A bicellular fluorescent ductal carcinoma in situ (DCIS)-like tumoroid to study the progression of carcinoma: practical approaches and optimization.

Recently, many types of 3D culture systems have been developed to preserve the physicochemical environment and biological characteristics of the original tumors better than the conventional 2D monolayer culture system. There are various types of models belonging to this culture, such as the culture based on non-adherent and/or scaffold-free matrices to form the tumors. Agarose mold has been widely used to facilitate tissue spheroid assembly, as it is essentially non-biodegradable, bio-inert, biocompatible, low-cost, and low-attachment material that can promote cell spheroidization. As no studies have been carried out on the development of a fluorescent bicellular tumoroid mimicking ductal carcinoma in situ (DCIS) using human cell lines, our objective was to detail the practical approaches developed to generate this model, consisting of a continuous layer of myoepithelial cells (MECs) around a previously formed in situ breast tumor. The practical approaches developed to generate a bi-cellular tumoroid mimicking ductal carcinoma in situ (DCIS), consisting of a continuous layer of myoepithelial cells (MECs) around a previously formed in situ breast tumoroid. Firstly, the optimal steps and conditions of spheroids generation using a non-adherent agarose gel were described, in particular, the appropriate medium, seeding density of each cell type and incubation period. Next, a lentiviral transduction approach to achieve stable fluorescent protein expression (integrative system) was used to characterize the different cell lines and to track tumoroid generation through immunofluorescence, the organization of the two cell types was validated, specific merits and drawbacks were compared to lentiviral transduction. Two lentiviral vectors expressing either EGFP (Enhanced Green Fluorescent Protein) or m-Cherry (Red Fluorescent Protein) were used. Various rates of a multiplicity of infection (MOI) and multiple types of antibodies (anti-p63, anti-CK8, anti-Maspin, anti-Calponin) for immunofluorescence analysis were tested to determine the optimal conditions for each cell line. At MOI 40 (GFP) and MOI 5 (m-Cherry), the signals were almost homogeneously distributed in the cells which could then be used to generate the DCIS-like tumoroids. Images of the tumoroids in agarose molds were captured with a confocal microscope Micro Zeiss Cell Observer Spinning Disk or with IncuCyte® to follow the progress of the generation. Measurement of protumoral cytokines such as IL-6, IL8 and leptin confirmed their secretion in the supernatants, indicating that the properties of our cells were not altered. Finally the advantages and disadvantages of each fluorescent approach were discussed. This model could also be used for other solid malignancies to study the complex relationship between different cells such as tumor and myoepithelial cells in various microenvironments (inflammatory, adipose and tumor, obesity, etc.). Although, this new model is well established to monitor drug screening applications and perform pharmacokinetic and pharmacodynamic analyses.

Yassir Alaa Muhammed Hassan Shubbar CORRELATION BETWEEN DIFFERENT CLINICOPATHOLOGICAL PARAMETERS AND MOLECULAR SUBTYPES OF FEMALE BREAST CARCINOMA IN SOUTH REGION OF IRAQ.

OBJECTIVE: The aim: To correlate variable clincopathological parameters with molecular subtypes of the breast carcinoma, which affect the prognosis and management of breast malignancy. PATIENTS AND METHODS: Materials and methods: In this study a total of 511 female patients with breast carcinoma were included, ranging from 32 to 85 years of age, with 35.8% premenopausal and 64.1% being post-menopausal. The sample slides were stained immunohistochemically for estrogen receptors (ER), progesterone receptors (PR), ki67 and HER2, the tumors were graded histologically using the Nottingham criteria system. RESULTS: Results: Most tumors (72.8%) ranged between 2 and 5 cm in size; the most common histological type of breast carcinoma (49.7%) was invasive ductal carcinoma of no special type, with grade 2 presented in 51.8% cases; most frequent stage at time of presentation was stage 3A, found in 39.9%; the most frequent molecular subtype was ER and/or PR+, Her2- with low proliferation rate ki67<14% subtype in 48.5%, and those group were more likely (statistically significant) to be older, have stage 3 breast cancer, present with tumor size between 2 and 5 cm and tend to be well differentiated histological grade (grade1), mostly with lymph node positive, and most likely have tumor type of invasive ductal carcinoma of no special type. CONCLUSION: Conclusions: the most common histological type of breast carcinoma in Iraq south was invasive ductal carcinoma of no special type and most cases showed (ER and/or PR+, HER 2-, low ki67) as the most common molecular subtype.

