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The role of SOX11 in mantle cell lymphoma
Leukemia Research, 11, 37, pages 1412 - 1419
The mechanism of SOX11 function has been widely published recently mainly focused on histone modifications. Besides diagnostic value in mantle cell lymphoma (MCL), SOX11 has also prognostic significance. Although it can also be observed in a fraction of other T and B-cell lymphomas, a monoclonal antibody, called SOX11-C1, may improve the function of SOX11 in both diagnosis and prognosis evaluation. In addition, detection of modified SOX11 cDNA by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) has a higher sensitivity than traditional CCND1 examination in minimal residual disease (MRD) detection, which is an appealing option for predicting disease outcome and status.
Keywords: Mantle cell lymphoma, SOX11, CCND1.
The Sry-related high-mobility-group (HMG) box (SOX) is a group of developmentally regulated transcription factors. There are approximately 20 SOX genes which can be further subdivided into eight groups (groups A to H), according to the degree of Sry, sharing both within and outside the HMG domain  . SOX C group is comprised of SOX4, SOX11, and SOX12  . They share two functional domains: a Sry-related SOX DNA-binding domain, located in the N-terminal, and a transactivation domain (TAD) at the C-terminal  . SOX11, a member of the SOX gene family, had been cloned and characterized by the partial cloning of both human and mouse SOX11 genes and mapped it to chromosome 2p25  . Many of the SOX genes, including SOX11, are widely expressed in the developing nervous system and may have a role in neurogenesis, neural cell survival and neurite outgrowth  . In contrast, it has been shown that many adult tissues are absent for SOX11  . In addition, SOX11 plays an important role in tissue remodeling and SOX11-deficient mice presented with various craniofacial and skeletal malformations, asplenia and hypoplasia of the lung and stomach, and SOX11 mutation is corresponding human malformation syndromes  . SOX11, which is expressed in virtually all aggressive mantle cell lymphoma (MCL), has recently been recognized as a diagnostic and prognostic antigen in MCL  . The mechanism of SOX11 function has been published recently , , and . DNA hypermethylation of SOX11 seems to be functionally inserted to SOX11 expression whereas histone modifications are much more important  and . In addition, SOX11 could target a lot of genes resulting in blocking of mature B-cell differentiation, modulations of cell fate, apoptosis and stem cell development  . Silence of SOX11 expression reduces tumor growth both in vitro and in vivo  . MCL is characterized by overexpression of CCND1 as the result of the translocation t(11;14) (q13;q32), and shows an aggressive clinical course with a frequent relapse pattern and a median survival of only 3–5 years  . However, approximately 10% of MCL lacked this specific translocation and did not express CCND1  . SOX11 is specifically expressed in almost all of MCL regardless of CCND1 status , , , and . Meanwhile, there is a subgroup of MCL does not express SOX11 and presents with an indolent behavior, which might not need therapy at diagnosis , , , and . All of these make SOX11 a differentiation biomarker of great importance. However, batch-to-batch variations of commercially available polyclonal antibody make the result of immunohistochemistry (IHC) controversial, which have hampered its routine clinical use  . At the same time, a fraction of other T and B-cell lymphomas were also reported expressing SOX11  and . Recently, a monoclonal mouse antibody, called SOX11-C1, plays robust activities in both IHC and flow cytometry (FCM)  , which may improve the performance of SOX11. A quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay, designed by Hamborg et al.  , using SOX11 cDNA with higher sensitivity than CCND1, seems to be an appealing option for minimal residual disease (MRD) detection.
