SATB2 Expression in Human Tumors: A Tissue Microarray Study on More Than 15 000 Tumors.

CONTEXT.­: Special AT-rich sequence-binding protein 2 (SATB2) induces local chromatin loops to facilitate transcription. SATB2 immunostaining is commonly used as a marker for colorectal adenocarcinoma and osteosarcoma. OBJECTIVE.­: To extend our knowledge on the diagnostic value of SATB2 analysis in a comprehensive set of human tumors. DESIGN.­: Tissue microarrays with 15 012 samples from 120 tumor types and 608 samples of 76 different normal tissues were analyzed. RESULTS.­: SATB2 positivity was found in 89 of 120 different tumor types (74%), including 59 of 120 (49%) with at least 1 moderately positive tumor and 38 of 120 tumor types (32%) with at least 1 strongly positive tumor. Expression was frequent in adenomas (44/42-47/44; 94%-96% positive), adenocarcinomas (1747 of 2023; 86%), and various subtypes of neuroendocrine neoplasms (3/7-12/12; 43%-100%) of the colorectum and appendix, Merkel cell carcinoma (25 of 34, 74%), osteosarcomas (15 of 25; 60%), and papillary renal cell carcinoma (RCC) (121 of 235; 52%). Associations to clinicopathologic tumor features were assessed in colorectal and kidney cancers. In colorectal cancer, weak SATB2 expression was linked to high pT (P < .001), nodal metastasis (P < .001), right-sided tumor location (P < .001), microsatellite instability (P < .001), and BRAF mutations (P = .02). In papillary RCC, low SATB2 expression was associated with high pT (P = .02), distant metastasis (P = .04), and reduced tumor-specific survival (P = .04). In clear cell RCC, low SATB2 expression was linked to high pT (P < .001), high Union for International Cancer Control stage (P < .001), high Thoenes grade (P = .02), and reduced recurrence-free survival (P = .02). CONCLUSIONS.­: Strong SATB2 expression argues for a colorectal origin within adenocarcinomas and neuroendocrine neoplasms. Weak SATB2 expression reflects progression and poor prognosis in colorectal and kidney cancer.

SATB1, genomic instability and Gleason grading constitute a novel risk score for prostate cancer.

Current prostate cancer risk classifications rely on clinicopathological parameters resulting in uncertainties for prognostication. To improve individual risk stratification, we examined the predictive value of selected proteins with respect to tumor heterogeneity and genomic instability. We assessed the degree of genomic instability in 50 radical prostatectomy specimens by DNA-Image-Cytometry and evaluated protein expression in related 199 tissue-microarray (TMA) cores. Immunohistochemical data of SATB1, SPIN1, TPM4, VIME and TBB5 were correlated with the degree of genomic instability, established clinical risk factors and overall survival. Genomic instability was associated with a GS ≥ 7 (p = 0.001) and worse overall survival (p = 0.008). A positive SATB1 expression was associated with a GS ≤ 6 (p = 0.040), genomic stability (p = 0.027), and was a predictor for increased overall survival (p = 0.023). High expression of SPIN1 was also associated with longer overall survival (p = 0.048) and lower preoperative PSA-values (p = 0.047). The combination of SATB1 expression, genomic instability, and GS lead to a novel Prostate Cancer Prediction Score (PCP-Score) which outperforms the current D'Amico et al. stratification for predicting overall survival. Low SATB1 expression, genomic instability and GS ≥ 7 were identified as markers for poor prognosis. Their combination overcomes current clinical risk stratification regimes.

Prognostic Significance of TLR2, SMAD3 and Localization-dependent SATB1 in Stage I and II Non-Small-Cell Lung Cancer Patients.

This study aimed to explore the prognostic value of SATB1, SMAD3, and TLR2 expression in non-small-cell lung carcinoma patients with clinical stages I-II. To investigate, we evaluated immunohistochemical staining to each of these markers using tissue sections from 69 patients from our cohort and gene expression data for The Cancer Genome Atlas (TCGA) cohort. We found that, in our cohort, high expression levels of nuclear SATB1n and SMAD3 were independent prognostic markers for better overall survival (OS) in NSCLC patients. Interestingly, expression of cytoplasmic SATB1c exhibited a significant but inverse association with survival rate, and it was an independent predictor of unfavorable prognosis. Likewise, TLR2 was a negative outcome biomarker for NSCLC even when adjusting for covariates. Importantly, stratification of NSCLCs with respect to combined expression of the three biomarkers allowed us to identify subgroups of patients with the greatest difference in duration of survival. Specifically, expression profile of SATB1n-high/SMAD3high/TLR2low was associated with the best OS, and it was superior to each single protein alone in predicting patient prognosis. Furthermore, based on the TCGA dataset, we found that overexpression of SATB1 mRNA was significantly associated with better OS, whereas high mRNA levels of SMAD3 and TLR2 with poor OS. In conclusion, the present study identified a set of proteins that may play a significant role in predicting prognosis of NSCLC patients with clinical stages I-II.