Prognostic Value of Intracellular Transcription of Factors HIF-1α and p53 and Their Relation to Estradiol and TNM Parameters of Breast Cancer Tissues in Women with Invasive Ductal Carcinoma in Thi-Qar Province, Iraq.

Breast cancer is the most common malignancy affecting women's health, with an increasing incidence worldwide. This study aimed to measure the intracellular concentration of the hypoxia-inducible factor 1 α (HIF-1α), tumor suppression protein p53, and estradiol (E2) in tumor tissues of adult females with breast cancer and their relation to tumor grade, tumor size, and lymph node metastases (LNM). The study was conducted on 65 adult female participants with breast mass admitted to the operating theater in Al-Hussein Teaching Hospital and Al-Habboby Teaching Hospital in Nasiriyah, Iraq, from January to November 2021. Fresh breast tumor tissues were collated and homogenized for intracellular biochemical analysis using the enzyme-linked immunosorbent assay method. In total, 44 (58%) out of 65 patients, in the age range of 18-42 years and the mean±SD age of 32.55±6.40 years, had fibroadenomas, and other 21 (42%) cases, in the age range of 32-80 years and the mean±SD age of 56±14.4 years had invasive ductal carcinoma (IDC) breast cancer. Intracellular levels of HIF-1α, p53, and E2 were elevated significantly (P<0.001) in IDC cases compared to the benign group. The most malignant tumors of IDC cases were in grade III and sizes T2 and T3. The tissue concentrations of HIF-1α, P53, and E2 were significantly elevated in patients with tumor stage T3 compared to T2 and T1. A significant elevation was found in the levels of HIF-1α, p53, and E2 in the positive LNM subgroup compared to the negative LNM group. Based on the obtained results, the prognostic value of the intracellular HIF-1α is considered to be a useful prognostic factor in Iraqi women with ICD and the combination of a HIF-1α protein with the nonfunctional p53 and E2 tends to indicate the proliferation, invasiveness, and metastases of the breast tumors.

14-3-3ζ promoted invasion and lymph node metastasis of breast invasive ductal carcinoma with HER2 overexpression.