2. SOX11 and tumors
The transcription factor SOX11, which is a member of the SOX family and plays a critical role in the regulation of cell cycle and differentiation in major developmental processes  , has recently been recognized as a diagnostic, prognostic and/or functional antigen in a variety of tumors including MCL , , , , , , and , epithelial ovarian cancer (EOC)  and  and gliomas  . Of major clinical interest, the expression of SOX11 in non-malignant tissues is limited to immature neurons  and tissue remodeling , , and . It is initially expressed throughout the central nervous system (CNS) and then down-regulated in the spinal cord  and , and expressed outside of the CNS , , , and . In contrast to its extensive occurrence during embryogenesis, there is little SOX11 expression in the adult  . The malformations showed by Sock et al.  in the SOX11-deficient mice indicated that an original function for SOX11 in tissue remodeling. In addition, SOX11 plays important roles in tumorigenesis. Sernbo et al.  reported that SOX11 is a functionally related protein in EOC with prognostic value for high-grade tumors. SOX11 is also highly expressed in malignant gliomas whereas normal in adult brain and other organs  . Human glioma-initiating cells (GICs) lost expression of SOX11, and overexpression SOX11 prevented their oncogenesis in vivo  . SOX4, the SOX family member with the highest homology to SOX11, is a prominent transcription factor in B and T cells  and is crucial for B-cell lymphopoiesis. In contrast, SOX11 has no known lymphopoietic function and it is not expressed in lymphoid progenitors or mature normal B-cells. However, it is expressed in virtually all aggressive MCL and at lower levels in some Burkitt lymphomas (BL) and acute lymphoblastic leukemia (ALL) but not in other lymphoid neoplasms , , and .
3. SOX11 and MCL
Non-Hodgkin lymphomas (NHL) can be divided into several subgroups according to their morphological and phenotypic properties refer to WHO classification  . MCL represents 5–10% of all NHL and predominates in males with advanced age. The clinical evolution is usually very aggressive with short responses to treatment, continuous relapses and a median survival of 3–5 years  , and patients cannot be cured with current therapies. The diagnosis of MCL is characterized by overexpression of CCND1 because of the translocation t(11;14) (q13;q32), and examination of CCND1 protein by IHC or evidence of CCND1/immunoglobulin heavy chain (IGH) fusion by fluorescence in situ hybridization (FISH). However, genomic expression profiling (GEP) studies have shown that approximately 10% of MCLs with an otherwise similar GEP lacked this specific translocation and did not express CCND1, which makes diagnosis of MCL difficult in these cases  . Since the clinical behavior of CCND1 negative MCL is also aggressive, it is important to find biomarkers that reliably identify this entity and separate them from the other B-cell NHLs (B-NHLs) in the routine clinical laboratory setting. Some cases of CCND1-negative MCL demonstrate CCND2 or CCND3 mRNA overexpression  ; however, GEP and performance of CCND2 or CCND3 qRT-PCR assays are not extensively feasible in clinical diagnostic laboratories and also prevented by the fact that both are also expressed in other B-NHL. The transcription factor SOX11, a neural transcription factor whose function in normal and neoplastic B-cell development is unknown, is specifically expressed in the nucleus of MCL compared with other lymphomas and benign lymphoid tissues. Whether SOX11 expression is correlated to CCND1 expression or CCND1 translocation is controversial. Recent studies show both SOX11 mRNA up-regulation and SOX11 protein overexpression in MCL regardless of CCND1 expression and CCND1 translocation status , , , and . Similar report showed no general co-regulation was found between CCND1 and SOX11 mRNA.
