The Utility of MYB Immunohistochemistry (IHC) in Fine Needle Aspiration (FNA) Diagnosis of Adenoid Cystic Carcinoma (AdCC).

Differentiating adenoid cystic carcinoma (AdCC) from other basaloid neoplasm in a fine needle aspiration (FNA) sample can be challenging. Activation of MYB in AdCC by the fusion transcript MYB-NFIB has been recently demonstrated in salivary gland and other organs. The aim of this study is to evaluate the utility of MYB immunohistochemistry (IHC) in distinguishing AdCCs and other basaloid neoplasm in cytology specimens. Eighteen FNA cases, from salivary gland and other sites, and their subsequent surgical resection specimens were included in the study. Eight cases were confirmed AdCC on resection. MYB IHC was performed on slides made from cytology cell block and surgical resection paraffin blocks. Percentage and intensity of nuclear staining in tumor cells was scored as 0 to 3. The staining results were concordant between cytology specimens and their corresponding surgical resection tumors. Strong diffuse nuclear staining (score 3, N = 5) was exclusively observed in AdCC, both in cytology and surgical specimens. Only one pleomorphic adenoma and one poorly differentiated basaloid carcinoma were positive for MYB staining (score 1 to 2). Any degree of nuclear MYB labeling was seen in 100% AdCC cases (N = 8/8) compared with of 20% (N = 2/10) of all other non-AdCC cases (P = < 0.001). The sensitivity and specificity of any degree MYB positivity for AdCC in cytology specimen is 100% and 78%. The sensitivity and specificity of strong diffuse MYB labeling (score 2 to 3) for AdCC is 83% and 100% in cytology specimen. Strong diffuse nuclear staining of MYB is valuable in supporting a cytologic diagnosis of AdCC. However, weak and focal labeling of MYB should be interpreted with caution as it can be seen in benign and other malignant basaloid lesions.

Consistent LEF-1 and MYB Immunohistochemical Expression in Human Papillomavirus-Related Multiphenotypic Sinonasal Carcinoma: A Potential Diagnostic Pitfall.

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC) is a distinct, newly-described sinonasal tract neoplasm characterized by a salivary gland tumor-like appearance with myoepithelial and ductal cells, frequent surface squamous dysplasia, and relatively indolent behavior. When considering a diagnosis of HMSC, aggressive high-grade salivary gland carcinomas, particularly those with a basaloid morphology such as basal cell adenocarcinoma and adenoid cystic carcinoma, enter the differential diagnosis. The full morphologic and immunophenotypic profile of HMSC continues to be unraveled. In this series of ten cases, we demonstrate that this tumor has consistent, strong immunohistochemical expression of LEF-1 yet lacks nuclear expression of ß-catenin, and also has consistent yet variable expression of MYB protein. While LEF-1 expression may be a useful diagnostic adjunct, it can also be a pitfall, as other salivary tumors such as basal cell adenocarcinoma have been previously shown to express LEF-1. Additionally, MYB protein expression is not a discriminatory marker when trying to separate HMSC from adenoid cystic carcinoma.

MYBL1 rearrangements and MYB amplification in breast adenoid cystic carcinomas lacking the MYB-NFIB fusion gene.

Breast adenoid cystic carcinoma (AdCC), a rare type of triple-negative breast cancer, has been shown to be driven by MYB pathway activation, most often underpinned by the MYB-NFIB fusion gene. Alternative genetic mechanisms, such as MYBL1 rearrangements, have been reported in MYB-NFIB-negative salivary gland AdCCs. Here we report on the molecular characterization by massively parallel sequencing of four breast AdCCs lacking the MYB-NFIB fusion gene. In two cases, we identified MYBL1 rearrangements (MYBL1-ACTN1 and MYBL1-NFIB), which were associated with MYBL1 overexpression. A third AdCC harboured a high-level MYB amplification, which resulted in MYB overexpression at the mRNA and protein levels. RNA-sequencing and whole-genome sequencing revealed no definite alternative driver in the fourth AdCC studied, despite high levels of MYB expression and the activation of pathways similar to those activated in MYB-NFIB-positive AdCCs. In this case, a deletion encompassing the last intron and part of exon 15 of MYB, including the binding site of ERG-1, a transcription factor that may downregulate MYB, and the exon 15 splice site, was detected. In conclusion, we demonstrate that MYBL1 rearrangements and MYB amplification probably constitute alternative genetic drivers of breast AdCCs, functioning through MYBL1 or MYB overexpression. These observations emphasize that breast AdCCs probably constitute a convergent phenotype, whereby activation of MYB and MYBL1 and their downstream targets can be driven by the MYB-NFIB fusion gene, MYBL1 rearrangements, MYB amplification, or other yet to be identified mechanisms. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