Subcellular dynamics of estrogen-related receptors involved in transrepression through interactions with scaffold attachment factor B1.

Estrogen-related receptor (ERR), a member of the nuclear receptor superfamily, consists of three subtypes (α, ß, γ) and has strong homology with estrogen receptor. No endogenous ligands have been identified for ERRs, but they play key roles in metabolic, hormonal, and developmental processes as transcription factors without ligand binding. Although subnuclear dynamics are essential for nuclear events including nuclear receptor-mediated transcriptional regulation, the dynamics of ERRs are poorly understood. Here, we report that ERRs show subcellular kinetic changes in response to diethylstilbestrol (DES), a synthetic estrogen that represses the transactivity of all three ERR subtypes, using live-cell imaging with fluorescent protein labeling. Upon DES treatment, all ERR subtypes formed discrete clusters in the nucleus, with ERRγ also displaying nuclear export. Fluorescence recovery after photobleaching analyses revealed significant reductions in the intranuclear mobility of DES-bound ERRα and ERRß, and a slight reduction in the intranuclear mobility of DES-bound ERRγ. After DES treatment, colocalization of all ERR subtypes with scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein, was observed in dot-like subnuclear clusters, suggesting interactions of the ERRs with the nuclear matrix. Consistently, co-immunoprecipitation analyses confirmed enhanced interactions between ERRs and SAFB1 in the presence of DES. SAFB1 was clarified to repress the transactivity of all ERR subtypes through the ERR-response element. These results demonstrate ligand-dependent cluster formation of ERRs in the nucleus that is closely associated with SAFB1-mediated transrepression. Taken together, the present findings provide a new understanding of the pathophysiology regulated by ERR/SAFB1 signaling pathways and their subcellular dynamics.

Identification of retinal ganglion cell types expressing the transcription factor Satb2 in three primate species.

In primates, the retinal ganglion cells contributing to high acuity spatial vision (midget cells and parasol cells), and blue-yellow color vision (small bistratified cells) are well understood. Many other ganglion cell types with large dendritic fields (named wide-field ganglion cells) have been identified, but their spatial density and distribution are largely unknown. Here we took advantage of the recently established molecular diversity of ganglion cells to study wide-field ganglion cell populations in three primate species. We used antibodies against the transcription factor Special AT-rich binding protein 2 (Satb2) to explore its expression in macaque (Macaca fascicularis, M. nemestrina), human and marmoset (Callithrix jacchus) retinas. In all three species, Satb2 cells make up a low proportion (1.5-4%) of the ganglion cell population, with a slight increase from central to peripheral retina. Intracellular dye injections revealed that in macaque and human retinas, the large majority (over 80%) of Satb2 cells are inner and outer stratifying large sparse cells. By contrast, in marmoset retina the majority (over 60%) of Satb2 expressing cells were broad thorny cells, with smaller proportions of recursive bistratified (putative direction-selective), large bistratified, and outer stratifying narrow thorny cells. Our findings imply that Satb2 expression has undergone rapid species specific adaptations during primate evolution, because expression is not conserved across Old World (macaque, human) and New World (marmoset) suborders.

YJ5 as an immunohistochemical marker of osteogenic lineage.