4. How does SOX11 work in tumorigenesis
SOX11 has a synergistic effect with WT1, a regulator of Wnt4 promoter, in the regulation of Wnt4-promoted nephrogenesis  . Knockdown of SOX11 in neuroblastoma cells increased the expression of the pro-apoptotic gene BNIP3 (BCL2 interacting protein-1 NIP3) and decreased the expression of the anti-apoptotic gene TANK (TNF receptor-associated factor family member-associated NF-κB activator)  . Therefore, it is hypothesized that SOX11 may contribute to the pathogenesis and progression of MCL by regulating genes involved in cell proliferation and apoptosis  . SOX11 up-regulation has been detected in various types of solid tumors including medulloblastomas, gliomas and epithelial ovarian tumors , , and . On the other side, Hide et al.  revealed that overexpression of SOX11 prevented tumorigenesis of in mouse glioma cell line (NSCL61s) by inducing their neuronal differentiation accompanied with decreased levels of plagl1, which was originally shown to regulate both cell cycle arrest and apoptosis. Plagl1 plays an important role in tumorigenesis of GICs, which has been shown to regulate the expression of several imprinted genes those are involved in tumorigenesis and embryonic growth  . Experiments in the induced NSCL61 cell line have disclosed that overexpression of SOX11 blocked their tumourigenicity and recurrent glioblastomas. In addition, downregulation of SOX11 mRNA resulted in diminished patient survival  . Using global gene expression analysis, Gustavsson et al.  showed that the CCND1-related Rb-E2F pathway  and  was affected by the increased level of SOX11. Besides, a significant positive relationship was documented between SOX11 and p-STAT-3 expression, which is a promoter of neurite growth  and induces astrocytic differentiation during CNS development  . Similarly, silencing of SOX11 in MCL cell lines caused a deregulation of STAT-1 transcription  . However, whether a similar interaction may exist between SOX11 and STAT-3 is presently unknown.
Although SOX11 does not seem to play a role in hematopoiesis, its expression was observed in various aggressive B-cell neoplasms, suggesting that this protein plays a role in the pathogenesis of these tumors. In particular, SOX11 is highly expressed in MCL, ALL and some BL , , , , and . In contrast, patients with an indolent variant of MCL  and other mature B-cell neoplasias such as chronic lymphocytic leukemia (CLL), follicular lymphoma (FL) or diffuse large B-cell lymphoma (DLBCL) do not express SOX11 , , and .
Chromosomal abnormalities, like translocations or gene amplifications, comprise one of the main mechanisms resulting in deregulated gene expression in lymphomas  . In the case of SOX11, chromosomal modifications affecting band 2p25 have not been identified in MCL, BL or ALL , , , , and . The relationship between methylation status of SOX11 promoter region and SOX11 expression is controversial. The expression of SOX11 is inversely correlated to specific promoter methylation in hematopoietic malignancies  . Vegliante et al.  analyzed DNA methylation status of SOX11 by microarray and qRT-PCR and reported that SOX11 was generally unmethylated in MCL cell lines and some types of lymphoid neoplasias like TEL-AML1 positive-ALL whereas all non-MCL cell lines were strongly methylated  . A significant inverse correlation between SOX11 promoter methylation and gene expression had been recognized by looking into the correlation between gene expression and DNA methylation  . However, in many samples (embryonic/adult stem cells, normal B cells and some indolent MCL (iMCL), some CLL and FL), SOX11 expression was repressed in spite of its unmethylated status. These findings indicated that SOX11 expression did not depend exclusively on the DNA methylation status of the gene. Analyzing together with SOX11 expression, DNA methylation and histone marks in the same cells suggested that SOX11 expression was associated with activating histone marks and absence of DNA methylation. In contrast, absence of SOX11 expression was associated with silencing histone marks regardless of simultaneous presence of DNA methylation ( Fig. 1 )  . These results suggest that histone marks, rather than DNA methylation, are the major epigenetic mechanism controlling SOX11 expression. Wasik et al.  reported that the expression of the SOXC genes is highly correlated in SOX11 positive MCL and the SOX11 promoter region is hypomethylated in both MCL and normal B-lymphocytes. Interestingly, low methylation of the SOX11 promoter was found both in SOX11 positive cell lines and primary MCL, as well as in SOX11 negative MCL cell lines, which confirmed the findings by Vegliante et al.  . Further detection revealed that SOX11 silencing in cell lines was reversed by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) ( Fig. 1 ) but not the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (AZA)  . These suggest that, although DNA hypermethylation of SOX11 is frequent in lymphomatosis, it seems to be functionally inserted, as SOX11 is already silenced in the hematopoietic system.