BACKGROUND: HER2 was a recognized oncogene that promoted the development and metastasis of breast cancer, but its positive expression rate in invasive ductal carcinoma (IDC) was much lower than that in ductal carcinoma in situ (DCIS). The correlation between the occurrence and development of breast cancer and the amplification and overexpression of HER2 gene alone was still controversial. 14-3-3ζ had a strong protein binding ability and a variety of functions, mainly through the interaction with other proteins to exert its unique biological activities. However, influence and interaction relationship of the two proteins on the development of IDC was not clear. Furthermore, the mutual effect mechanism of synergy effect on lymph node metastasis of IDC was not known well too. METHODS: Immunohistochemistry experiment was performed to detect expression status of 14-3-3ζ, HER2, TGF-ß, p53 and Gli2 in paraffin-embedded samples respectively, including 30 cases of normal breast tissue, 30 cases of usual ductal hyperplasia (UDH), 30 cases of atypical ductal hyperplasia (ADH), 30 cases of DCIS and 120 cases of IDC. RESULTS: The positive expression rates of 14-3-3ζ/HER2 in Normal group, UDH group, ADH group, DCIS group and IDC group were 30%/0.00%, 26.7%/0.00%, 53.3%/33.3%, 46.7%/53.3% and 50%/24.2%, respectively. Compared with Normal group or UDH group, the expression of 14-3-3ζ was significantly increased in ADH, DCIS and IDC groups. 14-3-3ζ was overexpressed in only 4 of the 16 DCIS cases with HER2 overexpression (25.0%, 4/16), but it was overexpressed in 7 of the 9 IDC cases with DCIS (77.8%, 7/9). Among HER2 overexpression cases, 14-3-3ζ overexpression was significantly different between DCIS group and IDC with DCIS group (P = 0.017). In 18 IDC cases with lymph node metastasis and HER2 overexpression, 14-3-3ζ was overexpressed in 15 cases (83.3%, 15/18), while in the 11 IDC cases without lymph node metastasis, 14-3-3ζ and HER2 were overexpressed in only 5 cases (45.5%, 5/11). Co-overexpression of 14-3-3ζ and HER2 was positively correlated with occurrence of lymph node metastasis (P = 0.048). TGF-ß was overexpressed in both precancerous lesion group and IDC group compared with normal group. Compared with the IDC group without lymph node metastasis, TGF-ß expression was significantly increased in the IDC group with lymph node metastasis (P = 0.015). In IDC cases with 14-3-3ζ and HER2 co-overexpression, the expression of p53 in IDC with lymph node metastasis was significantly decreased (P = 0.010), while the expression of Gli2 was significantly increased compared with IDC cases without lymph node metastasis (P = 0.038). The co-overexpression of 14-3-3ζ and HER2 was positively correlated with ER negative expression (P < 0.001) and PR negative expression (P = 0.038), respectively. CONCLUSION: 14-3-3ζ synergistic with HER2 could promote the occurrence and development of breast IDC and induce the lymph node metastasis of IDC, suggesting that combined overexpression of 14-3-3ζ and HER2 would lead to higher invasion and metastasis risk of breast cancer. It was speculated that the combined detection of 14-3-3ζ and HER2 would be one of the key factors affecting the clinical treatment decision and prognosis.

Neuroglobin: A New Possible Marker of Estrogen-Responsive Breast Cancer.

The expression of the α-subtype of Estrogen Receptor (ERα) characterizes most breast cancers (more than 75%), for which endocrine therapy is the mainstay for their treatment. However, a high percentage of ERα+ breast cancers are de novo or acquired resistance to endocrine therapy, and the definition of new targets for improving therapeutic interventions and the prediction of treatment response is demanding. Our previous data identified the ERα/AKT/neuroglobin (NGB) pathway as a common pro-survival process activated in different ERα breast cancer cell lines. However, no in vivo association between the globin and the malignity of breast cancer has yet been done. Here, we evaluated the levels and localization of NGB in ERα+ breast ductal carcinoma tissue of different grades derived from pre-and post-menopausal patients. The results indicate a strong association between NGB accumulation, ERα, AKT activation, and the G3 grade, while no association with the menopausal state has been evidenced. Analyses of the data set (e.g., GOBO) strengthen the idea that NGB accumulation could be linked to tumor cell aggressiveness (high grade) and resistance to treatment. These data support the view that NGB accumulation, mainly related to ER expression and tumor grade, represents a compensatory process, which allows cancer cells to survive in an unfavorable environment.

Can AI-assisted microscope facilitate breast HER2 interpretation? A multi-institutional ring study.

The level of human epidermal growth factor receptor-2 (HER2) protein and gene expression in breast cancer is an essential factor in judging the prognosis of breast cancer patients. Several investigations have shown high intraobserver and interobserver variability in the evaluation of HER2 staining by visual examination. In this study, we aim to propose an artificial intelligence (AI)-assisted microscope to improve the HER2 assessment accuracy and reliability. Our AI-assisted microscope was equipped with a conventional microscope with a cell-level classification-based HER2 scoring algorithm and an augmented reality module to enable pathologists to obtain AI results in real time. We organized a three-round ring study of 50 infiltrating duct carcinoma not otherwise specified (NOS) cases without neoadjuvant treatment, and recruited 33 pathologists from 6 hospitals. In the first ring study (RS1), the pathologists read 50 HER2 whole-slide images (WSIs) through an online system. After a 2-week washout period, they read the HER2 slides using a conventional microscope in RS2. After another 2-week washout period, the pathologists used our AI microscope for assisted interpretation in RS3. The consistency and accuracy of HER2 assessment by the AI-assisted microscope were significantly improved (p < 0.001) over those obtained using a conventional microscope and online WSI. Specifically, our AI-assisted microscope improved the precision of immunohistochemistry (IHC) 3 + and 2 + scoring while ensuring the recall of fluorescent in situ hybridization (FISH)-positive results in IHC 2 + . Also, the average acceptance rate of AI for all pathologists was 0.90, demonstrating that the pathologists agreed with most AI scoring results.

