Decreased MYC-associated factor X (MAX) expression is a new potential biomarker for adverse prognosis in anaplastic large cell lymphoma

MYC-associated factor X (MAX) is a protein in the basic helix-loop-helix leucine zipper family, which is ubiquitously and constitutively expressed in various normal tissues and tumors. MAX protein mediates various cellular functions such as proliferation, differentiation, and apoptosis through the MYC-MAX protein complex. Recently, it has been reported that MYC regulates the proliferation of anaplastic large cell lymphoma. However, the expression and function of MAX in anaplastic large cell lymphoma remain to be elucidated. We herein investigated MAX expression in anaplastic large cell lymphoma (ALCL) and peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) and found 11 of 37 patients (30%) with ALCL lacked MAX expression, whereas 15 of 15 patients (100%) with PTCL-NOS expressed MAX protein. ALCL patients lacking MAX expression had a significantly inferior prognosis compared with patients having MAX expression. Moreover, patients without MAX expression significantly had histological non-common variants, which were mainly detected in aggressive ALCL cases. Immunohistochemical analysis showed that MAX expression was related to the expression of MYC and cytotoxic molecules. These findings demonstrate that lack of MAX expression is a potential poor prognostic biomarker in ALCL and a candidate marker for differential diagnosis of ALCL and PTCL-NOS.

clinical characteristics according to MAX expression in ALcL patients. We compared clinical characteristics between ALCL patients with MAX expression (MAX-positive ALCL) and ALCL patients without MAX expression (MAX-negative ALCL). As shown in Table 1, there were no significant differences in clinical features between MAX-positive and MAX-negative ALCL patients, such as invasion site (p = 0.295 to 1.000), clinical stage (p = 0.940), IPI (p = 0.940), and serum lactate dehydrogenase (p = 0.908). Furthermore, no significant difference in MAX expression was observed in cALCL (p = 0.391). However, serum soluble interleukin-2 receptor level was higher in MAX-negative ALCL than in MAX-positive ALCL, although the difference was not statistically significant.
clinical outcomes according to MAX expression in ALcL patients. We analyzed clinical outcomes of 37 ALCL patients. The 3-year progression-free survival (PFS) and overall survival (OS) rates of all ALCL patients were 55% and 64%, respectively. The 3-year PFS rate was significantly lower in MAX-negative ALCL than in MAX-positive ALCL (18% vs 64%, p = 0.044). The 5-year OS rate in MAX-negative ALCL (n = 11) was also significantly lower than that in MAX-positive ALCL (n = 26) (30% vs 77%, p = 0.019) (Fig. 3A). As cALCL is known to have a good prognosis and is usually classified as an independent entity from systemic ALCL, we analyzed PFS and OS again in only systemic ALCL patients (Fig. 3B). In these patients, 3-year PFS and OS rates in MAX-negative ALCL (n = 10) were significantly lower than those in MAX-positive ALCL (n = 19) (PFS, 23% vs 63%, p = 0.016; OS, 19% vs 66%, p = 0.016, respectively). Because MYC-MAX heterodimerization is essential for MYC-driven oncogenesis, we stratified patients according to MYC expression and conducted Cox analysis. In our cohort, there were no cases of MYC(-) and MAX(-). MYC-positive ALCL had a poorer prognosis than MYC-negative ALCL, although the difference was not statistically significant (Fig. 3C). Of note, MAX-positive ALCL had a better prognosis than MAX-negative ALCL, regardless of MYC expression (Fig. 3D).
To evaluate the possibility that MAX expression serves as an independent prognostic factor in ALCL, we conducted univariate and multivariate Cox regression analyses for PFS and OS using the following variables: sex, IPI, MYC expression, MAX expression, and ALK expression. In univariate and multivariate analyses for PFS, significant differences were detected in both IPI status (multivariate: p = 0.013) and MAX expression (multivariate: p = 0.022) ( Table 2A). The long-term survival rate associated.