In contrast, the pathogenic role of SOX11 is concerned with its de novo expression in some aggressive lymphoid malignancies, which is regulated by a shift from inactivating to activate histone modifications. Conrotto et al.  showed an association between SOX11 level and cellular growth rate in tumorigenic cells and in NOD-SCID mice, knocking down of SOX11, resulting in an increased cellular growth dependent on autotaxin (ATX) which can be regulated by SOX11 ( Fig. 1 )  and . Both siRNA-mediated overexpression of SOX11 and knocking down lead to altered proliferation, so they suggested that SOX11 is not an onlooker but an active and central regulator of cellular growth. Similar interpretation existed, using ChIP-chip analysis combined with GEP upon SOX11 knocking down, Vegliante et al.  recognized target genes and transcriptional programs including the block of mature B-cell differentiation, modulation of cell cycle, apoptosis and stem cell development regulated by SOX11. PAX5, an essential transcription factor regulating early B-cell development  and late B-cell differentiation  , emerges as one of the major SOX11 direct targets  . PAX5 repression, owing to SOX11 silencing, induces BLIMP1 expression and promotes the shift from a mature B-cell into the initial plasmacytic differentiation phenotype in both vivo and in vitro ( Fig. 1 ). The induction in luciferase activity was detected in the co-expression with SOX11 but not with SOX4 or truncated SOX11 proteins lacking the HMG domain (ΔHMGSOX11) or the C-terminal TAD domain (ΔTADSOX11), both required for its transcriptional activity  and . These findings indicate the specificity of SOX11 in regulating the transcription of PAX5. SOX11 silencing reduced tumor growth rate compared to SOX11-positive control and significant alteration in tumor size. Concordant with the in vitro results, in vivo SOX11-silenced tumors demonstrated SOX11 and PAX5 down-regulation and BLIMP1 up-regulation compared to SOX11-positive tumors ( Fig. 1 ). However, significant changes in cell density proliferation, cycle phases or viability after SOX11 silencing in cell lines were not observed. SOX11-positive primary MCL had a GEP significantly enriched in SOX11-upregulated genes whereas SOX11-negative ones presented an enrichment in SOX11-downregulated genes. Conversely, SOX11 overexpression in aggressive MCL may block the cells in a mature B-cell stage and prevent their further differentiation  . Resistance of MCL to new treatments with proteasome inhibitors has been linked to the development of plasmacytic differentiation, indicating that this finding may also have implications in the design of new therapeutic strategies. Above all, SOX11 is not a bystander but an active and central regulator of cellular growth.
5. Diagnostic and prognostic role of SOX11 in MCL
5.1. iMCL is a new subgroup of MCL
In the last few years, there has been recognition that a subgroup of patients with MCL have a significantly longer survival (often more than 7–10 years) and a more indolent disease course who did not receive upfront chemotherapy at the time of diagnosis but were instead managed with a ‘watch and wait’ strategy  . Although diagnostic criteria for the recognition of these patients are not currently available, there is evolving identification of clinicopathological differences identifying this group from the group of patients with classical MCL (cMCL). It is becoming clear that many, although by no means all, patients with indolent disease present with a leukemic picture rather than nodal disease  .
5.2. The features of iMCL
These patients show a non-progressive or slowly progressive course with the characteristics as follows ( Table 1 ): (a) more frequent immunoglobulin heavy chain variable gene (IGHV) mutations; (b) a significant proportions had a subset of CD23 positive cells (usually negative in MCL) and kappa light chain restriction, as opposed to the more typical lambda light chain restriction that usually found in conventional MCL  ; (c) lower proliferation rate (through Ki-67 staining), which has been validated within the MCL specific International Prognostic Index (sMIPI) score  ; (d) mild-moderate lymphocytosis, interstitial low-level bone marrow involvement and simple karyotype; (e) expression of CCND1 and lack of SOX11  ; (f) less presented with B symptoms, lower ECOG score, non-nodal presentation and low serum LDH level  .
|Clinical and pathological data||iMCL||cMCL|
|IGHV mutation status||More frequent||Less frequent|
|Bone marrow involvement||More frequent||Less frequent|
|Serum LDH level||Low||High|
|Performance status: ECOG ≥ 2||Scarcely||Common|
|IG light chain expression status||Kappa light chain restriction||Lambda light chain restriction|
Abbreviations: iMCL: indolent mantle cell lymphoma; cMCL: classical MCL; IGHV: immunoglobulin heavy chain variable region; LDH: lactate dehydrogenase; ECOG: Eastern Cooperative Oncology Group; IG: immunoglobulin.