GATA3 and MYB Expression in Cutaneous Adnexal Neoplasms.

Knowledge of staining pattern of certain immunostains might be useful in the classification of cutaneous adnexal tumors that can have clinical importance. We studied GATA3 and MYB expression in archival materials of 220 adnexal tumors comprised of sebaceous carcinomas, follicular tumors, apocrine carcinoma, predominantly apocrine tumors, predominantly eccrine tumors, and others including adenoid cystic carcinomas. Nuclear GATA3 expression was seen in 70% (153/220) of cases, including sebaceous carcinoma (93%), apocrine carcinoma (93%), follicular neoplasms (100%), and predominantly apocrine neoplasms (69%), yet only 38% of predominantly eccrine neoplasms. Nuclear MYB expression was seen in 43% (81/188) of cases, including adenoid cystic carcinoma (90%), predominantly apocrine tumors (66%), follicular neoplasms (49%), apocrine carcinomas (14%), predominantly eccrine tumors (11%), and sebaceous carcinomas (4%). GATA3 and MYB expression were noted in 43% (9/21) and 24% (5/21) of cutaneous metastases, respectively. Expression of both GATA3 and MYB was noted in 33% (60/184) of primary adnexal tumors versus 19% (4/21) of cutaneous metastases. GATA3 preferentially labels tumors with follicular, sebaceous, and apocrine differentiation. MYB is potentially a helpful stain in the distinction of desmoplastic trichoepithelioma versus basal cell carcinoma. The coexpression of GATA3 and MYB might be helpful in the distinction of primary cutaneous adnexal carcinoma versus metastatic breast, salivary gland, or urothelial carcinoma.

Nuclear trapping controls the position-dependent localization of CAPRICE in the root epidermis of Arabidopsis.

Cell fate determination and differentiation are central processes in the development of multicellular organisms, and the Arabidopsis (Arabidopsis thaliana) root epidermis provides a model system to study the molecular basis of these processes. A lateral inhibition mechanism mediated by an R3 single-repeat MYB protein, CAPRICE (CPC), has been proposed to explain the specification of the two types of root epidermal cells (hair cells and nonhair cells). However, it is not clear how CPC acts preferentially in the H-position cells, rather than the N-position cells, where its gene is expressed. To explore this issue, we examined the effect of misexpressed CPC on cell fate specification and CPC localization in the root epidermis. We show that CPC is able to move readily within the root epidermis when its expression level is high and that CPC can induce the hair cell fate in a cell-autonomous manner. We provide evidence that CPC is capable of moving from the stele tissue in the center of the root to the outermost epidermal layer, where it can induce the hair cell fate. In addition, we show that CPC protein accumulates primarily in the nuclei of H-position cells in the early meristematic region, and this localization requires the H-cell-expressed ENHANCER OF GLABRA3 (EGL3) basic helix-loop-helix transcription factor. These results suggest that cell-cell movement of CPC occurs readily within the meristematic region of the root and that EGL3 preferentially traps the CPC protein in the H-position cells of the epidermis.

Localization of c-MYB in differentiated cells during postnatal molar and alveolar bone development.