Optical tissue measurements of invasive carcinoma and ductal carcinoma in situ for surgical guidance.

BACKGROUND: Although the incidence of positive resection margins in breast-conserving surgery has decreased, both incomplete resection and unnecessary large resections still occur. This is especially the case in the surgical treatment of ductal carcinoma in situ (DCIS). Diffuse reflectance spectroscopy (DRS), an optical technology based on light tissue interactions, can potentially characterize tissue during surgery thereby guiding the surgeon intraoperatively. DRS has shown to be able to discriminate pure healthy breast tissue from pure invasive carcinoma (IC) but limited research has been done on (1) the actual optical characteristics of DCIS and (2) the ability of DRS to characterize measurements that are a mixture of tissue types. METHODS: In this study, DRS spectra were acquired from 107 breast specimens from 107 patients with proven IC and/or DCIS (1488 measurement locations). With a generalized estimating equation model, the differences between the DRS spectra of locations with DCIS and IC and only healthy tissue were compared to see if there were significant differences between these spectra. Subsequently, different classification models were developed to be able to predict if the DRS spectrum of a measurement location represented a measurement location with "healthy" or "malignant" tissue. In the development and testing of the models, different definitions for "healthy" and "malignant" were used. This allowed varying the level of homogeneity in the train and test data. RESULTS: It was found that the optical characteristics of IC and DCIS were similar. Regarding the classification of tissue with a mixture of tissue types, it was found that using mixed measurement locations in the development of the classification models did not tremendously improve the accuracy of the classification of other measurement locations with a mixture of tissue types. The evaluated classification models were able to classify measurement locations with > 5% malignant cells with a Matthews correlation coefficient of 0.41 or 0.40. Some models showed better sensitivity whereas others had better specificity. CONCLUSION: The results suggest that DRS has the potential to detect malignant tissue, including DCIS, in healthy breast tissue and could thus be helpful for surgical guidance.

Histopathologic features of breast cancer in Li-Fraumeni syndrome.

Breast cancer is the most common malignancy in female patients with Li-Fraumeni syndrome (LFS), a rare autosomal dominant hereditary syndrome characterized by germline TP53 mutations. Recent studies have shown that the majority of these tumors are estrogen receptor (ER) positive with frequent HER2 co-expression. However, the morphologic features of these tumors have not been as well studied as other germline-associated breast cancers. We evaluated the pathologic features of 27 invasive and in situ carcinomas from patients with known germline TP53 mutations collected through the Li-Fraumeni Consortium. Overall, 60% of cases were HER2 positive and 44% showed ER co-expression. Most DCIS was high nuclear grade with central necrosis and associated periductal fibrosis and lymphocytic response. Invasive carcinomas were mostly of ductal type (NOS), modified Scarff-Bloom-Richardson (mSBR) high grade, with marked nuclear atypia and high mitotic rate. Prominent tumor infiltrating lymphocytes, syncytial growth pattern, or pushing borders were not seen in these tumors. High p53 IHC expression was seen in tumors from individuals with germline TP53 missense mutations whereas little or no protein expression (<1% nuclear expression, null pattern) was seen in tumors from carriers of non-missense mutations. In this study, we report in detail the morphologic features of invasive and in situ carcinomas in LFS. We found that these tumors share features with cancers harboring somatic TP53 mutations but are distinct from BRCA-associated breast cancers.

