TMA Blocks Research Articles

GLUT1 and ASCT2 expression and their prognostic value in colorectal carcinoma.

Investigation of new molecular markers expressed in colorectal carcinoma can help to select patients getting benefits from new target therapeutic modalities. Investigation of expression of GLUT1 and ASCT2 in colorectal carcinoma. Sixty three colorectal resection specimens for cases diagnosed with colorectal carcinoma were included in the study. Full sections were examined for histopathological data including tumor type, grade, stage, and lymphovascular invasion were recorded. TMA blocks were constructed and immunostained with polyclonal antibodies for both GLUT1 and ASCT2. GLUT1 was expressed in 82% of cases while ASCT2 was expressed in 76% of cases. Statistically significant correlation was found between both GLUT1 and ASCT2. A statistically significant correlation was found between either marker with both disease stage and lymph node metastases. No significant correlation was found between either GLUT1 or ASCT2 and any of the clinical parameters as well as with disease-free survival. GLUT1 and ASCT2 are more prevalent in poorly differentiated and advanced stage colorectal carcinoma. Their expression in high percentage of cases can suggest the possible role of their target therapies in colorectal carcinoma.

DIPG-66. MULTIPLEXED IMMUNOFLUORESCENCE MAPPING AND AI MODEL PREDICTION OF TUMOR MICROENVIRONMENT IN DIFFUSE MIDLINE GLIOMAS

Abstract BACKGROUND Diffuse midline glioma (DMG) is a fatal childhood brain cancer with a survival rate of less than one year from diagnosis. Pharmacological approaches and immunotherapy have failed to make a clinical impact. Incomplete understanding of the tumor microenvironment (TME) and tumor associated antigens have contributed to the observed poor prognosis. Therefore, mapping TME is urgently needed. METHODS Whole brains were collected at autopsy from 80 pediatric subjects, including patients diagnosed with DMG (n=50), GBM (n=9), non-malignant controls (n=10), ependymoma (n=5), ATRT (n=2). Up to four brain anatomical sites were selected: primary tumor, metastatic and adjacent healthy control sites, and processed for staining with hematoxylin and eosin (H&E). After reviewing the tumor and healthy regions, three punch cores were obtained resulting in a total of 918 core punches and two TMA blocks. Multiplexed immunofluorescence (MxIF) technology was used to probe 43 biomarkers on a single TMA slide, focusing on immune cell types and activation thereof. Clinical data including genomics alterations were obtained from each patient for downstream analyses. RESULTS Highest expressed immune markers across all patients were CD8 and CD3 (T-cells), CD68, Iba1 and CD163 (microglia). In DMGs, T-cells were mostly detected and increased in ONC201 and immunotherapy treated patients compared to only immunotherapy treated patients. Analysis of Iba1 confirmed a higher difference in activation in primary tumor compared to metastatic and adjacent healthy tissue, whereas in contrast, CD68 was significantly increased in metastatic sites. Patient plasma and RNA transcriptome analysis confirmed biomarkers and immune related pathways. Further, immune competent murine models were used to validate immune cell infiltration. AI deep learning technology modeling is ongoing now to, in future, be able to predict cell type composition and neighborhoods mapping. CONCLUSION We posit that this comprehensive data and characterization of the DMG TME will help to identify biology-informed targeted treatments.

Alveolar rhabdomyosarcoma: novel surrogate markers associated with oncogenic translocation

Anomalies of the FOXO1 gene in alveolar rhabdomyosarcoma are associated with a worse clinical prognosis, which determines the high value of studying the status of this gene when choosing a therapy strategy. The «gold standard» for determining FOXO1 gene rearrangements is currently the fluorescent in situ hybridization (FISH) technique. Study of the relationship between canonical FOXO1 translocation and immunohistochemical expression of new surrogate markers in alveolar rhabdomyosarcoma to determine their predictive value. 139 cases of rhabdomyosarcoma were retrospectively studied. The study used tissue matrix technology (TMA). On sections obtained from TMA blocks, the FISH technique was implemented using the locus-specific probe MetaSystems XL FOXO1 Break Apart (Metasystems, Germany). Immunohistochemical studies were performed on similar sections from TMA blocks with OLIG2 (Cell Marque Antibodies, clone 211F1.1) and MUC4 (Cell Marque Antibodies, clone 8G7) antibodies. The final expression analysis and statistical processing using a 2x2 contingency table and Fisher's exact test passed 111 cases (76 without FOXO1 rearrangement and 35 with rearrangement). The specificity of OLIG2 and MUC4 expression for FOXO1-rearranged alveolar rhabdomyosarcoma was 85.53% and 80.26%, respectively (p<0.01). The present study confirms the high predictive value of the expression of surrogate markers OLIG2 and MUC4 in determining the genetic status of alveolar rhabdomyosarcoma, which makes it possible to predict with high specificity the detection of the FOXO1 gene rearrangement.