The MYB family of transcription activators has been associated with a high proliferation rate and an undifferentiated state of cells in a number of tissues. Recently emerging data suggest that these molecules may also play a role in differentiation. In this study, the pattern of expression of c-MYB was followed during postnatal stages of mouse molar odontogenesis using immunohistochemistry on serial sections. Along with an abundance of the c-MYB protein in proliferating zones, we confirmed the presence of this protein in differentiated ameloblasts, odontoblasts, and osteoblasts. In addition, c-MYB was also found in cementoblasts and alveolar fibroblasts. These findings suggest integration of c-MYB into regulatory networks during hard-tissue differentiation and mineralization.

Celastrol inhibits the growth of estrogen positive human breast cancer cells through modulation of estrogen receptor α.

Human estrogen receptor α (ERα) is a nuclear transcription factor that displays a major therapeutic target for breast cancer. The transcriptional activity of ERα can be regulated by particular estrogen receptor modulators. Celastrol, a quinine methide triterpene extracted from a Chinese medicine (Trypterygium wilfordii Hook F.), has been reported to have therapeutic efficacy against various cancer cells, including prostate cancer, leukemia, and melanoma cells. However, ERα regulation by Celastrol has not been reported. In this study, we investigated the effects of Celastrol on the growth of breast cancer cells. We observed that Celastrol decreased expression of ERα at both the mRNA and the protein levels in MCF7 and T47D human breast cancer cells. Results from a luciferase assay showed that Celastrol decreased the transcriptional activity of ERα. Also, Celastrol treatment inhibited ERα target gene expression, including expressions of cyclin D(1), progesterone receptor (PR), and c-Myb leading to cell cycle arrest and growth inhibition of breast cancer cells. We propose that Celastrol, an anti-cancer drug extracted from natural sources, induces inhibition of cell growth through modulation of ERα in estrogen positive breast cancer cells and is a candidate for use in cancer chemotherapy for human breast cancer.

Estrogen prevents sustained COLO-205 human colon cancer cell growth by inducing apoptosis, decreasing c-myb protein, and decreasing transcription of the anti-apoptotic protein bcl-2.

The proto-oncogene c-myb is overexpressed in human colon cancer cells. c-myb is known to be affected by estrogen in some breast cancers and leukemias. However, the mechanism of c-myb regulation via estrogen in colon cancer requires further investigation. Human COLO-205 colon cancer cells were cultured and treated with beta-estradiol for 24 h. Apoptosis was quantified using acridine orange/propidium iodide labeling and confirmed with DNA fragmentation gel electrophoresis. Expression of c-myb protein was assessed via SDS-PAGE and immunoblotting and RT-PCR was used to quantify bcl-2 RNA. Protein and RNA expression levels were also assayed after c-myb siRNA treatment for 24 h. We demonstrate an increase in apoptosis after 24 h of beta-estradiol treatment of human COLO-205 colon cancer cells. Estrogen treatment also decreases c-myb protein levels as well as expression of its transcriptional target bcl-2. Suppression of c-myb protein also results in increased apoptosis and decreases bcl-2 expression. These results indicate that estrogen has a protective effect from sustained colon cancer cell growth at least partly through suppression of c-myb and bcl-2.

In utero exposure to benzene increases embryonic c-Myb and Pim-1 protein levels in CD-1 mice.

Benzene is a known human leukemogen, but its role as an in utero leukemogen remains controversial. Epidemiological studies have correlated parental exposure to benzene with an increased incidence of childhood leukemias. We hypothesize that in utero exposure to benzene may cause leukemogenesis by affecting the embryonic c-Myb/Pim-1 signaling pathway and that this is mediated by oxidative stress. To investigate this hypothesis, pregnant CD-1 mice were treated with either 800 mg/kg of benzene or corn oil (i.p.) on days 10 and 11 of gestation and in some cases pretreated with 25 kU/kg of PEG-catalase. Phosphorylated and total embryonic c-Myb and Pim-1 protein levels were assessed using Western blotting and maternal and embryonic oxidative stress were assessed by measuring reduced to oxidized glutathione ratios. Our results show increased oxidative stress at 4 and 24 h after exposure, increased phosphorylated Pim-1 protein levels 4 h after benzene exposure, and increased Pim-1 levels at 24 and 48 h after benzene exposure. Embryonic c-Myb levels were elevated at 24 h after exposure. PEG-catalase pretreatment prevented benzene-mediated increases in embryonic c-Myb and Pim-1 protein levels, and benzene-induced oxidative stress. These results support a role for ROS in c-Myb and Pim-1 alterations after in utero benzene exposure.