Overexpression of WLS, an upstream protein in the Wnt pathway, has been implicated in several non-osteogenic tumours. This study represents the first attempt at evaluating WLS expression in various bone and soft tissue tumours using YJ5, a monoclonal antibody specific to WLS, with the aim of elucidating its utility in discerning tumours with aberrant Wnt signalling and as a marker of osteogenic lineage in challenging cases. Tumour tissue sections of 144 bone mass lesions and 63 soft tissue mass lesions were immunostained with the YJ5 antibody following standardised protocols. Subsequent assessment of immunoreactivity segregated cases into one of three groups: absent/weak, moderate, or strong YJ5 immunoreactivity. For the bone tumours, strong YJ5 immunoreactivity was seen in almost all osteosarcomas and chondroblastomas, all osteoblastomas and osteoid osteomas. In contrast, all other cartilaginous tumours, chordomas, aneurysmal bone cysts, chondromyxoid fibromas, most fibrous dysplasias and most giant cell tumours exhibited absent/weak YJ5 immunostaining. For the soft tissue tumours, a more heterogeneous pattern of YJ5 immunoreactivity was observed. Because diffuse and strong YJ5 expression is identified in almost all benign and malignant bone tumours with osteoblastic activity, it can be potentially utilised as an immunohistochemical marker to support osteogenic lineage. If interpreted in the appropriate context, this marker is useful in determining whether a malignant bone tumour is an osteosarcoma, particularly in those subtypes with no or minimal osteoid or unusual morphological features. This marker can also complement SATB2 to denote osteogenic lineage.

Hepatocellular carcinomas can be Special AT-rich sequence-binding protein 2 positive: an important diagnostic pitfall.

Special AT-rich sequence-binding protein 2 (SATB2) is a sensitive and specific marker for tumors originating with the colon and appendix. It is now commonly used in surgical pathology, while working up carcinomas of unknown primary. We had anecdotally encountered occasional hepatocellular carcinomas (HCCs) that were SATB2 positive. Immunohistochemical expression of SATB2 in HCC has not yet been examined in detail. In this study, we evaluated SATB2 expression in 46 HCCs. Nineteen (41%) of 46 HCCs were positive for SATB2. SATB2 expression in HCCs was more commonly seen in poorly differentiated tumors (11 of 13 cases, 85%) than well and moderately differentiated tumors (8 of 33 cases, 24%), p value = 0.0001. No other statistically significant correlations were observed (p > 0.05). There were no other statistically significant correlations between SATB2 expression and age, gender, background liver disease, and cirrhosis (p > 0.05). Results of our study show that a significant subset (41%) of HCCs can be SATB2 positive. Awareness of this phenomenon is important as SATB2 expression in a liver tumor does not completely exclude a diagnosis of HCC.

An Integrative Morphomolecular Classification System of Gastric Carcinoma With Distinct Clinical Outcomes.

A robust morphomolecular classification system for gastric carcinoma is required. A 4-tier morphologic classification is proposed, including diffuse, intestinal, tubular, and lymphoid types. A tissue microarray for mismatch repair immunohistochemistry and Epstein-Barr virus (EBV) in situ hybridization were performed in 329 gastric carcinomas. DNA flow cytometry was used to detect aneuploidy in formalin-fixed paraffin-embedded samples. Lymphoid histology was the third most common histologic pattern at our institute and strongly associated with EBV infection and PMS2/MLH1-deficiency (both P<0.001). HER2 overexpression and SATB2 expression more frequently occurred in intestinal histology (both P<0.001). Loss of ARID1A expression was strikingly associated with lymphoid histology (P<0.001) and negative E-cadherin expression was correlated with diffuse histology (P=0.001). Programmed death-ligand 1 expression was most frequently present in lymphoid-type gastric carcinoma than other histologic subtypes and correlated with the molecular features of PMS2/MLH1-deficiency and EBV infection (all P<0.001). Aneuploidy was detected in 53% of gastric carcinomas and was highly correlated with intestinal type and the least with the lymphoid type (P<0.001). Notably, lymphoid-type gastric carcinoma showed the best outcome, whereas tubular type showed the worst survival rate (P<0.001). We integrated aneuploidy with morphologic patterns to propose a morphomolecular classification scheme, which served as a successful and independent prognostic factor in multivariate 5-year disease-free survival analysis (P<0.001). Overall, we describe an integrated morphomolecular classification system for gastric carcinomas to effectively predict patient outcomes. This system is cost-effective and reliable and can help select target therapeutics and facilitate clinical management.

Histomorphometric Analysis of Pre- and Post-Denosumab-Treated Giant Cell Tumor of Bone.