Expression of CD10 and CD15 in colorectal mucinous and signet ring adenocarcinomas and its relation to clinicopathological features and prognosis.

CD10 and CD15 expression has been reported in several tumors. Whether CD10 and CD15 have a role in colorectal mucinous and signet ring adenocarcinoma (MSA) tumorigenesis is not yet known. We aimed to investigate the role of CD10 and CD15 expression in mucinous colorectal adenoma-carcinoma sequence (ACS) and determine if there is any clinical and prognostic significance associated with their expression. Seventy-five cases of colorectal MSA, and 9 cases of adenoma samples were collected. Manual TMA blocks were constructed and immunohistochemistry for CD10 and CD15 was done. Compared to adenomas, CD15 expression was significantly higher in MSA (p= 0.002), in contrast to CD10 expression. CD15 positivity was significantly associated with microsatellite stable (MSS) tumors (p= 0.018). The association between CD10 positivity and fungating tumor growth showed marginal significance. Unlike CD10, CD15 positivity showed significant association with overall survival of colorectal MSA patients. CD15 expression seems to have a role in mucinous colorectal ACS, with significant impact on the survival of MSA patients. Further studies are suggested to identify any genetic alterations that may underlie a potential association with disease progression.

Prognostic significance of E-Cadherin, β-Catenin and cyclin D1 in oral squamous cell carcinoma: a tissue microarray study.

To study the prognostic significance of E-cadherin, β-catenin, and cyclin D1 expression in oral squamous cell carcinoma. The study included 65 subjects with histologically confirmed squamous cell carcinoma. TMA blocks were prepared for immunohistochemical quantification of the expression of the three markers using IHC profiler and Immune ratio plugin of Image J. E-cadherin expression was significantly correlated with histological grades and the metastasis status (p<0.05), whereas β-catenin expression was significantly correlated with smoking and tumor recurrence (P<0.05). Cyclin D1 expression was significantly correlated with depth of invasion and tumor recurrence. (p<0.05). Advanced tumor stage and depth of tumor invasion increases the risk of recurrence or death by 2.5 times (OR= 2.53 and 0.84 respectively). High expression of β-Catenin and Cyclin D1 are significantly correlated with tumor recurrence and old age. Depth of invasion, low histological grade and old age were a significant predictor for the risk of having tumor recurrence and cancer related death.

Accuracy Analysis of a Next-Generation Tissue Microarray on Various Soft Tissue Samples of Wistar Rats

This study aimed to investigate accuracy in different sectional planes of the TMA Grand Master (3DHISTECH) Workstation in various soft tissue samples collected from Wistar rats. A total of 108 animals were sacrificed and 963 tissue specimens collected from 12 soft-tissue types. A total of 3307 tissue cores were punched and transferred into 40 recipient TMA blocks. Digital image analysis was performed. Core loss showed a significant correlation with tissue type and was highest in skin tissue (p &lt; 0.001), renal medulla and femoral artery, nerve, and vein bundle (p &lt; 0.01). Overall, 231 of 3307 tissue cores (7.0%) were lost. Hit rate analysis was performed in 1852 punches. The target was hit completely, partially and missed totally by 89.4%, 7.2% and 2.2%. A total of 54.5% of punches had good accuracy with less than 200 µm deviation from the centre of the targeted region and 92.6% less than 500 µm. Accuracy decreases with greater sectional depth. In the deepest sectional plane of roughly 0.5 mm median depth, almost 90% of cores had a deviation below 500 µm. Recommendations for automated TMA creation are given in this article. The ngTMA®-method has proven accurate and reliable in different soft tissues, even in deeper sectional layers.