Univariate and multivariate analyses for OS also showed a statistically significant difference in both IPI status (univariate: p = 0.037, multivariate: p = 0.034, respectively) and MAX expression (univariate: p = 0.029, multivariate: p = 0.044, respectively). Multivariate analysis for OS also showed that both IPI status and MAX expression were independent factors for ALCL (Table 2B). ALK expression was not an independent prognostic factor in this www.nature.com/scientificreports/ study and there was no statistically difference in PFS and OS between ALK-positive and ALK-negative ALCL. This may be due to similarity of age in these two groups. Although ALK-positive ALCL cases usually have better prognosis than that of ALK-negative ALCL, this difference may be due to the fact that ALK-positive ALCL occurs more frequently at a young patient 17 . These results indicate that decreased MAX expression might be a biomarker of poor prognosis in ALCL.  morphological patterns of all ALCL patients. ALCL is known to have several morphological patterns. Ninetytwo percent of MAX-positive ALCL patients were classified into the so-called common type and only 64% of MAX-negative ALCL patients were classified into this type (p = 0.037) ( Table 3). The remaining MAX-negative patients exhibited non-common patterns such as lymphohistiocytic pattern, small cell pattern, and Hodgkinlike pattern (Fig. 4A). We also explored the expression of lymphoma-associated markers including CD markers, cytotoxic molecules, and MYC protein. fiSH results. We analyzed TP63 and DUSP22 rearrangement for ALCL patients without ALK rearrangement by FISH. The two ALCL patients with TP63 rearrangement were included in the MAX-negative ALCL

Discussion
In this study, we found that decreased MAX expression is a potential adverse prognostic factor in ALCL patients. Our results are comparable to those of a previous report of lymphoblastic lymphoma, in which lack of MAX expression was shown as a worse prognostic factor 16 . MYC translocation or amplification is associated with an aggressive clinical course in ALCL [10][11][12]20 . As MAX is an essential molecule for the oncogenic activity of MYC to form a heterodimer with MYC protein, it is conceivable that MAX expression affects MYC-driven oncogenic activity in ALCL. Indeed, MYC-positive ALCL patients tended to have a worse prognosis than MYC-negative ALCL patients, although the difference was not statistically significant in our cohort. Moreover, MAX-negative ALCL patients had a worse prognosis than MAX-positive ALCL patients, regardless of MYC expression. This result is rational because MYC transcriptional activity is dependent on MAX. MAX has a biphasic effect on MYCrelated transcription activity. Abundant MAX expression generates more MAX-MAX homodimer availability and represses MYC activity through the occupation of DNA binding sites (E-box) of MYC-MAX heterodimer by the homodimer. Decreased MAX protein permits MYC to heterodimerize with MAX instead of MAX-MAX homodimer and to upregulate MYC transcription activity (Fig. 5). In fact, significantly lower MAX expression was observed in ALCL than in PTCL-NOS, while MYC expression levels were similar between groups both in our study and other data. Interestingly, MAX mRNA levels in ALCL were lower than those in other mature T-cell lymphomas, regardless of MYC expression. From these results, this peculiar relationship between MYC and MAX as mentioned above may be characteristic for ALCL. Additionally, the detection of MAX expression may aid in the differential diagnosis between ALCL and PTCL-NOS. From the immunohistochemical results, decreased MAX expression correlated with the expression of cytotoxic molecules such as TIA-1 and granzyme B. Recent reports have shown that expression of cytotoxic molecules may be independent prognostic factors in mature T-cell neoplasms including ALCL [21][22][23] . Thus, the prognostic difference between MAX-positive and MAX-negative ALCL may be a result of the expression of these molecules. Most of the p63-positve cases and CD56-positive cases were included in MAX-negative group. The high expression of p63, CD56, and high rates of Ki-67 are also known as prognostic markers of ALCL 18,19 . Additionally, several reports recently described that cytotoxic molecules are expressed in ALCL with TP63 rearrangement but not in ALCL with DUSP22 rearrangement 4,6 . This finding is in agreement with our results, which showed that the expression of these molecules was detected in 2 ALCLs with TP63 rearrangement (both of them were MAX  Supplementary  Table S4B online). These results indicated that DUSP22 or TP63 rearrangement might have a partial influence on the expression of cytotoxic molecules through MAX expression in ALCL.
