36 %) and sensitivity (91 %) to predict C-MYC breaks (area = 0.599, 95 % confidence interval 0.484–0.715, P = 0.063).
- (d)From a practical point of view, we would recommend the following approach (Table 1):
If c-myc is expressed in ≥40 to <75 % and bcl2 in ≥70 % of tumor cells, it is classified as DLBCL with double hit score 2 (DHS-2).
If c-myc is expressed in >75 % of tumor cells, FISH is recommended to identify C-MYC rearranged cases as well as to identify cases with c-myc upregulations resulting from mechanisms other than translocations. Only if C-MYC is rearranged and bcl2 is expressed in >70 % of tumor cells is BCL2 FISH recommended, since double BCL2/C-MYC translocated cases (genotypic double-hits) have a very poor prognosis . An additional BCL6 translocation does not seem to have a significant prognostic impact [8, 27], although this remains controversial [54, 55]. Moreover, BCL6 is often found to be involved in so-called triple-hit lymphomas together with BCL2 and C-MYC translocations .
If c-myc is expressed in 40 to 74 % of the tumor cells, and BCL2 in <70 %, FISH can be omitted, since the probability of detecting rearranged BCL2 is very low, and “C-MYC-only” rearranged cases with low c-myc protein expression levels seem to have a prognosis similar to non-rearranged cases .
The main goal of this approach is the identification of patients who may benefit from different therapeutic strategies. In the past, patients with C-MYC translocation did very poorly  with “CHOP-only” therapeutic regimens; while they improved with R-CHOP regimens, the results are still poor compared to C-MYC unrearranged patients. Moreover, recent studies have shown that R-CHOP is not an optimal regimen for patients with phenotypic DHS 2 DLBCL, and is particularly inefficient in C-MYC/BCL2 genotypic double-hit DLBCL .
In the light of the favorable outcome of the C-MYC-driven BL and the impact of C-MYC on proliferation, it is appealing to consider the more aggressive therapies used in BL for this group of DLBCL patients. However, there is as yet no published data demonstrating that patients with “double-hit” DLBCL profit from more aggressive therapies . Currently, the US Intergroup is studying a dose-adjusted R-EPOCH protocol (rituximab plus etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin) for C-MYC-driven DLBCL, including patients with double-hit biology. This regimen has shown efficacy in BL  and in isolated C-MYC-rearranged cases, but not in genotypic double-hit DLBCL . More importantly, the median age of these patients is 69 years, meaning that the majority will not be able to tolerate this regimen because of toxicity . Therefore, while extremely promising, much work remains to be done toward the goal of improving the prognosis of these patients.
B cell lymphoma, unclassified
The current WHO classification system defines this category as a lymphoma that has both morphological and genetic features of DLBCL and BL, but cannot be decisively attributed to either disease entity. This includes morphology (cells not typical of BL) as well as immunohistochemistry (lacking the full BL phenotype) and genetics. C-MYC FISH is mandatory in this category, since 30–50 % show a C-MYC gene translocation. Excluded from this category are typical BL without detectable C-MYC-rearrangement, phenotypic double-hit DLBCL, and otherwise clearly defined entities such as blastoid mantle cell lymphomas. However, according to the definition, transformed follicular lymphomas (FL) can also fall into this category , which is problematic since transformed FL are genetically and biologically different from aggressive lymphomas arising independently from underlying low-grade lymphomas. In contrast to de novo DLBCL, FL present with STAT6, ARID1A, and FAS mutations and or deletions, as well as aberrations of MLL2, CREBBP, and BCL2 . Approximately 35–50 % of these cases have C-MYC translocations, especially non-IG/C-MYC translocations .
According to the data of Hummel et al. , rare DLBCL (7/114, 6 %) may show a true BL-signature on GEP and may be missed by the current definition based on morphology, immunohistochemistry, and FISH diagnostic/cytogenetics. Finally, there are cases classified as BL or BCL-U that lack C-MYC-translocation but present with chromosome 11q aberrations, characterized by interstitial gains including 11q23.2-q23.3 and telomeric losses of 11q24.1-qter , indicating a molecularly distinct subset of B cell lymphomas reminiscent of BL.
BCL-U are considered aggressive lymphomas with a shortened overall survival time [29, 59], and recent data suggests that C-MYC is most probably the main cause for this influence on prognosis, while BCL-U cases without C-MYC alterations do not differ from DLBCL cases . Similar observations have been made in DLBCL based on expression of bcl2 and c-myc, which abrogate the prognostic significance of the germinal center type/non-germinal center type DLBCL categories based on GEP data .
The central role of the C-MYC oncogene in the pathogenesis of aggressive lymphomas warrants further clinical research. However, the c-myc protein itself appears to be an undruggable target given that efforts to reduce C-MYC expression or interfere with its interaction partner, MAX, have not been successful to date. Novel approaches will target BRD4, a member of the bromodomain and extraterminal superfamily proteins (BET) [60, 61]. BRD4 regulates c-mycexpression directly and indirectly via miRNA expression , and interference with BRD4 by small molecules (JQ1 and iBET) has shown significant downregulation of c-myc . Such an interfering small molecule GSK525762 is currently being tested in a phase I study for relapsed hematologic diseases (NCT01943851). If the initial findings regarding BRD4 and C-MYC can be translated into clinical practice, C-MYC status could become not only a prognostic, but also a predictive marker for BL, DLBCL, and other C-MYC-driven lymphoproliferative diseases, e.g., multiple myeloma and plasmablastic lymphoma.
The risk stratification of the clinically heterogeneous group of DLBCL is still ongoing. Recently, C-MYC and BCL2 have emerged as promising prognostic (immunohistochemical and genetic) markers that clearly stratify DLBCL patients into different risk groups. Unresolved issues include the fact that double-hit DLBCL (on the genetic level) and DHS 2 DLBCL (on the protein expression level) are not the same despite significant overlap between these two groups, and it remains to be determined whether risk stratification by FISH is superior to immunohistochemistry or vice versa. From our point of view, immunohistochemistry has advantages such as practicability, short turn-around time. and low cost, but FISH analysis remains an indispensable tool in some of the above-described situations and cannot be completely replaced by immunohistochemistry. Considering that immunohistochemical stainings with the anti-c-myc antibody is a very recent methodology with limited experience, we recommend the use of a combined FISH and immunohistochemical model (Table 1). In light of the upcoming therapeutic options such as small molecules that downregulate C-MYC, pathologies should be ready for adequate determination of C-MYC status.
Conflict of interest
The authors declare that they have no conflict of interest.
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