In primary effusion lymphoma cells, MYB transcriptional repression is associated with v-FLIP expression during latent KSHV infection while both v-FLIP and v-GPCR become involved during the lytic cycle.

Primary effusion lymphoma (PEL) is a rare, distinct subtype of non-Hodgkin lymphoma, which is associated with Kaposi sarcoma-associated herpesvirus (KSHV) infection. Although MYB levels are high in most neoplastic B cells, we found that, unexpectedly, both PEL cells and uncultured PEL patients' samples contained very low levels of MYB mRNA when compared to B-cell leukaemia samples obtained from KSHV(-) patients. These results were further confirmed at the protein level. Both latent viral FLICE inhibitory protein (v-FLIP) and early lytic viral G protein coupled receptor (v-GPCR) KSHV proteins were found to activate nuclear factor (NF)-kappaB and transrepress a MYB promoter reporter construct. In contrast, a dominant negative inhibitor of NF-kappaB (IkappaB-alpha) mutant prevented v-FLIP and v-GPCR from inhibiting MYB functions while a v-GPCR mutant that was impaired for NF-kappaB activation could not repress the MYB construct. Transduction of a v-FLIP expressing vector or stable transfection of v-GPCR both resulted in a marked downregulation of the endogenous MYB protein expression. However, MYB expression transactivated the lytic switch Replication and Transcription Activator (RTA) promoter in transient transfection assays. Taken together, our results demonstrate that, contrary to a number of other haematological malignancies, MYB expression is not required for PEL cell proliferation. Repressing MYB expression also helps in maintaining the virus in latency.

Collagen loss and impaired wound healing is associated with c-Myb deficiency.

Collagen type I serves as an abundant structural and signalling component of skin. It is also an established target gene of the transcription factor, c-Myb. When c-myb-/- embryos were examined it was observed that their skin was markedly thinner than normal. Importantly, immunohistochemical investigation showed complete absence of collagen type I. Although these homozygous knock-out embryos fail to develop beyond day 15, fibroblasts established from these embryos (mouse embryonic fibroblasts [MEFs]) show defective proliferative responses. Furthermore, in vitro scratch wound assays demonstrated that these c-myb-/- MEFs also exhibit slower closure than their wild-type counterparts. Embryonic lethality has meant that examination of the role of c-Myb in adult mouse skin has not been reported to date. However, in view of the abundance of collagen type I in normal skin, its role in skin integrity and the in vitro data showing proliferative and migration defects in c-myb-/- MEFs, we investigated the consequences of heterozygous c-myb loss in adult mice on the complex process of skin repair in response to injury. Our studies clearly demonstrate that heterozygous c-myb deficiency has a functional effect on wound repair, collagen type I levels and, in response to wounding, transforming growth factor-beta1 (an important collagen stimulating factor) induction expression is aberrantly high. Manipulation of c-Myb may therefore provide new therapeutic opportunities for improving wound repair while uncontrolled expression may underpin some fibrotic disorders.

Proper levels of c-Myb are discretely defined at distinct steps of hematopoietic cell development.

The definitive hematopoietic cell lineages have been proposed to originate from hemogenic endothelial cells during mouse embryogenesis. c-Myb is a transcription factor that is essential for the development of definitive hematopoiesis. To investigate the functional role of c-Myb in hematopoietic cell development from endothelial cells, we introduced a c-myb transgene expressed under the control of a tetracycline-regulated promoter into the c-myb(-/-) embryonic stem (ES) cell line, with the aim of inducing c-Myb expression at any stage and at any level. Induction of c-Myb expression after replating c-myb(-)(/)(-) endothelial cells rescued the generation and proliferation of definitive hematopoietic progenitor cells, suggesting that c-Myb expression in developing endothelial cells is not a prerequisite for their hematogenic potential. Overexpression of c-Myb, however, prevented the terminal differentiation of erythrocytes and megakaryocytes and completely abolished B-lymphocyte development. Our results indicate that c-Myb is a major factor that controls differentiation as well as proliferation of hematopoietic progenitor cells derived from hemogenic endothelial cells, and that appropriate levels of c-Myb protein are strictly defined at distinct differentiation steps of each hematopoietic cell lineage.