5.3. Different prognostic ideas of SOX11
The prognostic role of SOX11 protein expression in MCL has been controversial ( Table 2 ). Several publications have suggested that lack of SOX11 expression is a feature of MCL with a nonaggressive clinical course , , , , , and , even though exceptions were present  and . Most reports have indicated that a subset of SOX11 negative MCL carrying the typical t(11;14) translocation but with a non-nodal, leukemic presentation has an indolent clinical course , , , , , and , consistent with SOX11-silenced cells in the xenograft experiments  . In contrast, Wang et al. reported that in MCL with lymph node presentation (nodal MCL), absence of nuclear SOX11 was associated with a shorter overall survival (OS)  . Dreyling et al.  identified 13 genes (including SOX11) that were strongly expressed in 13 of the 15 cMCL samples and significantly under expressed in all iMCL samples. They studied an independent series of MCL by IHC and compared negative nuclear SOX11 staining with moderate-strong staining, and found positive SOX11 expression was correlated with poor outcome. In a multivariate analysis, SOX11 expression and Ki-67 were the most important variables for OS. Fernandez et al.  found that whereas both indolent and classical forms of MCL share a common gene expression profile, which differs from that of other leukemic variants of lymphoid neoplasms, there are significant differences between the two. In particular, the 13 genes, similar to Dreyling, were also found to be differentially expressed: all were underexpressed in patients with indolent disease and overexpressed in those with classical disease. SOX11 is one of these and potentially has a critical role in both the pathogenesis of MCL and in the identification of indolent disease  . They reported that patients with SOX11-negative had a longer OS than positive ones  , consistent with the result of Dreyling. However, Nygren and co-worker indicated that lack of SOX11 expression was associated with shorter OS and patients with SOX11-negative MCL had more frequent non-nodal presentation, splenomegaly, and higher WBC and lymphocyte counts than patients with SOX11-positive ones and with a high proliferation index (Ki-67 > 50%) similar to SOX11-positive cases  . The problem is that the majority of the SOX11 negative cases in that study strongly expressed p53, which may impair survival in that cohort. Strong expression of p53 is generally an indication of a mutation in the TP53 gene, resulting in a non-functioning p53 protein that is not properly degraded  . In addition, strong p53 positivity was seen in 69% of SOX11 negative cases and in 16% of SOX11 positive ones in Nygren's study and which was strongly associated to shorter OS. SOX4, another member of the SOXC group of transcription factors (consist of SOX4, SOX11 and SOX12), closely related to SOX11, competing for transcribing of the same target genes  , has been indicated to interact with p53  . It is probable that high p53 expression among SOX11 negative cases contributed to the shorter survival and it could be hypothesized that SOX11 may have similar functions but this remains to be investigated  . Ondrejka et al.  reported that leukemic MCL limited to blood and BM is an indolent variant characterized by mild-moderate lymphocytosis, low level BM involvement, simple karyotype, kappa light chain expression, CCND1 expression with lack of SOX11, and slow or absent clinical progression. As postulated by Ondrejka et al.  , some of the cases of leukemic MCL may in fact be considered a form of MCL-type monoclonal B-cell lymphocytosis rather than MCL.