Expression of apoptotic protease activating factor-1 in adenoid cystic carcinoma of the salivary glands and its clinicopathological relevance

Background/Aim. Apoptotic protease-activating factor-1 (Apaf-1) is a key molecule in the intrinsic or mitochondrial pathway of apoptosis. Some pathological conditions, such as cancer, stroke, and neurodegenerative diseases, are the result of dysregulation in the intrinsic apoptosis pathway. The aim of this study was to analyze the immuno-histochemical expression of Apaf-1 in adenoid cystic carcinoma (ACC) cells of the salivary glands and its correlation with clinicopathological parameters of patients (gender, age, localization, histological type, and overall survival). Methods. Formalin-fixed, paraffin-embedded tissues of ACC of the salivary glands from 50 male and female patients with an average age of 58 years, were used for the study. We used the technique of tissue microarray (TMA blocks). Sections from the TMA mold, 5 ?m thick, were stained with the streptavidin-biotin immunohistochemical technique using primary antibodies specific for Apaf-1 (Leica Biosystems, Newcastle, UK). Stained tissue sections were analyzed by the light microscope (Olympus type BH-2). Based on the data collected, the database was created in SPSS software v. 22.0 (SPSS Inc., Chicago, ILL, USA), which was used for further statistical analysis. The statistical data analysis included methods of descriptive and analytical (inferential) statistics. Results. The results of the immunohistochemical analysis of Apaf-1 expression in the samples of patients with ACC of the salivary glands were compared with the clinicopathological parameters of these patients. The immunohistochemical expression of Apaf-1 showed no statistical significance with regard to the patients? gender (p = 0.552), age (p = 0.106), histological tumor type (p = 0.654), and localization of ACC in the salivary glands (p = 0.486). There was no statistically significant correlation observed between the overall survival of ACC patients and Apaf-1 expression in tumor cells (p = 0.340, Log-Rank test). Conclusion. With regard to ACC, Apaf-1 expression is not in correlation with clinicopathological parameters (gender, age, localization, histological tumor type, outcome of the disease, and overall survival). Therefore, we believe Apaf-1 cannot be regarded as an independent prognostic factor for course and outcome of ACC of the salivary glands.

An introduction of an easy-operating and economical technique for tissue microarray preparation

AimTissue microarray (TMA) is a powerful and effective tool for in situ tissue analysis. However, manual TMA construction methods showed varied qualities. This study aimed to raise a standardised TMA...

Spinal versus intracranial meningioma: Expression of E-cadherin and Fascin with relation to clinicopathological features.

E-cadherin and Fascin are adhesive proteins that are expressed in many tumors. It was supposed that loss of expression of these proteins is associated with increased aggressiveness of the tumor. Whether spinal and intracranial meningiomas express adhesion proteins in different rates is not yet known. We aimed to investigate the expression of E-cadherin and Fascin in a large number of meningioma specimens and determine if clinical and prognostic significance existsMETHODS: One hundred and thirty-four spinal and intracranial meningioma samples were collected. Manual TMA blocks were constructed and immunohistochemistry for E-cadherin and Fascin was done. Focal or diffuse staining was considered positive. Intracranial meningioma occurred in significantly younger age than spinal ones. Most of spinal meningiomas were of transitional histology. E-cadherin was expressed in 38.8% of cases. Spinal meningiomas showed statistically significant negative expression of E-cadherin than intracranial tumors. All atypical meningiomas showed negative E-cadherin expression. Fascin was expressed in 9% of cases with significant expression in atypical cases. Aggressive behavior of meningioma could be explained in part by loss of E-cadherin and overexpression of Fascin especially in spinal meningiomas. Further studies are suggested to explore the biological aspects of spinal and intracranial meningiomas for constructing tailored targeted therapies.

Spinal Versus Intracranial Meningioma: Aberrant Expression of CD10 and Inhibin with Relation to Clinicopathological Features and Prognosis.