Context. Denosumab is a monoclonal antibody against RANK ligand. Its administration in giant cell tumor of bone (GCTB) cases results in elimination of giant cells and new bone formation. Neoplastic stromal cells of GCTB harbor mutation of histone 3.3 and have pre-osteoblastic properties and thus express SATB2. Objectives. To (1) analyze histological changes in post-denosumab-treated GCTB, (2) analyze expression of H3.3G34W and SATB2 in pre- and post-denosumab-treated samples, and (3) to discuss why changes occur in the expression of not only H3.3G34W but also SATB2. Materials and Methods. Hematoxylin and eosin slides of 19 cases of denosumab-treated GCTB were reviewed. Immunohistochemical stains H3.3G34W and SATB2 were performed. The number of positive mononuclear cells were counted and graded. Results. Complete absence of osteoclast-like giant cells (OCLGCs) was noted in most cases along with a fibro-osseous component merging with peripheral shell of reactive bone. Irregular trabeculae of woven bone and osteoid with focal osteoblastic rimming was seen. Spindle cells were arranged predominantly in fascicular pattern. Morphometric analysis of H3.3G34W showed a mean of 68.8% positive stromal cells in pretreatment and a mean of 26.9% positive stromal cells in posttreated specimens with a statistically significant P value (.001). Mean percentage of SATB2-positive stromal cells in the pre- and posttreatment specimens was 36.46% and 20.8%, respectively. Conclusions. Our study validates that denosumab treatment results in marked reduction of OCLGCs with increased osteoblastic activity. Decreased expression of H3.3G34W in posttreatment may be a result of decreased antigenicity of neoplastic mononuclear cells. No significant change in SATB2 expression was noted.

Scaffold attachment factor B: distribution and interaction with ERα in the rat brain.

Scaffold attachment factor (SAFB) 1 and its homologue SAFB2 are multifunctional proteins that are involved in various cellular mechanisms, including chromatin organization and transcriptional regulation, and are also corepressors of estrogen receptor alpha (ERα). Both SAFBs are expressed at high levels in the brain. However, the distributions of SAFB1 and SAFB2 have yet to be characterized in detail and it is unclear whether both proteins interact with ERα in the brain. In this study, we investigated the expression and distribution of both SAFBs and their interaction with ERα in adult male rat brain. Immunohistochemical staining showed that SAFB1 and SAFB2 have a similar distribution pattern and are widely expressed throughout the brain. Double-fluorescence immunohistochemical and immunocytochemical analyses in primary cultures showed that the two SAFB proteins are localized in nuclei of neurons, astrocytes, and oligodendrocytes. Of note, SAFB2 was also found in cytoplasmic regions in these cell lineages. Both SAFB proteins were also expressed in ERα-positive cells in the medial preoptic area (MPOA) and arcuate and ventromedial hypothalamic nuclei. Co-immunoprecipitation experiments revealed that both SAFB proteins from the MPOA reciprocally interact with endogenous ERα. These results indicate that, in addition to a role in basal cellular function in the brain, the SAFB proteins may serve as ERα corepressors in hormone-sensitive regions.

CDX2, SATB2, GATA3, TTF1, and PAX8 Immunohistochemistry in Krukenberg Tumors.

Twenty-six Krukenberg tumors (16 lower gastrointestinal, 4 upper gastrointestinal, and 6 of unknown origin) and their primaries when known were stained with CDX2, SATB2, GATA3, TTF1, and PAX8 using a tissue microarray containing predominantly or exclusively signet ring cells. The most common primary was appendiceal mixed adenoneuroendocrine carcinoma. CDX2 and SATB2 were positive in all known lower gastrointestinal primary tumors and negative in nearly all known upper gastrointestinal primary tumors. Primaries showed identical immunophenotypes to their metastases. Among cases of unknown primary origin, 3 were positive and 3 were negative for CDX2 and SATB2. Chest images, upper endoscopies, colonoscopies, appendectomies, and mammogram were performed with negative results in all, 4, 2, 2, and 1 cases, respectively. No cystoscopies were attempted. PAX8, GATA3, and TTF1 were negative in all cases. The literature was reviewed with emphasis on immunohistochemistry of signet ring cell-containing carcinomas from the appendix, colon, stomach, breast, lung, and bladder. Three quarters of gastric primaries stain for CDX2 and only rare examples stain for SATB2. Colorectal primaries (most of them) and appendiceal primaries (all of them) are positive for CDX2 and SATB2. GATA3 stains almost all breast primaries and approximately half of bladder primaries. All pulmonary primaries are positive for TTF1. PAX8 is negative in the gastric, colorectal, and appendiceal primaries reported. This study shows that the panel of immunostains is useful in confirming the site of origin of a metastatic Krukenberg tumor when one is known and has limited diagnostic value for diagnosing metastases of unknown origin.

SATB2 protein expression by immunohistochemistry is a sensitive and specific marker of appendiceal and rectosigmoid well differentiated neuroendocrine tumours.