|Studies||The prognostic significance of SOX11 negative cases|
|Ondrejka at el ||Leukemic picture, mild-moderate lymphocytosis, interstitial low level bone marrow involvement, simple karyotype, kappa light chain expression, CCND1 expression and slow or absent clinical progression|
|Wang et al. ||A shorter survival than patients with MCL with nuclear SOX11 expression|
|Orchard et al. ||Mutated IGHV genes, good prognosis, and non-nodal disease|
|Fernandez et al. ||A non-nodal leukemic disease with predominantly hypermutated IGHV and lack of genomic complexity|
|Kimura et al. ||Indolent lymphoma characterized by bone marrow involvement, splenomegaly, and a low Ki-67 labeling index|
|Carvajal et al. ||Indolent clinical behavior and managed without any treatment for more than 2 years, a less tendency to progress|
|Nygren et al. ||SOX11 negativity is not associated with an indolent clinical course|
|Dreyling et al. ||A non-nodal presentation with predominantly hypermutated IGHV, lack of genomic complexity, low LDH level and MIPI and long OS|
|Navarro et al. ||MCL with mutated IGHV, SOX11- negativity, and non-nodal presentation correspond to a subtype of the disease with more indolent behavior|
Abbreviations: MCL: mantle cell lymphoma; IGHV: immunoglobulin heavy chain variable region; LDH: lactate dehydrogenase; MIPI: mantle cell lymphoma international prognostic index.
Although it remains to be confirmed whether SOX11-negative cases identify an indolent rare subtype  and  or conversely, patients with a shorter survival  , there is no doubt that iMCL does exist. In addition, it is well recognized within patients presenting with non-nodal and leukemic disease. SOX11 and other genes are likely to become useful in the recognition of these patients at diagnosis and this will ultimately provide clinicians with the confidence to deliver less intensive treatment approaches.
5.4. SOX11 and ‘in situ MCL’
It is well recognized that MCL has an extensive spectrum of growth patterns. In most conditions, a vaguely nodular and/or diffuse growth pattern is observed, while very rare cases have a follicular growth pattern, and a larger minority has a mantle zone growth model in which the lymphoma grows like an expanded ring around reactive germinal centers  . Although controversial, one study indicated that a mantle zone growth model was associated with a better outcome  . More recently, CCND1-positive MCL-like cells restricted to the mantle zone of hyperplastic follicles in otherwise reactive lymph nodes have been described by isolated reports , , and . The restricted distribution pattern of the atypical cells indicates that these lesions may represent an early stage in the development of MCL, and the process has been termed ‘in situ MCL’.
As is known that ‘in situ MCL’ usually represents with a very indolent behavior  , in this condition, SOX11 could be a useful biomarker to identify two subtypes of MCLs with different clinicopathologic features and prognosis. These findings strongly suggest that the lack of SOX11 in a CCND1-positive lymphoma may recognize a subtype of MCL with a different biological activity than conventional SOX11-positive MCL.
For this reason, these cases must be told apart from conventional MCL because they may not need therapeutic intervention. It is intriguing, although not statistically significant, that the SOX11-negative ‘in situ MCL’ lesions were primarily found in women whereas SOX11-positive ones and mantle zone pattern cases were more common in men. Both SOX11-negative and -positive ‘in situ MCL’ had a very indolent biological behavior  . Similarly, leukemic MCL limited to blood and BM is an indolent subset characterized by absent of SOX11  and . Some ‘in situ MCL’ lesions expressing SOX11, progresses to an overt MCL, and the higher number of cases of SOX11-positive MCL with a mantle zone model suggest that SOX11 is already differentially expressed in the early development of ‘in situ MCL’ lesions and indicate that lesions expressing SOX11 may have a greater tendency to progress  . In these cases they indicated that a diagnosis of ‘in situ involvement by MCL-like cells’ should be made, with a note suggesting that this does not build a diagnosis of lymphoma, and that treatment should not be undertaken depended exclusively on the basis of this finding. In such cases, surveillance of the patients and evaluation for the presence of progressive disease should be considered  .
