Primary Invasive Breast Carcinomas Research Articles

5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines.

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive therapy that can target local metastatic bone lesions. This paper presents an in vitro method to study the PDT effect in adherent cell lines. To this end, we demonstrate a step-by-step approach to subject both primary (giant cell bone tumor) and human bone metastatic cancer cell lines (derived from a primary invasive ductal breast carcinoma and renal carcinoma) to 5-aminolevulinic acid (5-ALA)-mediated PDT. After 24 h post 5-ALA-PDT irradiation (blue light-wavelength 436 nm), the therapeutic effect was assessed in terms of cell migration potential, viability, apoptotic features, and cellular growth arrest (senescence). Post 5-ALA-PDT irradiation, musculoskeletal-derived cell lines respond differently to the same doses and exposure of PDT. Depending on the extent of cellular damage triggered by PDT exposure, two different cell fates-apoptosis and senescence were noted. Variable sensitivity to PDT therapy among different bone cancer cell lines provides useful information for selecting more appropriate PDT settings in clinical settings. This protocol is designed to exemplify the use of PDT in the context of musculoskeletal neoplastic cell lines. It may be adjusted to investigate the therapeutic effect of PDT on various cancer cell lines and various photosensitizers and light sources.

Unusual staining of immunohistochemical markers PAX8 and CDX2 in breast carcinoma: a potential diagnostic pitfall

Knowing the sensitivity and specificity of tissue-specific immunohistochemical markers is crucial for accurate determination of the primary tumor site. PAX8 has been used as a diagnostic marker for carcinomas of the gynecologic tract, kidney, and thyroid gland, and CDX2 has been used as a marker of gastrointestinal carcinoma. Neither is considered a marker for breast carcinoma (BC). However, we have encountered BCs that express PAX8 or CDX2, some of which caused diagnostic confusion. We investigated the immunohistochemical staining frequency of PAX8 and CDX2 in BC. We identified 237BCs for which PAX8 staining results were reported (102 primary and 135 metastatic BCs); seven primary and four metastatic BCs (4.6%) were positive for PAX8, with various intensities and staining patterns. CDX2 staining results were reported for 271BCs (78 primary and 193 metastatic); four primary BCs and one metastatic BC (1.8%) were positive for CDX2, ranging from focal and weak to diffuse and strong. We also stained primary invasive BCs with PAX8 and CDX2 using tissue microarrays. None of the 332 PAX8-stained cases was positive, while one of 143 CDX2-stained cases was positive. Four PAX8-positive and three CDX2-positive cases were stained with TRPS1, and all were positive for TRPS1. In addition, we reviewed the literature for PAX8 and CDX2 expression in BCs and found 5.5% PAX8-positive BCs (90/1625) in 17 studies and 0.8% CDX-2 positive BCs (7/909) in 20 studies. PAX8 and CDX2 are infrequently expressed in BC by immunohistochemistry, and in rare cases, the staining can be strong and diffuse. Additional diagnostic markers are necessary and helpful in distinguishing breast from other primary origins.

TRPS1, GATA3, and SOX10 expression in triple-negative breast carcinoma

A diagnostic dilemma can be encountered when primary triple-negative breast carcinoma (TNBC) without an in situ component or metastatic TNBCs lose the currently used organ-specific marker such as GATA3, raising concerns about metastatic carcinoma from other sites. In the current study, we compared the newly identified breast marker TRPS1 with currently used breast markers GATA3 and SOX10 in whole-tissue sections from 315 cases of various subtypes of TNBC. TRPS1 was highly expressed in 100% of triple-negative primary and metastatic invasive lobular carcinomas, 99% of triple-negative primary and metastatic invasive breast carcinoma of no special type (IBC-NST), and 95% of metaplastic breast carcinomas. In contrast, GATA3 and SOX10 were expressed in 94% and 0% of invasive lobular carcinomas, 63% and 74% of IBC-NST, and 50% and 49% of metaplastic breast carcinomas, respectively. For special-type TNBCs, both TRPS1 and GATA3 were negative in acinic cell carcinomas, most cribriform adenoid cystic carcinomas, and neuroendocrine carcinomas, but positive in secretory carcinomas. Triple-negative apocrine carcinoma was the only subtype of TNBC with positive GATA3 but negative TRPS1. These data indicate that TRPS1 is a highly sensitive marker for TNBCs with positivity not only in GATA3/SOX10-positive TNBCs but also in almost all GATA3/SOX10-negative TNBCs.