CD10 and inhibin are used mainly in CNS pathology to distinguish hemangioblastoma from metastatic clear cell renal cell carcinoma. Some meningiomas can mimic both tumors and so we aimed at this study to investigate the expression of both markers in a large number of meningioma cases. One hundred thirty-four meningioma samples were collected, 14 of them were spinal and 120 were intracranial. Manual TMA blocks were constructed using modified mechanical pencil tip method and immunohistochemistry for CD10 and inhibin was done. Intracranial meningioma occurred in significantly younger age than spinal ones. Most of spinal meningiomas were of transitional histology. CD10 was expressed in 14% of cases with significant positivity in spinal rather than intracranial cases. Transitional meningiomas showed the highest positivity for CD10 expression, while the least positive was the meningiotheliomatous type. Inhibin was expressed in 6% of cases with no significant relation to clinicopathological and histological features. There was no significant relationship between the expression of CD10 and inhibin expression in meningiomas. In conclusion, spinal meningiomas differ than intracranial ones in many clinicopathological and biological aspects. Among these differences is CD10 expression being more expressed in spinal meningiomas. However CD10 and inhibin are aberrantly expressed in a proportion of meningiomas, both have no relations to poor prognostic factors but more caution should be exerted during usage of these markers in diagnosis of hemangioblastoma and metastatic RCC. Further studies are suggested for exploring more biological differences between spinal and intracranial meningiomas.

Increased COX-2 Immunostaining in Urothelial Carcinoma of the Urinary Bladder Is Associated with Invasiveness and Poor Prognosis.

Background Urothelial carcinoma of the urinary bladder (UCB) is the commonest bladder tumor. Cyclooxygenase-2 (COX-2) mediates angiogenesis, cell survival/proliferation, and apoptosis. This study investigates the relation of COX-2 immunostaining in UCB to clinicopathological parameters in Saudi Arabia. Methods The study population includes 123 UCB and 25 urothelial mucosae adjacent to UCB. UCB samples were collected before any local or systemic therapy. Tissue microarrays were designed and constructed, and TMA blocks were sliced for further immunohistochemical staining. Immunohistochemical staining was done using a mouse anti-human COX-2 monoclonal antibody. A cutoff point of 10% was chosen as the threshold to determine low and high COX-2 immunostaining. Results COX-2 immunostaining is higher in UCB than in the adjacent urothelium (p = 0.033). High COX-2 immunostaining is associated with high-grade UCB (p = 0.013), distant metastasis (p = 0.031), lymphovascular invasion (p = 0.008), positive muscle invasion (p = 0.017), pT2 and above (p = 0.003), and high anatomical stages (stage II and above). High COX-2 immunostaining is an independent predictor of higher tumor grade (p < 0.001), muscle invasion (p = 0.015), advanced pathological T (p = 0.014), lymphovascular invasion (p = 0.011), and distant metastasis (p = 0.039). High COX-2 immunostaining is associated with lower overall survival rate (p = 0.019). Conclusion COX-2 immunostaining is associated with the invasiveness of UCB which may be used as an independent prognostic marker. COX-2 may be a significant molecule in the initiation and progression of UCB. Molecular and clinical investigations are required to explore the molecular downstream of COX-2 in UCB and effectiveness of COX-2 inhibitors as adjuvant therapy along with traditional chemotherapy.

Abstract P5-07-10: The prognostic significance of estrogen formation as a consequence of aromatase expression in tumor microenvironment