The role of TP53 pathogenic variants in early-onset HER2-positive breast cancer.

Breast cancer is the most frequent event in Li-Fraumeni syndrome associated with germline TP53 variants. Some studies have shown that breast cancers in women with Li-Fraumeni syndrome are commonly HER2-positive, suggesting that HER2 amplification or over-expression in a young woman may be a useful criterion to test for germline variants in the TP53 gene. We assessed the prevalence of germline TP53 variants by Sanger sequencing or next-generation sequencing in 149 women with HER2-positive breast cancer diagnosed until age 40. The pattern of HER2 amplification was evaluated with dual-probe FISH in a subset of breast carcinomas from patients with germline TP53 variants as compared with those of noncarriers. Among 149 women tested, three presented a deleterious TP53 germline variant (2%), with one patient diagnosed at age 31 and the other two with bilateral breast cancer at ages 29/33 and 28/32, respectively. Three of the 36 patients (8.3%) with the first breast cancer diagnosed at age 31 or younger presented a pathogenic TP53 variant. Additionally, all TP53 deleterious variant carriers had a first degree relative diagnosed with different early-onset cancers (frequently not belonging to the Li-Fraumeni syndrome tumor spectrum) diagnosed at age 45 or younger. Higher levels of HER2 amplification were found in breast carcinomas of TP53 pathogenic variant carriers than in those of noncarriers. Deleterious germline TP53 variants account for a small proportion of early-onset HER2-positive breast cancers, but these seem to have higher HER2 amplification ratios. All TP53 pathogenic variant carriers found in this study had the first breast carcinoma diagnosed at age 31 or younger and a first-degree relative with early-onset cancer. Further studies are needed to clarify if HER2 status in early-onset breast cancer patients, in combination with other personal and/or familial cancer history, is useful to update the TP53 testing criteria.

Pregnancy after breast cancer: Results from a prospective cohort of young women with breast cancer.

BACKGROUND: Many young women with newly diagnosed breast cancer are interested in future pregnancies. Prospective data regarding fertility interest and reproductive patterns after diagnosis are needed to counsel patients. METHODS: The Young Women's Breast Cancer Study is a multicenter, prospective cohort of women who were diagnosed with breast cancer at age ≤40 years between 2006 and 2016. Women complete surveys at baseline, every 6 months for 3 years, then annually. Here, the authors describe fertility interest and pregnancies within 5 years of diagnosis for women with stage 0 through III breast cancer. RESULTS: Of 1026 eligible participants, 368 (36%) reported interest in future biologic children at least once within 5 years after diagnosis, including 16% at 5 years after diagnosis. Among 130 women who attempted to become pregnant, 90 (69.2%) conceived; and, among 896 women who did not attempt to conceive, 18 (2.0%) became pregnant, with a total of 152 pregnancies resulting in 91 live births. Factors associated with pregnancy included younger versus older age at diagnosis (aged ≤30 vs 36-40 years: odds ratio [OR], 6.63; 95% CI, 3.18-13.83; P < .0001; aged 31-35 vs 36-40 years: OR, 5.86; 95% CI, 3.37-10.17; P < .0001) and being nulliparous versus parous (OR, 2.66; 95% CI, 1.56-4.53; P = .001). The receipt of endocrine therapy versus no endocrine therapy (OR, 0.35; 95% CI, 0.20-0.59; P = .001) was inversely associated with pregnancy. CONCLUSIONS: Many women remain interested in future fertility in the 4 years after a breast cancer diagnosis, indicating that longitudinal fertility discussions are needed. Although a minority of those interested in having children attempted to become pregnant in the first 5 years, most who attempted to conceive did so and had live births.

Intratumoral heterogeneity of second-harmonic generation scattering from tumor collagen and its effects on metastatic risk prediction.