Abstract PD3-03: EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression

Abstract Introduction: Triple negative breast cancers (TNBC) are frequently poorly differentiated with higher propensity for metastasis than all other subtypes. Enhancer of Zeste Homolog 2 (EZH2) is a lysine methyltransferase that mediates transcriptional repression of pro-differentiation genes in normal and neoplastic cells. The oncogenic role of EZH2 through H3K27me3 is well established. However, non-canonical H3K27me3-independent functions are unclear. We have reported that p38 phosphorylates T367 of EZH2, increasing TNBC metastasis. Recent reports show that EZH2 can regulate signaling pathways through direct methylation of proteins suggesting the hypothesis that EZH2 may methylate p38 in TNBC. Methods: Human tissue samples from 16 primary invasive breast carcinomas and matched distant metastasis arrayed in tissue microarrays were interrogated for pEZH2 T367 and p-p38 by immunohistochemistry. To study p38 methylation, we performed LC-MS/MS analyses GST-p38α, and GST-EZH2 were each incubated with or without recombinant PRC2 complex (EZH2/E ED/SUZ12/RbAp48/AEBP2) and S-adenosyl methionine methyl donor. MDA-MB-231, mouse and human primary TNBC cells and murine 4T1 cells were used in funcional assays in vitro and in vivo. EZH2 knockdown and rescue was carried out using lentiviral transduction of EZH2 with pBabe-myc-EZH2 (wild-type) or pBabe-myc-EZH2 (T367A). We developed p38 methylation mutants: HA-p38a K139A, HA-p38a K165A, and HA-p38a K130A/K165K. To investigate the importance of EZH2 and p38 enzymatic activities we used the EZH2 methyltransferase inhibitors GSK-343 and EPZ-6438 and the p38 inhibitor SB202190 in vitro and in vivo. For animal studies, MDA-MB-231 and 4T1 cells were orthotopically injected into the right inguinal mammary fat pad of eight-week old NOD/SCID or BALBc mice, respectively. When tumors reached 100 mm3 mice were treated 5 days/week by intraperitoneal injection with SB202190 (1mg/kg/day), EPZ-6438 (10mg/kg/day), combination of SB202190 and EPZ-6438, or control. We monitored tumor growth for 50 days, and harvested primary tumors, lungs, and sites of metastasis for histopathology, Western blot, and immunohistochemistry. Primary xenografts from the control and treatment groups were subjected to RNA sequencing. Results: EZH2 methylates p38 at lysines 139 and 165 leading to enhanced p38 stability and increased invasion, and p38 activation requires T367 phosphorylation of EZH2. Dual inhibition of EZH2 methyltransferase and p38 kinase activities downregulates pEZH2 T367, H3K27me3, and p-p38 in vivo, reduces TNBC growth and metastasis, and results in gene expression changes towards a better differentiated phenotype. pEZH2 T367 and p-p38 proteins are significantly coexpressed in human primary and metastatic breast cancer. Conclusions: We provide direct evidence that EZH2 methylates p38 with resultant p38 activation, and that EZH2 phosphorylation at T367 is important for this function. Dual targeting of EZH2 methyltransferase and p38 kinase activities with specific inhibitors reduces breast cancer growth and metastasis indicating a cooperation between EZH2 canonical and non-canonical mechanisms and suggesting therapeutic strategies. Citation Format: Maria E Gonzalez, Shilpa R. Tekula, Talha Anwar, Shoshana A. Leflein, Celina G. Kleer. EZH2 T367 phosphorylation activates p38 signaling through lysine methylation to promote breast cancer progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-03.

Evaluation of CD9 Expression of Tumour Cells and Stromal Immune Cells in Breast Carcinoma by Immunohistochemistry

Introduction: Cluster of Differentiation 9 (CD9), known as Motility Related Protein (MRP-1) regulates cell adhesion, motility, migration and proliferation. Many studies have stated conflicting results on prognostic significance of invasive breast carcinomas with CD9 expression that had performed on tumour tissues. Aim: To assess the inter-relationship of CD9 expression of tumour cells and stromal immune cells in breast carcinoma with clinicopathological parameters which include age, tumour size, grade, histological type, lymph nodes, tumour staging and molecular classification. Materials and Methods: An observational prospective (2020 July to 2021 June) and retrospective (2019 October to 2020 June) study was done in 71 cases of resected primary invasive breast carcinoma over a period of one and half year at Sri Devaraj Urs Medical College, Karnataka, India. Immunohistochemistry (IHC) staining was done using CD9 antibody. Tumour cells (T-CD9) expression was evaluated by Immunoreactivity scoring (IS= Intensity score × Extent of staining). The stromal cells (S-CD9) expression was evaluated by Percentage (%) of stromal area occupied by CD9 stained immune cells. Chi-square test was used as test of significance for qualitative data. The p-value of <0.05 was considered as statistically significant. Results: Out of 71 cases, T-CD9 expression was noticed in 40 cases (56.34%) and IS <4 considered as negative was observed in 31 cases (43.66%). However there was no association with age, tumour size, grade and molecular markers for the expression of both T-CD9 and S-CD9. Human Epidermal growth factor receptor 2 neu (HER2neu) negative was associated with T-CD9 expression (p-value=0.05). Hence CD9 can be used as prognostic marker for Her2neu negative cases. Conclusion: CD9 expression was not significantly associated with tumour cells (T-CD9) and stromal cells (S-CD9) in breast carcinoma cases. However it was significantly associated with Her2neu negative tumour cells. T-CD9 showed more positivity in Luminal A followed by Triple negative whereas S-CD9 showed more positivity in Luminal B. CD9 did not show association with any parameters except Her2neu negative.