Early-onset gastric cancers have a different molecular expression profile than conventional gastric cancers.

Many studies examine the molecular genetics of gastric cancer, but few look at young patients in particular and there is no comparison of molecular expression between early-onset gastric cancer (< or = 45 years old) and conventional gastric cancers. Expression of cycloxygenase-2 (COX-2) is elevated in gastric adenocarcinomas compared to non-neoplastic mucosa, and in light of studies showing reduced risk of gastric cancer in nonsteroidal anti-inflammatory drug users, we have chosen to investigate the expression of COX-2 and related molecules in 113 early-onset gastric cancers and compare it with 91 conventional gastric cancers, using tissue microarrays. These markers include molecules known to be important in conventional gastric carcinogenesis, such as E-Cadherin, p53, COX-2, Trefoil Factor-1 (TFF1), beta-catenin, p16 and c-myc; as well as molecules not yet described as being important in gastric cancer, such as the transcription factor c-jun, the COX-2 mRNA stabilizer HuR, and C/EBP-beta, a transcription factor for COX-2. All markers showed a statistically significant difference between early-onset gastric cancers and conventional gastric cancers, using a chi2 test. In particular, early-onset gastric cancers displayed a COX-2 Low, TFF1-expressing phenotype, whereas COX-2 overexpression and loss of TFF1 was found in conventional cancers, and this difference between early-onset gastric cancers and conventional cancers remained statistically significant when adjusted for location and histology (P<0.0001 and P = 0.002 respectively). We found that COX-2 overexpression correlates significantly with loss of TFF1 (P = 0.001), overexpression of C/EBP-beta (P<0.001) and cytoplasmic HuR (P = 0.016). COX-2 was significantly associated with p53 positivity (P = 0.003). Abnormalities in E-Cadherin correlated significantly with diffuse phenotype, whereas high expression of COX-2, loss of TFF1 and overexpression of C/EBP-beta correlated with the intestinal phenotype. Our results provide further evidence that early-onset gastric cancer exhibits a distinctive expression profile that may have practical implications.

Alterations and correlations of the components in the Wnt signaling pathway and its target genes in breast cancer.

Both cyclin D1 and c-myc are key molecules in breast cancer carcinogenesis, and their transcriptional level and stability are regulated through several signaling pathways, including the Wnt signaling pathway. We performed immunohistochemical and mutational analyses of Wnt signaling components to investigate the association of Wnt signaling alterations with breast cancer carcinogenesis using 49 surgically resected primary breast cancer samples. Positive staining of cyclin D1 and c-myc was observed in 55.1% and 30.6% of the 49 breast cancer samples, respectively. Aberrant cytoplasmic expression of beta-catenin, which indicates the existence of alterations in the Wnt signaling pathway, was observed in 38.8% of breast cancer samples, though no mutation was found in the beta-catenin and Axin 1 genes. Reduced expression of APC was observed in 34.7% of samples. Statistical analysis revealed strong correlations between overexpression of beta-catenin and that of cyclin D1 and c-myc (p=0.0001 and 0.0117, respectively). Furthermore, overexpression of beta-catenin was significantly correlated with reduced expression of APC (p=0.0127). Wnt signaling alterations were frequently observed in breast cancer from the results of beta-catenin immunohistochemistry, although no mutation in the components of the Wnt signaling pathway was found in the present study. Based on the statistical analyses, we speculated that reduced expression of APC leads to overexpression of beta-catenin, and aberrant expression of cyclin D1 and c-myc mainly depends on alterations in the Wnt signaling pathway in breast cancer.