Abstract Estrogen synthesis via aromatase in adipose tissue has an important role in progression of postmenopausal breast cancer. The increased local concentrations of estrogen in breast cancer via aromatase overexpression within the tumor tissue have been demonstrated by some investigators. Although aromatase inhibitor is the standard endocrine therapy for postmenopausal breast cancer patients, it is not uncommon for patients to have poor compliance to the drugs due to their side effects. This research is based on the hypothesis that if aromatase expression is related to prognosis and if therapeutic effect varies depending on the degree of aromatase expression, then this study may be able to suggest a new guideline in terms of choosing between aromatase inhibitors and tamoxifen. Methods: 154 postmenopausal breast cancer patients who underwent surgery and aromatase inhibitor therapy in Busan Paik Hospital, Inje University from January 2005 to December 2010 were enrolled. Patients with DCIS or stage IV breast cancer were excluded. Patients' clinicopathological data were collected and TMA blocks were created for immunohistochemistry studies to examine aromatase expression. Results: The recurrence has occured in 7 patients (6.9%). Stage, tumor size and number of lymph node metastasis were related to increased risk of recurrence (p=0.051, 0.043, 0.001). The aromatase expression in cancer cells had significant correlation with clinical stage (p=0.041). There was also a positive correlation between Ki67 and aromatase expression in cancer tissue (p=0.006). However, aromatase expression in cancer, stromal, and adipose tissue had no relationship with recurrence (p=0.410, 0.627, 0.552). Correlation between clinicopathologic factors and aromatase expression (Linear by linear association and Spearman's correlation coefficient test)Site of aromatase expressionERPRKi67P53BMIStageIn Cancer0.6060.5920.0060.4510.9560.041In Stroma0.2200.4710.5840.3290.3670.229In Adipose0.9880.2650.1590.1170.7701.000 Conclusions: Aromatase expression in cancer cells was correlated with clinical stage. This implies that aromatase expression might have a role of prognostic marker in addition to role of treatment indicator. There was no direct correlation between aromatase expression and recurrence. Citation Format: Kim TH, Lee A, Han JW, Jung SJ, Byun K-D. The prognostic significance of estrogen formation as a consequence of aromatase expression in tumor microenvironment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-07-10.

EGFR, COX2, p-AKT expression and PIK3CA mutation in distal extrahepatic bile duct carcinoma

Distal extrahepatic bile duct (EBD) carcinoma is a rare but highly aggressive malignant neoplasm. Some invitro studies have shown that EGFR and PI3K-Akt pathway play an important role in the carcinogenesis of bile duct carcinoma. The aim of the present study is to investigate the expression of EGFR, p-AKT, and COX-2 and the mutation of PIK3CA in distal EBD carcinoma and evaluate the association with clinicopathological factors. Ninety cases of distal extrahepatic bile duct (EBD) carcinoma specimens were studied. Immunohistochemistry (IHC) using antibodies against EGFR, p-AKT, and COX-2 was performed on TMA blocks. The PIK3CA mutation was evaluated using the PNAClamp Detection Kit from DNA samples extracted from formalin fixed, paraffin embedded tissue. EGFR expression of distal EBD carcinomas was 61.9%, 26.2%, 6.0% and 6.0% in the negative, weakly positive, moderately positive, and strongly positive groups, respectively. Positive EGFR expression showed significant relationships with high T stage (p=0.024). In Kaplan-Meier analysis, EGFR expression was associated with shorter cancer-specific overall survival (p=0.005). Multivariate analysis also showed that moderate or strong (2+ or 3+) EGFR expression was a significant prognostic factor in distal EBD carcinoma: HR 5.286; p=0.001. Ninety cases of EBD carcinoma tissue were analysed for hotspot mutations (exon 9 and 20) in the PIK3CA gene. Only one mutation was detected: a missense mutation of H1047 at exon 20. The expression levels of p-AKT and COX-2 showed no association with any clinicopathological parameters, including survival rate. Moderate and strong EGFR expressions demonstrate a direct link to poor prognosis. Although further study is warranted to understand the clinicopathological significance, our finding suggests EGFR is a useful prognostic marker of patients with distal EBD carcinoma. A low prevalence of PIK3CA mutation exists in the distal EBD carcinoma of Korean patients, indicating that mutation screening may not be useful in determining prognosis or in formulating a treatment response to targeted inhibition in Korea.

Abstract C155: Cyclin-dependent kinase 1 (Cdk1) is a promising therapeutic target to overcome ovarian cancer