HOTAIR Modulated Pathways in Early-Stage Breast Cancer Progression.

The long-non-coding HOX transcript antisense intergenic RNA (HOTAIR) was identified as significantly upregulated in breast ductal carcinoma in situ (DCIS). The aim of this study was to characterize the phenotypic effects and signaling pathways modulated by HOTAIR in early-stage breast cancer progression. We determined that HOTAIR induces premalignant phenotypic changes by increasing cell proliferation, migration, invasion and in vivo growth in normal and DCIS breast cell lines. Transcriptomic studies (RNA-seq) identified the main signaling pathways modulated by HOTAIR which include bioprocesses related to epithelial to mesenchymal transition, cell migration, extracellular matrix remodeling and activation of several signaling pathways (HIF1A, AP1 and FGFR). Similar pathways were identified as activated in primary invasive breast carcinomas with HOTAIR over-expression. We conclude that HOTAIR over-expression behaves as a positive regulator of cell growth and migration both in normal and DCIS breast cells involved with early-stage breast cancer progression.

Assessment of 5-Aminolevulinic Acid-Mediated Photodynamic Therapy on Bone Metastases: An in Vitro Study.

Simple SummaryBone metastases are typically associated with a short-term prognosis. Photodynamic therapy (PDT) emerges as a promising alternative treatment for targeting metastatic lesions. In this study we investigated the effect of 5-aminolevulinic acid-mediated PDT treatment on both primary and human bone metastatic cancer cell lines. We found that human cell lines have different sensitivity to the same doses and exposure of 5-ALA PDT resulting in two different cell fates, apoptosis or senescence, depending on the extent of the cellular damage. As such, PDT has potential applicability in bone metastases of invasive ductal carcinoma.Bone is a frequent site of metastases, being typically associated with a short-term prognosis in affected patients. Photodynamic therapy (PDT) emerges as a promising alternative treatment for controlling malignant disease that can directly target interstitial metastatic lesions. The aim of this study was to assess the effect induced by PDT treatment on both primary (giant cell bone tumor) and human bone metastatic cancer cell lines (derived from a primary invasive ductal breast carcinoma and renal carcinoma). After 24 h post light delivery (blue light-wavelength 436 nm) with 5-aminolevulinic acid, the effect on cellular migration, viability, apoptosis, and senescence were assessed. Our results showed that bone metastasis derived from breast cancer reacted with an inhibition of cell migration coupled with reduced viability and signs of apoptosis such as nuclei fragmentation following PDT exposure. A limited effect in terms of cellular viability inhibition was observed for the cells of giant cell bone tumors. In contrast, bone metastasis derived from renal carcinoma followed a different fate—cells were characterized by senescent features, without a notable effect on cell migration or viability. Collectively, our study illustrates that PDT could act as a successful therapy concept for local tumor control in some entities of bone metastases.

Axillary Lymph Node Involvement in Primary Invasive Breast Carcinoma in a Center in Baghdad Province

Introduction: Breast carcinoma is the most common malignancy in women and it has become a major health problem affecting women worldwide. In Iraq, breast carcinoma is still a major cause of health morbidity and mortality in women. Axillary lymph node metastasis is considered the most important factor affecting prognosis and management in patients with breast cancer. Objective: This study aimed to evaluate lymph node metastasis in cases of primary invasive breast carcinoma and to find if there was any association among lymph node involvement with type, grade, and tumor stage. Materials and Methods: This was cross-sectional study of 100 female patients with primary invasive breast carcinoma aged 25–88 years, which collected from the archive of the educational laboratory in Baghdad Medical City, Baghdad, Iraq from April 2018 to December 2020. Male patients were excluded from this study. Results: A total of 100 patients were included in this study; all were women with primary invasive breast cancer with different histopathological types (80% of cases were invasive ductal ca. NOS). The age range of the patients was between 25 and 88 years. A significant correlation was observed between lymph node stage and tumor pathological stage (tumor size). Conclusions: Most cases of primary invasive breast carcinoma had axillary lymph nodes metastasis, with a strong correlation between lymph node stage and tumor size. However, no association was found with tumor grade or tumor type that could be related to the small sample size of this study.