AIMS: Neuroendocrine neoplasms (NNs) range from well to poorly differentiated and indolent to highly aggressive. The site of origin in metastatic NNs has therapeutic and prognostic implications. SATB2 is a transcriptional regulator involved in osteoblastic and neuronal differentiation and is a sensitive and specific marker of colorectal epithelium. This study aimed to evaluate the expression of SATB2 in NNs from various primary sites and its utility as a marker in determining the site of origin of these neoplasms. METHODS AND RESULTS: SATB2 immunohistochemistry was performed on 266 NNs, including lung small cell carcinomas (n = 39) and carcinoids (n = 30), bladder (n = 21) and prostate (n = 31) small cell carcinomas, and gastrointestinal (GI)/pancreatic NNs of various primary sites (n = 145) consisting of well-differentiated neuroendocrine tumours (WDNET)s (n = 124) and poorly differentiated neuroendocrine carcinomas (PDNEC)s (n = 21). SATB2 was expressed in prostatic (10 of 31, 32%) and bladder (eight of 21, 38%) small cell carcinomas, lung carcinoid tumours (one of 30, 3%), and lung small cell carcinomas (eight of 39, 21%). Among primary GI NNs, SATB2 was expressed in 37 of 124 (30%) WDNETs and four of 21 (19%) PDNECs. Of the former, 15 of 15 (100%) rectal/rectosigmoid and 22 of 22 (100%) appendiceal neoplasms expressed SATB2. Using receiver operator characteristic analysis, SATB2 was a sensitive and specific marker for rectal (100.0%, 80.0%) and appendiceal (100.0%, 84.5%) WDNETs, respectively. CONCLUSIONS: In summary, SATB2 is a sensitive and specific marker for rectal/rectosigmoid and appendiceal WDNETs, and may represent a useful diagnostic tool when these sites of origin are considered in the differential diagnosis.

Metastatic colorectal carcinomas with high SATB2 expression are associated with better prognosis and response to chemotherapy: a population-based Scandinavian study.

Background: Survival and response to therapy in patients with metastatic colorectal cancer (mCRC) are very heterogeneous. There is an unmet need for better markers of prognosis and treatment benefit for mCRC patients. The homeobox 2 gene SATB2 has a highly specific expression in colorectal tissue and is associated with better prognosis in non-metastatic CRC.Material and methods: A population-based cohort of 798 mCRC patients was analysed. From primary tumour material, protein expression was assessed by immunohistochemistry. BRAF and KRAS mutation status was also determined. Associations with clinicopathological data, overall and progression-free survival and response to first-line chemotherapy were analysed.Results: Tumour tissue and clinical data were available from 467 patients. SATB2 was strongly expressed in 58% of cases, significantly more in left-sided, low-grade and wild-type BRAF tumours. Patients with high SATB2 tumours had longer overall survival compared with low SATB2 tumours (median 13 vs 8 months respectively, p < .001). Chemotherapy was given to 282 patients (63%). Patients with high SATB2 tumours had longer OS (median 22 vs 15 months respectively, p = .001) and more often responded to chemotherapy than those with low SATB2 (objective response 43% vs 29%, p = .02; clinical response 83% vs 67%, p = .004). Progression-free survival on first-line irinotecan chemotherapy was longer in high SATB2 cases (median 8 vs 4 months respectively, p = .019). Patients with both low SATB2 expression and mutated BRAF (n = 69) had particularly poor survival compared to the rest (median 8 and 12 months respectively, p = .001). In multivariable analysis, the SATB2 findings were independent of known clinicopathological prognostic markers, including BRAF mutation status.Conclusion: Patients with mCRC expressing high level of SATB2 have better prognosis and response to chemotherapy than those with low SATB2 expression. Patients with both low SATB2 expression and mutated BRAF had particularly poor prognosis and could thus benefit from more aggressive therapies.

Two types of primary mucinous ovarian tumors can be distinguished based on their origin.