We also tried to characterize morphological features of MAX-positive or MAX-negative ALCL. Our result that MAX-negative ALCL was related to histological features of non-common variants is consistent with the report of Lamant et al., who showed that ALCL with small cell variant or lymphohistiocytic variant had a worse prognosis than ALCL with common variant 24 .
In summary, we demonstrated that (1) decreased MAX expression could be a poor prognostic factor in ALCL, probably through cytotoxic molecules in coordination with MYC, (2) decreased MAX expression is related to histological non-common patterns of ALCL (e.g., patients that had a poor prognosis), and (3) decreased MAX expression might help to distinguish between ALCL and PTCL-NOS.
This study is limited because of a small number of cases so that further extensive studies will be necessary to determine whether the loss of MAX expression is an independent poor prognostic factor in ALCL including the functional analysis of MAX in ALCL.  Rt-pcR and mRnA expression. Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific) and reverse transcribed using the Super Script™ III First-Strand Synthesis SuperMix (Thermo Fisher Scientific) for PCR according to the manufacturer's protocol. MAX and GAPDH cDNA sequences were obtained from the National Center for Biotechnology Information GenBank database (https ://www.ncbi.nlm.  immunohistochemical analysis. Immunohistochemical analysis was performed as previously described 28 . Antibodies listed in Supplementary Table S1 were used for immunohistochemical detection. Immunohistochemical staining was performed using Ventana i-View DAB kit reagents (Ventana Medical Systems, Tucson, AZ, USA) and an automated immunostainer (Ventana ULTRA). Protein expression was blindly assessed by two pathologists (T.Y. and J.T.). Immunohistochemical results were defined as positive or negative according to the proportion of positive cells in 5 fields. Criteria used to indicate positive staining were as follows: all CD markers, TIA-1, and granzyme B, > 20% of cancer cells stained 29 ; MAX, ≥ 30% of cancer cells stained 16 ; MYC, ≥ 40% of cancer cells stained 25 ; and Ki-67 and p63, ≥ 70% of cancer cells stained 5,26 . Moreover, the intensity of the MAX-positive signal was scored from 0 to 5 + , and > 3 + was assessed as positive 16 . fluorescent in situ hybridization (fiSH). FISH probes for TP63 and DUSP22 were purchased from ZytoVision GmbH (ZytoLight SPEC IRF4, DUSP22 Dual Color Break Apart Probe, Bremerharven, Germany) and Empire Genomics (TP63 Break Apart FISH probe, Williamsville, NY, USA), respectively. For DUSP22, break apart probe labeled with Spectrum ZyOrange and ZyGreen labeled polynucleotide target sequences mapping to 6p25.3 distal and proximal to the DUSP22 gene region, respectively. For TP63, break apart probe consisted of distal and proximal regions to TP63 region in 3q28 and were labeled with Spectrum Orange and Spectrum Green, respectively. Images were obtained and analyzed according to routine institutional protocols. Cut-off levels for positive FISH signal were 10% and 4.5% for DUSP22 and TP63, respectively, as previously described 6,25,30 . Total counted numbers of target cells were approximately 100 cells for detection of fracture, and all cases were judged by two or more investigators.

Statistical analyses.
Comparisons between groups for immunohistochemical analysis were carried out using Fisher's exact test, the Mann-Whitney U test, or the Wilcoxon test. The Kaplan-Meier method and logrank test were used for comparison of overall survival and progression-free survival between groups separated by immunohistochemical results. Univariate and multivariate Cox regression analyses were performed to test the association between predicted prognostic factors and survival outcome. In all cases, results were considered significant at p < 0.05. Statistical testing was performed using JMP12 (SAS, Tokyo, Japan).
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