Prognostic Value of NLRP3 Inflammasome and TLR4 Expression in Breast Cancer Patients.

Inflammasome complexes play a pivotal role in different cancer types. NOD-like receptor protein 3 (NLRP3) inflammasome is one of the most well-studied inflammasomes. Activation of the NLRP3 inflammasome induces abnormal secretion of soluble cytokines, generating advantageous inflammatory surroundings that support tumor growth. The expression levels of the NLRP3, PYCARD and TLR4 were determined by immunohistochemistry in a cohort of primary invasive breast carcinomas (BCs). We observed different NLRP3 and PYCARD expressions in non-tumor vs tumor areas (p<0.0001). All the proteins were associated to more aggressive clinicopathological characteristics (tumor size, grade, tumor proliferative activity etc.). Univariate analyses were carried out and related Kaplan-Meier curves plotted for NLRP3, PYCARD and TLR4 expression. Patients with higher NLRP3 and TLR4 expression had worse 5-year disease-free survival (DFS) compared to patients with lower NLRP3 and TLR4 expression (p =0.021 and p = 0.009, respectively). In multivariate analysis, TLR4 was confirmed as independent prognostic factors for DFS (HR = 2.03, 95% CI 1.16–3.57, p = 0.014), and high NLRP3 expression showed a slight association with DFS (HR = 1.75, 95% CI 0.98–3.15, p = 0.06). In conclusion, we showed TLR4 expression as independent prognostic factors and we highlighted for the first time that high expression of NLRP3 is linked to a poor prognosis in BC patients. These results suggest that NLRP3 and TLR4 could be two new good prognostic factor for BC patients.

KI67/BCL2 index in invasive breast carcinoma (NST)

Background: B-cell lymphoma-2 (BCL2) is an anti-apoptotic protein that has an anti-proliferative effect and is a favourable prognostic marker in breast carcinoma. Ki67/BCL2 ratio is emerging as a superior prognostic marker than either marker alone. Aim: This study investigated the association of Ki67/Bcl2 index with hormonal receptor status and other clinicopathological parameters in premenopausal and postmenopausal primary invasive breast carcinomas (NST). Method: Clinicopathological data were obtained from 63 (42%) premenopausal and 87 (58%) postmenopausal carcinomas from a prospective study. Immunohistochemical assessment of ER, PR (Allred score), HER2 (ASCO/CAP 2013), Ki67 and BCL2 in the tumours were performed using routine protocols. BCL2 and Ki67 scores were obtained based on the percentage of positively stained cells (0=0–10%, 1=11–32%, 2=>33%). BCL2 staining score was subtracted from the Ki67 score to produce five categories (−2, −1, 0, 1, 2) and further categorised into two groups, low-index (-2, -1, 0) and high-index (1, 2). Association of Ki67/Bcl2 index with tumour grade, tumour size, T and N stage, nodal status, node ratio (<0.15), ER, PR and HER2 status and molecular subtype, were evaluated using the Chi-square test (SPSS-20). Associations were considered significant when p≤0.05. Results and conclusion: High Ki67/BCL2 index was significantly associated with negative ER (premenopausal χ2=4.701(1), p=0.030; postmenopausal χ2=23.104(1), p≤0.001) and negative PR (premenopausal χ2=12.106(1), p=0.001; postmenopausal χ2=12.956(1), p≤0.001) expression in both groups. It was also significantly associated with premenopausal grade ΙΙΙ tumours (χ2=8.168(2), p=0.017) and postmenopausal triple negative breast cancers (χ2=19.510(4), p=0.001). It is evident that high Ki67/BCL2 index, indicating a high proliferation rate, is associated with poor prognosticators in breast carcinoma and is suggestive of a poor clinical outcome.