BACKGROUND: Metastases are the leading cause of breast cancer-related deaths. The tumor microenvironment impacts cancer progression and metastatic ability. Fibrillar collagen, a major extracellular matrix component, can be studied using the light scattering phenomenon known as second-harmonic generation (SHG). The ratio of forward- to backward-scattered SHG photons (F/B) is sensitive to collagen fiber internal structure and has been shown to be an independent prognostic indicator of metastasis-free survival time (MFS). Here we assess the effects of heterogeneity in the tumor matrix on the possible use of F/B as a prognostic tool. METHODS: SHG imaging was performed on sectioned primary tumor excisions from 95 untreated, estrogen receptor-positive, lymph node negative invasive ductal carcinoma patients. We identified two distinct regions whose collagen displayed different average F/B values, indicative of spatial heterogeneity: the cellular tumor bulk and surrounding tumor-stroma interface. To evaluate the impact of heterogeneity on F/B's prognostic ability, we performed SHG imaging in the tumor bulk and tumor-stroma interface, calculated a 21-gene recurrence score (surrogate for OncotypeDX®, or S-ODX) for each patient and evaluated their combined prognostic ability. RESULTS: We found that F/B measured in tumor-stroma interface, but not tumor bulk, is prognostic of MFS using three methods to select pixels for analysis: an intensity threshold selected by a blinded observer, a histogram-based thresholding method, and an adaptive thresholding method. Using both regression trees and Random Survival Forests for MFS outcome, we obtained data-driven prediction rules that show F/B from tumor-stroma interface, but not tumor bulk, and S-ODX both contribute to predicting MFS in this patient cohort. We also separated patients into low-intermediate (S-ODX < 26) and high risk (S-ODX ≥26) groups. In the low-intermediate risk group, comprised of patients not typically recommended for adjuvant chemotherapy, we find that F/B from the tumor-stroma interface is prognostic of MFS and can identify a patient cohort with poor outcomes. CONCLUSIONS: These data demonstrate that intratumoral heterogeneity in F/B values can play an important role in its possible use as a prognostic marker, and that F/B from tumor-stroma interface of primary tumor excisions may provide useful information to stratify patients by metastatic risk.

miR­135b­5p enhances the sensitivity of HER­2 positive breast cancer to trastuzumab via binding to cyclin D2.

Trastuzumab has led to a marked improvement in the outcomes of patients with human epidermal growth factor receptor 2 (HER­2)­positive breast cancer. However, the effects of trastuzumab on HER­2­positive breast cancer are limited by the emergence of its cardiotoxicside effects. MicroRNA (miR)­135b­5p has been shown to inhibit tumor metastasis in breast cancer. The present study aimed to explore the effects of miR­135b­5p overexpression on the efficacy of trastuzumab in HER­2­positive breast cancer. Reverse transcription­quantitative PCR was performed to detect the levels of miR­135b­5p. Cell viability was evaluated with a Cell Counting Kit­8 assay. Annexin V/propidium iodide staining was employed to detect the number of apoptotic cells. Flow cytometry assay was performed to investigate the cell cycle. Western blotting was used to detect the expression levels of Bax, cleaved caspase­3, Bcl­2, cyclin D2, p27Kip1 and cyclin E1. Cell migration and invasion were detected by Transwell assay. Luciferase assays were conducted to identify the target gene of miR­135b­5p. In addition, an in vivo tumor xenograft model was established. miR­135b­5p agomir significantly enhanced the anti­proliferative effect of trastuzumab on HER­2­positive breast cancer cells via the induction of apoptosis, whereas the anti­metastatic effect of trastuzumab was enhanced by miR­135b­5p agomir treatment. Subsequently, luciferase assays indicated that cyclin D2 was the direct target of miR­135b­5p, whereas overexpression of the latter arrested cell cycleduring the G0/G1 phase. Moreover, miR­135b­5p agomir notably increased the antitumor effect of trastuzumab in vivo. The data demonstrated that miR­135b­5p sensitized HER­2­positive breast cancer cells to trastuzumab in vitro and in vivo by directly binding to cyclin D2. These results suggested that the combination of miR­135b­5p with trastuzumab may be a therapeutic strategy for patients with HER­2­positive breast cancer.