Abstract Purpose: Ovarian cancer shows heterogeneous characteristics which cause chemoresistance and recurrence. The goal of this study was to elucidate characteristics common to various ovarian cancers in order to evaluate their potential for targeted therapy. Methods: Microarray results for cell lines of serous, mucinous, and brenner types of ovarian cancers were reanalyzed and validated via ovarian cancer cell lines, GEO datasets and TMA blocks. RO-3306 which is an inhibitor of Cdk1 and si-cdk1 was used to measure the growth rate of ovarian cancer cell lines via FACS analysis. In addition, combination treatment with RO-3306 and cisplatin was administered in vitro and in in vivo xenograft mouse model. Results: Microarray reanalysis revealed that expression of Cdk1 and cyclinB1 was higher in 5 types of ovarian cancer cell lines, 4 GEO datasets and ovarian cancer TMA blocks than in HOSE cells. Ovarian cancer TMA particularly showed increased level of cytoplasmic Cdk1 protein, but not in nucleus. When Cdk1 was inhibited by siRNA and a potent Cdk1 inhibitor (RO-3306), growth of ovarian cancer cells decreased, while G2/M arrest or apoptosis increased. In addition, tumor growth was significantly lesser in a xenograft mouse model treated with a combination of RO-3306 and cisplatin than in mice treated with each drug alone. Conclusions: Cdk1 is a promising gene for targeted anticancer therapy. It is expected that combined treatment with Cdk1 inhibitor and chemotherapeutic agents would maximize the effects of ovarian cancer treatment. Citation Format: Hanbyoul Cho, Wookyeom Yang, Ha-Yeon Shin, Eun-ju Lee, Doo-Byung Chay, Jae-Hoon Kim. Cyclin-dependent kinase 1 (Cdk1) is a promising therapeutic target to overcome ovarian cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C155.

The Lauren classification highlights the role of epithelial-to-mesenchymal transition in gastric carcinogenesis: an immunohistochemistry study of the STAT3 and adhesion molecules expression.

Despite some recent advances, gastric cancer remains an important cause of death at world level. This indicates an absence of therapeutic options, stemming from the limited understanding of the molecular mechanisms involved in carcinogenesis. Nearly fifty years ago Lauren classified gastric cancers, according to the morphological aspect, as intestinal or diffuse. The phenotype of the cells indicates the presence of different molecular mechanisms, which can be approached in the light of recent data and identified with the help of current techniques. The best described are the germline/somatic mutations or the hypermethylations of the E-cadherin 1 CDH1 gene promotor. We analyzed 195 gastric tumors,120 intestinal and 75 diffuse type, using immunohistochemistry (tissue microarray TMA method) for pStat3Tyr705, E-cadherin, α-catenin and β-catenin; 985 spots of gastric tumors, distributed on 4 TMA blocks were analyzed. For pStat3Tyr705 we took the nuclear staining into account and for the adhesion molecules, membrane staining. In our study, in the diffuse type gastric cancer, pStat3Tyr705 nuclear expression was statistically significantly increased (p=0.003). Also we observed a decreased expression of the adhesion molecules in the same type of gastric cancer (E-cadherin p<0.0001, α-catenin p<0.0001, β-catenin p<0.0001), suggesting that epithelial-to-mesenchymal transition (EMT) may be involved not only in gastric carcinogenesis, but also in resistance to treatment. The Stat3 role has been recently highlighted in carcinogenesis of the diffuse type of gastric cancer. We found that the morphological features of the diffuse type also suggest the involvement of EMT in this type of gastric cancer. Therefore, targeting the key molecules involved in this process may interfere with EMT process in the diffuse type of gastric cancer.

βIII-Tubulin Overexpression Is an Independent Predictor of Prostate Cancer Progression Tightly Linked to ERG Fusion Status and PTEN Deletion

Evidence suggests that class III β-tubulin (βIII-tubulin) may represent a prognostic and predictive molecular marker in prostate cancer. βIII-Tubulin expression was determined by IHC in 8179 prostate cancer specimens in a TMA format. Results were compared with tumor phenotype, biochemical recurrence, v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) status, and deletions on PTEN, 3p13, 5q21, and 6q15. βIII-Tubulin expression was detectable in 25.6% of 8179 interpretable cancers. High βIII-tubulin expression was strongly associated with both TMPRSS2:ERG rearrangement and ERG expression (P<0.0001 each). High βIII-tubulin expression was tightly linked to high Gleason grade, advanced pT stage, and early prostate-specific antigen (PSA) recurrence in all cancers (P<0.0001 each), but also in the subgroups of ERG-negative and ERG-positive cancers. When all tumors were analyzed, the prognostic role of βIII-tubulin expression was independent of Gleason grade, pT stage, pN stage, surgical margin status, and preoperative PSA. Independent prognostic value became even more evident if the analysis was limited to preoperatively available features, such as biopsy specimen Gleason grade, preoperative PSA, cT stage, and βIII-tubulin expression (P<0.0001 each). βIII-Tubulin expression was associated with PTEN (P<0.0001) when all tumors were analyzed, but also in the subgroups of ERG-negative and ERG-positive cancers. βIII-Tubulin expression is an independent prognostic parameter. The significant associations with key genomic alterations of prostate cancer, such as TMPRSS2:ERG fusions and PTEN deletions, suggest interactions with several pivotal pathways involved in prostate cancer.