Overall, MCL with SOX11 negative corresponds to a subtype of the disease with more indolent clinicopathologic behavior and SOX11 positive ‘in situ MCL’ lesions should be treated with cautions. Analysis the immunogenetic features of MCL, Navarro et al.  revealed that the IGHV highly mutated tumors had less genomic complexity, were preferentially SOX11-negative, and showed more frequent non-nodal disease. In a multivariate analysis, IGHV gene status and SOX11 expression were independent risk factors. Although little is well documented about the function of SOX11 in MCL, considerable interest has been raised since its expression may correlate to disease course. Thus, several hematologists have suggested that the lack of SOX11 expression is a character of MCL with a nonaggressive clinical course.
6. Positive results of SOX11 in other subgroup of lymphomas
SOX11 expression has also been reported in a fraction of other T and B-cell lymphomas, including B and T-cell lymphoblastic lymphomas and a subset of BL  and , plasma cell neoplasms and hairy cell leukemia (HCL)  and . However, the most common B-cell lymphomas lack SOX11 expression  and  and the different morphological and phenotypic characters of these malignancies make it easy to differentiate them from CCND1-negative MCL  . Thus, using SOX11 as a diagnostic marker for MCL avoids misclassification of morphologically similar CD5+ marginal zone lymphomas (MZL) or CD23− CLL. A few single case reports described in the literature shown that rare cases of DLBCL, the most common type of lymphoma, can also express CCND1  and . Nevertheless, all of which were negative for SOX11  and . Thus, SOX11 immunostaining is useful in the differential diagnosis of CCND1-positive DLBCL, particularly the morphological variant. At all events, SOX11 can become a potential marker for the diagnosis of MCL regardless of the presence or absence of CCND1  .
7. A monoclonal mouse antibody of SOX11
Polyclonal antibodies targeting SOX11 have been extensively used in IHC on paraffin sections , , , , , and . However, batch-to-batch variations of commercially available reagents have hampered routine clinical use where standardized protocols are a prior condition. All of these controversies came from non-specific staining might be worked out by a monoclonal mouse antibody, called SOX11-C1, developed by Nordstrom et al.  . Unlike the polyclonal antibodies, this monoclonal antibody against a C-terminal peptide of SOX11, which has no homology to the closely related protein SOX4, gives rise to its high sensitivity and improved specificity and offers single epitope specificity and no batch-to-batch variation. Additionally, a hybridoma clone in murine or hamster cell lines enables large-scale production at low cost  . Nordstrom et al.  indicated that the previous cases detected with SOX11, including B and T lymphoblastic lymphomas, FL, CLL, HCL, tonsil sections and non-malignant BM show absence of nuclear SOX11 staining, except for BL where two of four cases showed moderate nuclear staining. The case of SOX11 negative MCL detected previously, with a clonal IGH-rearrangement by PCR and a t(11;14) (q13;q32) translocation by FISH, was confirmed to be negative. The monoclonal SOX11-C1 antibody enables improved use of SOX11 as a diagnostic antigen in MCL and provides high sensitivity and improved specificity in IHC. Using the novel SOX11-C1 antibody, none of the tested HCL cases previously reported SOX11 positive  and  showing nuclear staining, which is in concordance with previous mRNA data  . Furthermore, there is an increasing requirement for expanded use of FCM in immunoprofiling of tumors, which is both sensitive and specific to identify, and classify B cell lymphomas. In contrast to IHC, FCM capacitates quantitative measurements at the single cell level and provides the ability to analyze and quantify the co-expression of proteins in defined subgroups, in a manner superior to IHC. However, reagents targeting SOX11 in FCM have been lacking until now. The SOX11-C1 antibody which allowed detection of SOX11 by FCM showed strong or moderate to weak signals in SOX11 positive cell lines whereas negative cell lines presented only background staining, below the defined cut-off level, in accordance with mRNA level and Western blot data published previously  . This antibody can also separate primary samples of MCL from negative non-expressing lymphomas and non-malignant cell by FCM. Dilution experiments revealed that malignant cells, comprising as little as 1% of the total lymphocyte population were detectable. Above all, the SOX11-C1 antibody shows robust performance superior to previous polyclonal antibody in diagnosis of MCL and predicting the outcome of some subpopulations of MCL.