Procedimiento combinado de marcaje del ganglio axilar positivo biopsiado con semilla de yodo-125 y biopsia del ganglio centinela en pacientes con cáncer de mama tratadas con quimioterapia neoadyuvante / Combined procedure of marking axillary positive node with iodine-125 seed and sentinel lymph node biopsy in breast cancer patients treated with neoadjuvant chemotherapy

OBJETIVO: Presentar nuestra experiencia inicial en el procedimiento combinado de detección intraoperatoria del ganglio axilar positivo biopsiado marcado con semilla de 125I (GM) y biopsia del ganglio centinela (GC) después de quimioterapia neoadyuvante, en pacientes con cáncer de mama. MATERIAL Y MÉTODOS: Estudio prospectivo, enero de 2017 - marzo de 2019, 16 pacientes con cáncer de mama T1-3N1. Estadio TNM: II-A: 3, II-B: 10, III-A: 3. Tipo histológico ductal infiltrante: 14. Subtipos moleculares: luminal-A: 3, luminal-B: 9, HER2: 3, triple negativo: 1. El GM se marcó 227+/-36 días antes de iniciar la quimioterapia neoadyuvante (n: 10), o 1-6 días antes de la cirugía, sobre el ganglio previamente identificado con un marcador ecovisible tipo hidrogel (n: 3) o tridimensional-3D (n: 3). En 10 pacientes se realizó linfadenectomía axilar. RESULTADOS: GM y GC se identificaron en la cirugía en el 93,7% (15/16) de los casos, en 33,3% (5/15) GM no se encontraba entre los GC, y solo en una enferma (1/5) existió discrepancia entre el resultado de GM y GC (macrometástasis y negativo 0/2). Número medio ganglios GC: 2,2+/-0,9 (rango 1-3) y linfadenectomía axilar: 13,5+/-5,2 (rango 7-23). En todos los casos, el análisis anatomopatológico del GM, con semilla de 125I y/o marcador, predijo correctamente el estatus axilar posneoadyuvancia. En todas las pacientes se recuperó la semilla radiactiva de 125I. CONCLUSIONES: La colocación de semillas de 125I es una técnica factible para la localización intraoperatoria del ganglio positivo biopsiado en combinación con la biopsia del ganglio centinela. El resultado anatomopatológico del GM permite determinar el estatus axilar posneoadyuvancia

OBJECTIVE: To present our initial experience in the combined procedure of intraoperative detection of axillary positive node marked with 125I seed (ML) and sentinel node biopsy (SLN) after neoadjuvant chemotherapy (NACT), in breast cancer patients. MATERIAL AND METHODS: Prospective study, January 2017 - March 2019, 16 breast cancer patients T1-3N1. TNM stage: IIA: 3, IIB: 10, IIIA: 3. Histological type ductal invasive: 14. Molecular subtype: luminal A: 3, luminal B: 9, HER2: 3, basal like: 1. The ML was marked 227+/-36 days before neoadjuvant chemotherapy (n: 10), or 1-6 days before surgery, on previously identified node by ultrasound visibility marker, hydrogel (n: 3) or three dimensional-3D (n: 3). Axillary lymphadenectomy was undertaken in 10 patients. RESULTS: ML and SLN were identified in the surgery in 93.7% (15/16) of the cases, in 33.3% (5/15) ML was not among SLN, and in only one patient (1/5) was there a discrepancy between the result of ML and SLN (macrometastases vs. negative 0/2). Median number of lymph nodes SLN: 2.2+/-0.9 (range 1-3) and AD: 13.5+/-5.2 (range 7-23). In all cases, histopathological analysis of ML, 125I seed and/or marker within, correctly predicted axillary status after neoadjuvant chemotherapy. In all patients the 125I radioactive seed was recovered. CONCLUSIONS: Placing of 125I seeds is a feasible technique for intraoperative location of axillary positive node combined with SLN. The histopathological result of ML allows the axillary status to be determined after neoadjuvant chemotherapy

Nodal positivity decreases with age in women with early-stage, hormone receptor-positive breast cancer.