The origin of primary mucinous ovarian tumors is unknown. We explore the hypothesis that they originate from either Brenner tumors or teratomas and examine differences between the tumors that arise in these settings. A total of 104 Brenner tumor-associated mucinous tumors and 58 teratoma-associated mucinous tumors were analyzed. Immunohistochemistry for 21 antigens and fluorescence in situ hybridization for ERBB2 and MYC were performed. Genome-wide copy number analysis and mutation analysis for 56 cancer-related genes was carried out on a subset of mucinous ovarian tumors and their complementary Brenner tumor or teratoma. Patients with teratoma-associated mucinous tumors were significantly younger than patients with Brenner tumor-associated mucinous tumors (43 vs. 61 years). During progression from cystadenoma to atypical proliferative mucinous (borderline) tumor to carcinoma expression of typical gastrointestinal markers was increased in both Brenner tumor-associated and teratoma-associated mucinous tumors. Brenner tumor-associated mucinous tumors showed more frequently calcifications and Walthard cell nests, rarely expressed SATB2 and showed more often co-deletion of CDKN2A and MTAP. Teratoma-associated mucinous tumors were characterized by mucinous stromal dissection, SATB2 expression and RNF43 mutations. Other frequent mutations in both Brenner tumor-associated and teratoma-associated mucinous tumors were TP53 and KRAS mutations. Based on identical mutations or copy number profiles clonal relationships were indicated in two mucinous tumors and their associated Brenner tumor. Teratomas and Brenner tumors give rise to different subtypes of mucinous ovarian tumors. Subsequent progression pathways are comparable since both Brenner tumor-associated and teratoma-associated mucinous tumors develop a gastrointestinal immunophenotype during progression and show early mutations in KRAS and TP53. Teratoma-associated mucinous tumors may more closely resemble true gastrointestinal tumors, indicated by their expression of SATB2 and the presence of RNF43 mutations.

Loss of SATB2 Expression Is a Biomarker of Inflammatory Bowel Disease-associated Colorectal Dysplasia and Adenocarcinoma.

SATB2 is a sensitive immunohistochemistry marker of colorectal carcinoma and non-neoplastic colorectal epithelium that is complementary to CDX2. However, its expression is affected by molecular alterations. Inflammatory bowel disease-associated neoplasia demonstrates molecular alterations that are different from those in sporadic colorectal neoplasia. Given these differences, we examined SATB2 expression in 73 cases of inflammatory bowel disease-associated neoplasia including 37 dysplasia cases and 36 carcinomas and compared the expression patterns with 50 cases of nondysplastic colorectal mucosa in patients with active inflammatory bowel disease, 40 sporadic colonic polyps (20 conventional adenomas and 20 sessile serrated lesions/polyps), and 343 sporadic colorectal adenocarcinomas to assess SATB2 immunohistochemistry as a biomarker of inflammatory bowel disease-associated neoplasia. Loss of SATB2 expression was only identified in colorectal dysplasia arising in inflammatory bowel disease (15/37, 41%) and was not seen in nondysplastic colorectal mucosa with active inflammatory bowel disease or sporadic colonic polyps (P<0.001). Loss of SATB2 expression was identified in both endoscopically visible dysplasia (11/28, 39%) and invisible (4/9, 44%) dysplasia. Loss of SATB2 expression was identified in 67% (24/36) of inflammatory bowel disease-associated carcinomas and was significantly more frequent compared with sporadic colorectal carcinomas (47/343, 14%, P<0.001). There was no difference in positive CDX2 expression between inflammatory bowel disease-associated colorectal carcinoma and sporadic colorectal carcinoma (89% vs. 85%, P=1.0). In conclusion, loss of SATB2 expression is common in inflammatory bowel disease-associated colorectal dysplasia and adenocarcinoma and may be a helpful ancillary biomarker when evaluating for inflammatory bowel disease-associated dysplasia.

A combination of the immunohistochemical markers CK7 and SATB2 is highly sensitive and specific for distinguishing primary ovarian mucinous tumors from colorectal and appendiceal metastases.