O30: EVALUATION OF AXILLARY LYMPH NODE METASTATIC BURDEN BY PREOPERATIVE ULTRASOUND

Abstract Introduction Sentinel lymph node biopsy (SLNB) is the gold standard for determining axillary nodal status. There is growing interest in using preoperative axillary ultrasound (AUS) as a non-invasive means of assessing the axilla. However, AUS has limited sensitivity and is subject to operator dependency. This study aimed to quantify axillary nodal burden in preoperative AUS. Method This retrospective study used an institutional database of all primary invasive breast carcinomas from 2006–2019. Those with pathologically proven axillary metastatic disease were included. Patients were considered in two groups, low nodal burden/LNB(1-2LN) and high nodal burden/HNB(≥3LN) based on total positive lymph node count(SLNB+ALND). Preoperative AUS reports were assessed to determine those suspicious for axillary metastasis. Result Of the 347 patients (n=349 axillae), 77.9% had LNB and 22.1% had HNB. In patients with LNB, 228(83.8%) had a normal AUS versus 44(16.2%) suspicious AUS. In those with HNB 60(77.9%) had normal AUS findings versus 17(22.1%) suspicious findings. On multivariate analysis Nottingham Grade-3 was associated with suspicious AUS findings (p=0.02). However, receptor status, SLN macro-metastasis and extra-nodal extension were not associated with abnormal AUS. Conclusion As the surgical approach to the axilla becomes increasingly conservative, detection of axillary involvement by non-invasive means is an area of increasing research. In this cohort, AUS did not reliably identify patients with axillary metastasis. These results highlight the challenges in accurately assessing the axilla using preoperative AUS, which may result in axillary undertreatment if used as an alternative to surgical staging. Take-home message As the surgical approach to the axilla becomes increasingly conservative, detection of axillary involvement by non-invasive means is an area of increasing research. This study highlights the challenges in accurately assessing the axilla using preoperative AUS, which may result in axillary undertreatment if used as an alternative to surgical staging.

Glucose Transporter 4 and Interleukin 8 expression in hormone receptor negative/HER2 overexpressing breast carcinoma subtype: Correlation with biological behavior of the tumor cells and prognostic parameters

Background: Breast carcinoma (BC) is the most commonly occurring cancer. Nearly 15–20% of breast carcinomas are HER2 overexpressing subtype. Patients with this subtype have a poor prognosis with fewer therapeutic options. Increased glucose transporters expression is crucial for enhancing glucose uptake observed in several tumors. Overexpression of IL-8 has been found in neoplastic epithelial cells of various malignancies which are involved in tumor cell proliferation and invasion. Aim: Study the immunohistochemical (IHC) expression of GLUT4 and IL-8 in ER-, PR-/HER2+ BC subtype to correlate their expression with the biological behavior of tumor cells and prognosis. Materials & Methods: Fifty-eight ER-,PR-/HER2+ primary invasive ductal breast carcinoma NOS paraffin blocks were collected from archive of Patholohy department, Faculty of medicine, Tanta University and private labs. Cut sections were stained with primary anti-GLUT4, anti-IL-8, and anti-Ki67 antibodies. Results: High GLUT4 expression was significantly associated with advanced TN staging and lympho-vascular invasion (LVI). A significant relationship was found between high IL-8 immuno-expression and TN staging and LVI. A significant positive correlation was observed between high GLUT4 and IL-8 expressions on one hand and positive Ki67 immunoreactivity in tumor cells on the other hand. No statistically significant association was detected between either GLUT4 or IL-8 expression and patient age, tumor grade, or M stage. Conclusion: GLUT4 and IL-8 expression in ER-,PR-/HER2+ BC were associated with poor prognostic parameters, thus downregulation of GLUT4 and IL-8 might provide alternative therapeutic choices for patients with this subtype.

GATA3 as an Adjunct Prognostic Factor in Breast Cancer Patients with Less Aggressive Disease: A Study with a Review of the Literature.

Background: GATA binding protein 3 (GATA3) expression is positively correlated with estrogen receptor (ER) expression, but its prognostic value as an independent factor remains unclear. Thus, we undertook the current study to evaluate the expression of GATA3 and its prognostic value in a large series of breast carcinomas (BCs) with long-term follow-up. Methods: A total of 702 consecutive primary invasive BCs resected between 1989 and 1993 in our institution were arranged in tissue microarrays, immunostained for ER, progesterone receptor (PR), ki-67, HER2, p53, and GATA3, and scored. Clinico-pathological data were retrospectively collected. Results: GATA3 was evaluable in 608 (87%) of the 702 cases; it was positive in 413 (68%) cases and negative in 195 (32%) cases. GATA3 positivity was significantly associated with lower grade (p < 0.0001), size (p = 0.0463), stage (p = 0.0049), ER+ (p < 0.0001), PR+ (p < 0.0001), HER2− (p = 0.0175), and p53 wild-type pattern (p < 0.0001). The median follow-up was 183 months, GATA3 positivity was associated with better overall survival (HR 0.70, p = 0.001), and its prognostic value was retained in a multivariate analysis. The association with better overall survival was stronger in patients with grade 1–2, pT1–2, pN0, stage I–II, ER+, PR+, ki-67 < 20%, HER2−, a wild-type p53 immunohistochemical pattern, and in luminal B BC. Conclusions: Our findings indicate that GATA3 is a positive prognostic marker in BC patients, especially in patients with biologically less aggressive BC. Incorporating GATA3 immunohistochemistry into routine practice could help further stratify BC patients for their risk.