8. SOX11 is a better MRD marker in MCL
The ability of MRD measurements in MCL is now well identified as the overexpression of CCND1 has been shown to be a dependable predictor of the disease outcome  . Another MRD target in MCL was addressed in a study conducted by Anderson et al.  , where the persistence of CCND1/IGH translocation and clonally rearranged IGH genes in patients with MCL after autologous stem cell transplantation was related to a poor outcome compared to patients with no PCR detectable MRD. However, since CCND1 have high background in normal tissues and CCND1/IGH are very laborious to analyze, there is still a desire for alternative MRD markers. Of special interest, the expression of SOX11 was shown to be independent of t(11;14) translocation and CCND1 overexpression  . In addition, Mozos et al.  found that CCND1-negative cases were positive for SOX11 overexpression. Simultaneously, given the extensive application of qPCR assays for disease progression surveillance and responses to therapy in other hematological malignancies such as acute myeloid leukemia (AML)  , the construction of a qPCR assay for SOX11 would seem to be an appealing option. However, quantitative studies previously on the expression of SOX11 in MCL employed qRT-PCR assays were not mRNA specific  . One of the scopes with the current assay, besides diagnostic use, was to use it for MRD measurements in MCL. Previous studies  and  quantifying SOX11 used DNase treatment prior to cDNA synthesis could be inadequate when using the assay for tracing minimal amounts of transcripts for MRD usage and might lead to incorrect measurements  . In this condition, Hamborg et al.  designed a specific SOX11 qRT-PCR assay, a locked nucleic acid (LNA)-modified hydrolysis probe that could amplify and detect SOX11 cDNA efficiently, which overcame the risk of contamination from genomic DNA. This potent assay was available to compare the expression of SOX11 and CCND1 between MCL patients and healthy donors. The results showed the SOX11 expression was much lower in healthy donors and remission MCL patients than patients at diagnosis and relapse, whereas no significant difference was observed in CCND1 expression although lower level was also seen in healthy donors and remission MCL individuals  . In addition, the sensitivity of the SOX11 qPCR assay was 2 × 10−4, while the CCND1qPCR assay was found to be 2 × 10−3 because of the normal background in healthy individuals and in remission samples. Overall, SOX11 is a robust molecular marker for detecting MRD in MCL using both peripheral blood and BM.
In summary, SOX11 is a useful marker special to MCL regardless of CCND1 status. Beside diagnostic value, SOX11 also has prognostic significance. SOX11 negative MCL often documents an indolent behavior, prolonged survival and may even not need therapy at diagnosis. Although it can also be observed positive results in a fraction of other T and B-cell lymphomas, the monoclonal antibody, called SOX11-C1, may create a new situation. In addition, detection of a modified SOX11 cDNA by qRT-PCR has a higher sensitivity than traditional CCND1 examination, which is an appealing option for predicting disease prognosis and status. In short, SOX11 plays an important role in MCL from generation to progression. Large-scale investigation is still needed to explore detail function of SOX11 in lymphoma development. Drugs target SOX11 would be attractive in future therapeutic strategies.
Conflict of interest statement
The authors declare no conflict of interest.
This study was supported by National Natural Science Foundation of China (30971296, 81170485, 81170488, 81200360), Natural Science Foundation of Jiangsu Province (BK2010584, BK2012484), Key Projects of Health Department of Jiangsu Province (K201108), Jiangsu Province's Medical Elite Program (RC2011169), Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institute (JX10231801), National Public Health Grand Research Foundation (201202017), the Program for Development of Innovative Research Team in the First Affiliated Hospital of NJMU, Jiangsu Province, Higher Education Institute Foundation of Science and Technology Innovation Team Program, the Project for State Key Clinical Department Construction.
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