Primary ovarian mucinous tumors can be difficult to distinguish from metastatic gastrointestinal neoplasms by histology alone. The expected immunoprofile of a suspected metastatic lower gastrointestinal tumor is CK7-/CK20+/CDX2+/PAX8-. This study assesses the addition of a novel marker SATB2, to improve the diagnostic algorithm. A test cohort included 155 ovarian mucinous tumors (105 carcinomas and 50 borderline tumors) and 230 primary lower gastrointestinal neoplasms (123 colorectal adenocarcinomas and 107 appendiceal neoplasms). All cases were assessed for SATB2, PAX8 CK7, CK20, and CDX2 expression on tissue microarrays. Expression was scored in a 3-tier system as absent, focal (1-50% of tumor cells) and diffuse ( >50% of tumor cells) and then categorized into either absent/present or nondiffuse/diffuse. SATB2 and PAX8 expression was further evaluated in ovarian tumors from an international cohort of 2876 patients (expansion cohort, including 159 mucinous carcinomas and 46 borderline mucinous tumors). The highest accuracy of an individual marker in distinguishing lower gastrointestinal from ovarian mucinous tumors was CK7 (91.7%, nondiffuse/diffuse cut-off) followed by SATB2 (88.8%, present/absent cut-off). The most effective combination was CK7 and SATB2 with accuracy of 95.3% using the 3-tier interpretation, absent/focal/diffuse. This combination outperformed the standard clinical set of CK7, CK20 and CDX2 (87.5%). Re-evaluation of outlier cases confirmed ovarian origin for all but one case. The accuracy of SATB2 was confirmed in the expansion cohort (91.5%). SATB2 expression was also detected in 15% of ovarian endometrioid carcinoma but less than 5% of other ovarian histotypes. A simple two marker combination of CK7 and SATB2 can distinguish lower gastrointestinal from ovarian primary mucinous tumors with greater than 95% accuracy. PAX8 and CDX2 have value as second-line markers. The utility of CK20 in this setting is low and this warrants replacement of this marker with SATB2 in clinical practice.

SATB2 and CDX2 are prognostic biomarkers in DNA mismatch repair protein deficient colon cancer.

DNA mismatch repair protein deficient colon cancer frequently displays reduced CDX2 expression, and recent literature has suggested that negative CDX2 expression is a poor prognostic biomarker in colon cancer. We have recently demonstrated that SATB2 is an immunohistochemical marker that is complementary to CDX2. Using a tissue microarray approach, we evaluated SATB2 and CDX2 immunohistochemical expression in 514 patients with colonic adenocarcinoma including 146 with mismatch repair protein deficient tumors and correlated expression with histopathologic variables, molecular alterations, and survival. Overall, SATB2-negative and/or CDX2-negative expression was identified in 33% of mismatch repair protein deficient tumors compared with only 15% of mismatch repair protein proficient tumors (p < 0.001) and in 36% of BRAF V600E mutated compared with only 13% of BRAF wild-type tumors (p < 0.001). Both SATB2-negative and CDX2-negative colonic adenocarcinomas more often displayed lymphatic invasion, venous invasion, and perineural invasion (all with p < 0.05). SATB2-negative expression was also more frequently identified in tumors with mucinous or signet ring cell differentiation (p < 0.01 for both). In a multivariable analysis of survival in patients with mismatch repair protein deficient tumors (n = 131), only tumor stage (p = 0.01) and SATB2-negative and/or CDX2-negative expression (p = 0.009) independently predicted disease-specific survival. Of the 99 patients with stage II or III mismatch repair protein deficient tumors, death from disease only occurred in patients with either SATB2-negative or CDX2-negative tumors, and no patients with SATB2-positive/CDX2-positive tumors developed recurrence or died of disease. SATB2 and CDX2 expression had no effect on patient survival in mismatch repair protein proficient, BRAF-mutated, or KRAS-mutated tumors. In summary, our results suggest that SATB2 and CDX2 are prognostic biomarkers in patients with mismatch repair protein deficient colon cancer and that inclusion of SATB2 and CDX2 immunohistochemistry may be helpful as part of a comprehensive pathologic risk assessment in mismatch repair protein deficient colon cancer.

A COEUR cohort study of SATB2 expression and its prognostic value in ovarian endometrioid carcinoma.

The aim of this study was to describe the expression of special AT-rich sequence-binding protein 2 (SATB2) in ovarian endometrioid carcinoma (EC). SATB2 is a nuclear matrix-associated transcription factor that is associated with abnormal expression in certain cancers but has not been reported for ovarian carcinoma. SATB2 mRNA and protein expression was first assessed in a pilot cohort of 26 samples by Affymetrix microarray and by routine immunohistochemistry on a small tissue microarray. A large multicenter validation cohort representing the well-characterized cases of 235 ovarian EC from the Canadian Ovarian Experimental Unified Resource (COEUR) was then used to validate this result and to assess the prognostic impact of SATB2 expression. SATB2 staining was scored as negative, weak, moderate, and strong intensity, and by percentage of stained cells. No SATB2 expression was observed in clear cell carcinomas but 10% (n = 3) of the ECs in the pilot cohort showed SATB2 expression. In the validation cohort, strong expression was observed in 11% of ECs, while weak or moderate expression levels were detected in 12% of cases. Evaluation of SATB2 expression with clinicopathological parameters revealed an association with patient age and Federation International of Gynecology and Obstetrics grade but not with disease stage or postoperative residual disease. Any expression of SATB2, independent of intensity, was also associated with longer survival and improved progression-free survival with hazard ratio (HR) = 0.14 (95% CI 0.03-0.56) and HR = 0.16 (95% CI 0.02-1.24) respectively. A greater beneficial effect was observed in patients with stage III/IV disease compared to patients with stage I/II disease. Furthermore, direct comparison of SATB2 with other reported prognostic biomarkers such as progesterone receptor, CDX2 and ß-catenin within this cohort showed that SATB2 had the strongest association with survival. Given the current lack of accurate prognostic factors for these patients, SATB2 has promising clinical utility and warrants further study.