Triple-negative breast lobular carcinoma: a luminal androgen receptor carcinoma with specific ESRRA mutations

Primary triple-negative invasive lobular breast carcinomas (TN-ILCs), which do not express hormone receptors and HER2 at diagnosis, are rare and poorly known. In this study, we analyzed the largest TN-ILC series ever reported in the literature, in comparison to phenotypically similar breast tumor subtypes: triple-negative invasive ductal carcinoma (TN-IDC) and hormone receptor-positive invasive lobular carcinoma (HR + ILC). All primary TN-ILCs registered in our database between 2000 and 2018 (n = 38) were compared to tumors from control groups, matched by stage and Elston/Ellis grade, with regard to clinical, pathologic, and immunohistochemical characteristics. A comparative molecular analysis (whole-exome and RNA sequencing using next-generation technology) was also performed. We found that TN-ILC patients were older than those with HR + ILC (P = 0.002) or TN-IDC (P < 0.001). Morphologically, TN-ILCs had aggressive phenotypes, with more pleomorphism (P = 0.003) and higher nuclear grades than HR + ILCs (P = 0.009). Immunohistochemistry showed that TN-ILCs less frequently expressed basal markers (CK5/6, EGFR and SOX10) than TN-IDCs (P < 0.001), while androgen receptor (AR) positivity was more prevalent (P < 0.001). Survival curves analysis did not show differences between TN-ILC and TN-IDC patients, while overall and distant metastasis-free survival were significantly worse compared to those with HR + ILCs (P = 0.047 and P = 0.039, respectively). At a molecular level, we found that TN-ILCs had particular transcriptomic profiles, characterized by increased AR signaling, and associated with frequent alterations in the PI3K network and ERBB2. Interestingly, whole-exome analysis also identified three specific recurrent ESRRA hotspot mutations in these tumors, which have never been described in breast cancer to date and which were absent in the other two tumor subtypes. Our findings highlight that TN-ILC is a unique aggressive breast cancer associated with elderly age, which belong to the luminal androgen receptor subtype as determined by immunohistochemistry and transcriptomic profiling. Moreover, it harbors specific molecular alterations (PI3K, ERBB2 and ESRRA) which may pave the way for new targeted therapeutic strategies.

Pathohistological and Immunohistochemical Analysis of Primary Invasive Ductal Breast Carcinoma with Signet-Ring Cell Differentiation- Differential Diagnosis, Prognosis and Complex Treatment

Primary signet ring cell carcinoma (PSRCC) of the breast is a rarely diagnosed neoplasm. We present a 76-year-old woman with a tumor formation in the left mammary gland, who has been self-medicating for a year. Pathohistological and immunohistochemical analysis proved rare primary invasive ductal carcinoma with focal (over 90%) signet ring cell differentiation, size 4 cm / 3.5 cm / 2 cm, moderately differentiated (G2). Complex oncological treatment, including radical mastectomy with axillary dissection, 6 courses of systemic adjuvant chemotherapy, radiotherapy of the chest wall and regional lymph nodes with TD 46 Gy and antiestrogenic hormone therapy, was performed. The diagnosis and the differential diagnosis of this rare tumor require precise pathohistological and immunohistochemical analysis. The prognosis and complex treatment depend on the clinical stage, hormonal and HER2 status. In locally advanced PSRCC of the breast with moderately differentiation, the combination of surgery, systemic chemotherapy, postoperative radiotherapy and antiestrogenic hormone therapy achieves long-term local tumor control without distant metastases for nearly two years.

Metastatic adenocarcinoma to the breast from the lung simulates primary breast carcinoma—a clinicopathologic study