Molecular subtypes of ductal carcinoma in situ in African American and Caucasian American Women: Distribution and correlation with pathological features and outcome

Background: Molecular subtypes of breast cancer have been extensively studied in invasive carcinoma. They were shown to have a different distribution within the various ethnic populations. Few studies have applied the same classification to Ductal Carcinoma in Situ (DCIS). We report the distribution of the molecular breast cancer subtypes in DCIS between African American (AA) and Caucasian American (CA) women, their association with pathological features and outcome. Materials and methods: Tissue microarrays were constructed from paraffin blocks of 94 DCIS cases (67 AA and 27 CA) selected from a cohort of AA and CA patients diagnosed with DCIS between 1996 and 2000; mean age at diagnosis was 61±12 for the AA and 58±11 years for the CA group. TMA blocks were labeled with antibodies for ER, PR, HER2, Ki-67, and CK5/6. The cases were subtyped as Luminal A (ER+ and/or PR+; HER2−), Luminal B (ER+ and/or PR+; HER2+), HER2+ (ER−, PR−; HER2+), basal-like (BL) (ER−, PR−, HER2−; CK5/6+) or unclassified triple negative (UTN) (ER−, PR−, HER2−, CK5/6−). Information on grade, size and follow-up were obtained. Results: (1) Most DCIS cases were Luminal A, comprising 80% of the DCIS cases in AA and 92.6% in CA patients. (2) HER2+, BL and UTN DCIS subtypes were not seen in the CA population, and formed 9% of the DCIS cases in the AA population; these cases were all high grade. (3) In the cases with recurrence (8 AA and 1 CA patients), DCIS was Luminal A in 6 AA and 1 CA and Luminal B in 2 AA patients. Conclusion: The distribution of the molecular subtypes of DCIS did not show a significant difference between the two ethnic groups in our study. In addition, the risk of recurrence might not be higher in the non-luminal subtypes than in Luminal A and B.

Inflammatory cells in node-negative breast cancer

Background. To study the impact of inflammatory cells in a clinically well-defined cohort of women with node-negative breast cancer in a nested case-control study design. Material and methods. The cohort was comprised of 190 women who died from breast cancer and 190 women still alive at the date of death for the corresponding breast cancer patients were used as controls. The inclusion criteria included; a tumour size ≤ 50 mm, no lymph node metastases and no initiation of adjuvant chemotherapy. Immunohistochemical stainings for CD3, CD4, CD8, FoxP3, CD20, tryptase and CD68 were performed on TMA blocks, evaluated and correlated to each other and to age, tumour size, histological grade, ER, PgR, Ki67 and cyclin A. Results. There was no difference regarding the amount or content of inflammatory cells in the cases compared to controls. T- and B-cells were highly correlated to each other but these cell types correlated to a lesser extent to macrophages and not at all to mast cells. A weak tendency of correlations between all the subsets of inflammatory cells and histological grade, Ki67 and cyclin A was observed, although a negative correlation was seen for mast cells. Conclusion. The amount or content of inflammatory cells in invasive breast cancer did not appear to influence death in node-negative breast cancer.

P4-10-01: Tumor Markers Predicting Recurrence Type after a Primary Ductal Carcinoma In Situ.