SATB2 Versus CDX2: A Battle Royale for Diagnostic Supremacy in Mucinous Tumors.

CONTEXT.­: Metastatic mucinous tumors present a diagnostic challenge for pathologists as tumor histomorphology is often nonspecific and optimal immunoprofiles are still under investigation. OBJECTIVE.­: To present a head-to-head comparison of special AT-rich sequence-binding protein 2 (SATB2) and caudal type homeobox 2 (CDX2) expression in a diverse array of primary mucinous tumors. DESIGN.­: SATB2 and CDX2 immunohistochemical stains were performed on whole sections from 44 mucinous colorectal carcinomas and 175 noncolorectal mucinous tumors. A nuclear scoring system measuring intensity (0-3+) and percentage staining (0 = <5%, 1 = 5%-49%, 2 = ≥50%) was implemented, producing an additive histologic score (H-score). RESULTS.­: SATB2 demonstrated acceptable accuracy at low to moderate expression levels (H-scores of 1-4). With these H-score cutoffs, overall accuracy was greater than 90%. In contrast, CDX2's accuracy rivaled that of SATB2 only at an H-score of 5 (89.0%), as its specificity suffered at lower expression levels (<70.0% at H-scores of 1-4). Using a moderate H-score cutoff of 3 or higher, significant differences for both sensitivity and specificity were identified between SATB2 and CDX2 (P = .01 for sensitivity and P < .001 for specificity), though these stains were near equivalent when each was interpreted as positive at its respective optimal H-score (SATB2 ≥ 3 and CDX2 = 5). CONCLUSIONS.­: SATB2 is a more accurate marker of colorectal origin across a variety of expression levels compared with CDX2 when applied to mucinous tumors from a host of primary sites. However, these stains are near equivalent when each is interpreted at its optimal expression level.

Colitis-associated colorectal adenocarcinomas are frequently associated with non-intestinal mucin profiles and loss of SATB2 expression.

The special AT-rich sequence binding protein 2 (SATB2) is a sensitive and specific diagnostic marker for colorectal adenocarcinoma and reduced expression of SATB2 is associated with a poor prognosis. Colitis-associated colorectal adenocarcinoma often shows distinct morphologic and molecular phenotypes compared to sporadic cases. However, the SATB2 expression profile in colitis-associated carcinoma has not been defined. We performed immunohistochemistry for SATB2 as well as CDX2, MUC5AC, MUC6 and mismatch repair proteins on 60 consecutive colitis-associated carcinomas from 58 inflammatory bowel disease patients and compared the expression profile to a control group of 32 sporadic colorectal carcinomas. Only 26 (43%) colitis-associated carcinomas expressed SATB2, compared to 29 (91%) sporadic colorectal carcinomas (p < 0.0001). MUC5AC expression was more frequently observed in colitis-associated carcinomas than sporadic colorectal caracinomas (52% and 25% respectively; p = 0.013). Eight (13%) cases of colitis-associated carcinoma showed loss of CDX2 expression, which was retained in all of the sporadic controls (p = 0.047). In colitis-associated carcinoma, 50% of SATB2 negative cases had lymph node metastasis compared to only 15% of SATB2 positive cases (p = 0.007). Loss of SATB2 was particularly frequent in mucinous-type tumors, occurring in 83% of these cases. There was no significant association between SATB2 expression and mismatch repair protein status. These data show that the immunoprofile of colitis-associated carcinoma is different than that seen in sporadic cases. In particular, SATB2 is significantly less sensitive in colitis-associated carcinoma and it should be interpreted cautiously as a marker of colorectal origin in colitis patients. The association between loss of SATB2 and lymph node metastasis suggests that it may have similar prognostic value in the setting of inflammatory bowel disease as in sporadic cases.