BackgroundRare extra-mammary metastases of adenocarcinoma to the breast closely mimic primary invasive breast carcinoma (PBC), and specifically without an aware of clinical history, pose a difficult diagnostic issue.MethodsWith the aim to improve differential diagnosis of lung adenocarcinoma metastasis and primary breast carcinoma in the breast, we retrieved 41 breast metastases from lung adenocarcinoma, seven of which were from the archived pathologic files of Cancer Hospital, Chinese Academy of Medical Science (CHCAMS) between 2001 and 2019, and the other 34 cases were collected from the published literatures. Clinicopathological features were collected and analyzed for differential diagnosis of primary lung malignancy, triple negative breast pathology and breast lesions without ipsilateral axillary lymphadenopathy or with contralateral axillary lymphadenopathy. Supplementary breast (GCDFP-15, or GATA-3) and lung-lineage (TTF-1) immunostaining plus genetic alternation analysis were also recorded and analyzed.ResultsAmong the 41 cases, there were 37 females and four males, with a median age of 63 (range, 40–81) years at diagnosis of the breast lesion. Twenty-four cases (58.5%, 24/41) were detected metachronously to the counterpart of the lung. Strikingly, 13 cases (31.7%, 13/41) were initially misdiagnosed as primary breast cancer, and differential diagnostic factors were compared and analyzed between the correct and misdiagnosed cases, among which a documentation of lung cancer history showed significant difference. Pathologist initially misinterpreted six cases (46.2%, 6/13) as PBC on needle biopsy of breast mass with an unknown lung cancer history. The clinical diagnosis was considered two cases (15.4%, 2/13) to be either a primary breast tumor with lung and pleural metastasis or two synchronous primary tumors. Three cases (23.1%, 3/13) were initially misinterpreted as PBC by breast ultrasonography. TTF-1 immunostaining was found to be critical for a correct diagnosis of metastatic lesion (84.6%, 11/13) from the initially misdiagnosed cases as PBC.ConclusionsMetastatic lung adenocarcinoma to the breast, although rare, should be considered in the differential diagnosis of primary breast carcinoma, especially when the breast lesion exhibits as a “triple-negative invasive carcinoma”. A documented lung cancer history combined with the clinicoradiological assessment and pathological evaluation are essential to make a correct differential diagnosis. TTF-1 immunostaining is crucial in approaching the diagnosis.

Interobserver Variation of PD-L1 SP142 Immunohistochemistry Interpretation in Breast Carcinoma: A Study of 79 Cases Using Whole Slide Imaging.

The Ventana programmed death ligand-1 (PD-L1) SP142 immunohistochemical assay (IHC) is approved by the US Food and Drug Administration as the companion diagnostic assay to identify patients with locally advanced or metastatic triple-negative breast cancer for immunotherapy with atezolizumab, a monoclonal antibody targeting PD-L1. To determine interobserver variability in PD-L1 SP142 IHC interpretation in invasive breast carcinoma. The pathology database was interrogated for all patients diagnosed with primary invasive, locally recurrent, or metastatic breast carcinoma on which PD-L1 SP142 IHC was performed from November 2018 to June 2019 at our institution. A subset of cases was selected using a computerized random-number generator. PD-L1 IHC was evaluated in stromal tumor-infiltrating immune cells using the IMpassion130 trial criteria, with positive cases defined as immunoreactivity in immune cells in 1% or more of the tumor area. IHC was interpreted on whole slide images by staff pathologists with breast pathology expertise. Interobserver variability was calculated using unweighted κ. A total of 79 cases were assessed by 8 pathologists. Interobserver agreement was substantial (κ = 0.727). There was complete agreement among all 8 pathologists in 62% (49 of 79) of cases, 7 pathologists or more in 84% (66 of 79) of cases, and 6 pathologists or more in 92% (73 of 79) of cases. In 4% (3 of 79) of cases, all of which were small biopsies, pathologists' interpretations were evenly split between scores of positive and negative. The findings show substantial agreement in PD-L1 SP142 IHC assessment of breast carcinoma cases among 8 pathologists at a single institution. Further study is warranted to define the basis for discrepant results.

Recurrent diffuse large B-cell lymphomamimicking primary breast cancer

Breast lymphoma can represent 0.8%–2.2% of extranodal lymphomas and 0.1%–0.5% of primary breast neoplasms. Imaging findings are not specific, and its distinction from primary invasive breast carcinoma should be based on clinical data and histopathological analysis. We present the case of a 62-year-old woman who showed an unusual pattern of recurrent diffuse large B-cell lymphoma (DLBCL) mimicking primary breast cancer on imaging studies, including mammography, ultrasound, magnetic resonance imaging (MRI), and positron emission tomography-computed tomography (PET-CT).

Association of estrogen and progesterone receptors with clinicopathological prognostic factors in breast cancer