Abstract Background: The risk for recurrence after a primary ductal carcinoma in situ (DCIS) is approximately 8% after ten years. If a recurrence occurs, the risk of the recurrence being invasive is approximately 50%. However, the knowledge of determinants for type of recurrence is limited. The purpose of this study was to investigate the predictable effect of specific biologic markers on the type recurrence after a primary DCIS. Material and Methods: All women diagnosed with a primary DCIS between 1986 and 2004 in Uppsala and Västerås, Sweden were included (n=458). We also included all women from the SweDCIS Trial with a known recurrence (n= 166). TMA blocks were constructed and immunohistochemistry (IHC) was used to evaluate the status of estrogen receptor (ER), HER2, epidermal growth factor receptor (EGFR/HER1), cytokeratin 5/6 (CK 5/6), forkhead box A1 (FOXA1), forkhead box C1 (FOXC1), Ki 67, CD 10 and transacting T-cell-specific transcription factor GATA-3 (GATA-3) in the primary DCIS tumors. Logistic regression was employed to calculate the difference of expression status of biological markers between groups. Odds ratios (ORs) and 95% confidence interval (CI) were calculated, with adjustment for age, free margins, surgical type and molecular subtype by IHC. Results: Of the 624 patients, 358 without recurrence were grouped as controls. Of the 266 with recurrence, 130 developed an in situ recurrence, while the remaining 136 developed invasive recurrence. Mastectomy compared to breast conserving surgery (BCS) and free margins were associated with less recurrences (OR 0.07, 95% CI 0.03 — 0.15) and (OR 0.39, 95% CI 0.26 — 0.58). As compared with the most common molecular subtype Luminal A, the subtype Luminal B had statistically significant higher risk of recurrence (OR 2.01, 95% CI 1.12 — 3.59). Higher risk of all recurrences was also observed for FOXA1 (OR 3.16, 95% CI 1.48 — 4.74) and FOXC1 (OR 2.39, 95% CI 1.39 — 4.09). In the analyses of type of recurrence, ER-negative, HER2−positive and EGFR-positive tumors recurred more often as in situ; (OR 2.34, 95% CI 1.17 — 4.71), (OR 2.08, 95% CI 1.11 — 3.88) and (OR 2.20, 95% CI 1.11 — 4.36). The molecular subtype HER2+ was also associated with a lower risk of invasive recurrence (OR 0.27, 95% CI 0.11 — 0.67). Conclusion: As expected, mastectomy and free margins were associated with a lower risk of recurrence. Molecular subtype Luminal B and tumor markers FOXA1 and FOXC1 had a higher risk of recurrence. Recurrences after molecular subtype HER2+, ER-negative and EGFR-positive DCIS were more often of the in situ type. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-10-01.

Long-term survival of women with basal-like ductal carcinoma in situ of the breast: a population-based cohort study

BackgroundMicroarray gene-profiling of invasive breast cancer has identified different subtypes including luminal A, luminal B, HER2-overexpressing and basal-like groups. Basal-like invasive breast cancer is associated with a worse prognosis. However, the prognosis of basal-like ductal carcinoma in situ (DCIS) is still unknown. Our aim was to study the prognosis of basal-like DCIS in a large population-based cohort.MethodsAll 458 women with a primary DCIS diagnosed between 1986 and 2004, in Uppland and Västmanland, Sweden were included. TMA blocks were constructed. To classify the DCIS tumors, we used immunohistochemical (IHC) markers (estrogen-, progesterone-, HER2, cytokeratin 5/6 and epidermal growth factor receptor) as a surrogate for the gene expression profiling. The association with prognosis was examined for basal-like DCIS and other subtypes using Kaplan-Meier survival analyses and Cox proportional hazards regression models.ResultsIHC data were complete for 392 women. Thirty-two were basal-like (8.2%), 351 were luminal or HER2-positive (89.5%) and 9 unclassified (2.3%). Seventy-six women had a local recurrence of which 34 were invasive. Another 3 women had general metastases as first event. Basal-like DCIS showed a higher risk of local recurrence and invasive recurrence 1.8 (Confidence interval (CI) 95%, 0.8-4.2) and 1.9 (0.7-5.1), respectively. However, the difference was not statistically significant. Also, no statistically significant increased risk was seen for triple-negative or high grade DCIS.ConclusionsBasal-like DCIS showed about a doubled, however not statistically significant risk for local recurrence and developing invasive cancer compared with the other molecular subtypes. Molecular subtyping was a better prognostic parameter than histopathological grade.