Background: The detection of the estrogen receptor (ER) and progesterone receptor (PgR) in women with breast cancer (BC) is considered a crucial step for prognostic evaluation and treatment choice in clinical practice. Objectives: This study aimed to evaluate the expression of the hormonal receptors (ER and PgR), their distribution, and their association with clinicopathologic prognostic parameters in a sample of Iraqi women with BC. Materials and Methods: The paraffin-embedded blocks from a total of 80 women diagnosed with primary invasive breast carcinomas with BC were examined by immunohistochemistry to assess the expression of ER and PgR status. Demographic and clinical data were collected from each patient in the preformed questionnaire. The association of steroid receptor expression with clinicopathologic parameters was assessed using binary logistic regression. Results: Among 80 BC women involved in the study, 54 (67.5%) and 39 (48.75%) had high expression of ER and PgR, respectively. High ER expression was significantly associated with older age (odds ratio [OR] = 4.8, 95% confidence interval [CI] = 1.16-19.81, P = 0.03), postmenopausal status (OR = 3.6, 95% CI = 1.25–10.33, P = 0.015), smaller tumor (OR = 0.27, 95% CI = 0.09–0.75, P = 0.01), and with noninvolvement of lymph node (OR = 0.11, 95% CI = 0.02–0.51, P = 0.005). High PgR expression, on the other hand, was significantly associated with older age (OR = 4.44, 95% CI = 1.02–19.39) and postmenopausal status (OR = 3.13, 95% CI = 1.24–7.88, P = 0.016). Concomitant overexpression of both receptors was significantly associated with postmenopausal status and noninvolvement of lymph nodes. Conclusion: High expression of ER and PgR seems to be a good prognostic indicator because it was associated with less aggressive tumors.

Abstract PO-087: Tumor and immune cell profiling in breast cancer using highly multiplexed imaging mass cytometry single-cell technology demonstrates tumor heterogeneity and immune phenotypic abnormality in Ethiopian women

Abstract Background: Tumor heterogeneity represents a complex challenge to cancer treatment, disease recurrence, and patient survival. Imaging mass cytometry (IMC) is an emerging proteomic tool for cancer profiling in tumor tissue samples. IMC enables digital image analysis by multiplexed immunostaining of cells and proteins within tissue and preserves spatial relations within tumor environment. We have applied IMC based approach to study the heterogeneity of invasive breast carcinoma protein expression pattern in formalin fixed paraffin embedded tissues. Methods: A total of 10 region of interest (ROI) derived from 5 patients with primary invasive breast carcinoma representing three molecular subclasses (HR+/HER2-,HER2+/HR- and TNBC) were stained with a 30-marker IMC metal labeled antibody panel (α-SMA, EGFR, p53, CD33, CD16, CD163, CD11b, PDL1, CD31, CD45, D44,Vimentin, FoxP3, CD4, ECadherin, CD68, CD20, CD8a, Cytokeratin7, PD1, GranzymeB, Ki67, ColTypeI, CD3, CD45RO, HLADR, DARC &amp; CD11c). Tissue imaging was done by quantifying the abundance of bound antibody with a Hyperion IMC. MCD Viewer was used for visualization purpose and to export raw 16-bit tiff images for segmentation on CellProfiler. Segmentation masks were combined with the individual tiff files to extract single-cell information from each individual image. HistoCAT was applied to perform unbiased clustering of cell populations using the PhenoGraph algorithm and clustered cell populations was overlaid on t-SNE plot. The relative marker expression was used to generate heat-maps and each cluster was manually assigned a phenotype based on its expression profile. Results: The t-SNE generated from each ROI revealed different distinct cell populations and we report the presence of diverse tumor and immune cell populations in our samples. The (min, max) number of PhenoGraph clustered tumor cell populations in HR+/HER2-, HER2+ and TNBC Cases were (5,8) (7,9) and (5,7) respectively. Similarly, the (min, max) number of PhenoGraph clustered immune cell populations in HR+/HER2-, HER2+ and TNBC Cases were (5,8) (7,9) and (5,7) respectively. We also document the presence of inter and intra-tumor heterogeniety in expression of PD1 and PDL1 in all the tumor subtypes studied. Additionally, we report a phenotypic abnormality in the immune cell populations identified with dual or triple markers expression of the canonical CD antigens of T-Cells, B-Cells and macrophages. Conclusion: The current study demonstrates high-dimensional visualization with the simultaneous analysis of epithelial, immune, and stromal components using IMC can be used to explore cell populations in tumor tissue to quantify tumor heterogeneity or identification of novel clustering patterns that has potential for translational research and clinical practice. Significance: This study presents the potential of Imaging Mass Cytometry and single cell analysis algorithms in multiplex high throughput tumor tissue studies. Citation Format: Maheteme Bekele, Aisha Jibril, Daniel Seifu, Markos Abebe, Abebe Bekele, Wondemagegnhu Tigneh, Yonas Bokretsion, Christina Karlsson, Mats G. Karlsson, Rachel Martini, Olivier Elemento, Clayton Yates, Paula Ginter, Lisa Newman, Melissa Davis, Endale Hadgu Gebregzabher. Tumor and immune cell profiling in breast cancer using highly multiplexed imaging mass cytometry single-cell technology demonstrates tumor heterogeneity and immune phenotypic abnormality in Ethiopian women [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-087.