BACKGROUND: Despite data demonstrating the safety of omitting axillary surgery in older women with early-stage breast cancer, the incidence of axillary surgery remains high. It was hypothesized that the prevalence of nodal positivity would decrease with advancing age. METHODS: The National Cancer Data Base was used to construct a cohort of adult women with early-stage, clinically node-negative, estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative breast cancer treated between 2013 and 2015. Multivariable logistic regression was used to assess the relationship between age and nodal positivity, and this was stratified by the axillary surgery category. Modified Poisson regression was used to estimate the proportion of women receiving adjuvant therapy according to age and nodal status. RESULTS: The incidence of axillary surgery among women aged 70 and older (n = 51,917) remained high nationwide (86%). There was a significant decrease in nodal positivity with advancing age in women with early-stage, ER+, clinically node-negative breast cancer from the youngest cohort up to patients aged 70 to 89 years, and this was independent of histologic subtype (ductal vs lobular), race, comorbidities, and socioeconomic factors. Overall, less than 10% of women aged 70 or older who underwent surgery had node-positive disease, regardless of axillary surgery type, and almost 95% of node-positive patients aged 70 or older were at pathological stage N1mi or N1. CONCLUSIONS: Axillary surgery may be safely omitted for many older women with ER+, clinically node-negative, early-stage breast cancer. Nodal positivity declines with advancing age, and this suggests varied biology in older patients versus younger patients.

Using the Chemical Noise Background in MALDI Mass Spectrometry Imaging for Mass Alignment and Calibration.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is an established tool for the investigation of formalin fixed paraffin embedded (FFPE) tissue samples and shows a high potential for applications in clinical research and histopathological diagnosis. The applicability and accuracy of this method, however, heavily depends on the quality of the acquired data, and in particular mass misalignment in axial time-of-flight (TOF) MSI continues to be a serious issue. We present a mass alignment and recalibration method that is specifically designed to operate on MALDI peptide imaging data. The proposed method exploits statistical properties of the characteristic chemical noise background observed in peptide imaging experiments. By comparing these properties to a theoretical peptide mass model, the effective mass shift of each spectrum is estimated and corrected. The method was evaluated on a cohort of 31 FFPE tissue samples, pursuing a statistical validation approach to estimate both the reduction of relative misalignment, as well as the increase in absolute mass accuracy. Our results suggest that a relative mass precision of approximately 5 ppm and an absolute accuracy of approximately 20 ppm are achievable using our method.

Combined procedure of marking axillary positive node with iodine-125 seed and sentinel lymph node biopsy in breast cancer patients treated with neoadjuvant chemotherapy. / Procedimiento combinado de marcaje del ganglio axilar positivo biopsiado con semilla de yodo-125 y biopsia del ganglio centinela en pacientes con cáncer de mama tratadas con quimioterapia neoadyuvante.

OBJECTIVE: To present our initial experience in the combined procedure of intraoperative detection of axillary positive node marked with 125I seed (ML) and sentinel node biopsy (SLN) after neoadjuvant chemotherapy (NACT), in breast cancer patients. MATERIAL AND METHODS: Prospective study, January 2017 - March 2019, 16 breast cancer patients T1-3N1. TNM stage: IIA: 3, IIB: 10, IIIA: 3. Histological type ductal invasive: 14. Molecular subtype: luminal A: 3, luminal B: 9, HER2: 3, basal like: 1. The ML was marked 227±36 days before neoadjuvant chemotherapy (n: 10), or 1-6 days before surgery, on previously identified node by ultrasound visibility marker, hydrogel (n: 3) or three dimensional-3D (n: 3). Axillary lymphadenectomy was undertaken in 10 patients. RESULTS: ML and SLN were identified in the surgery in 93.7% (15/16) of the cases, in 33.3% (5/15) ML was not among SLN, and in only one patient (1/5) was there a discrepancy between the result of ML and SLN (macrometastases vs. negative 0/2). Median number of lymph nodes SLN: 2.2±0.9 (range 1-3) and AD: 13.5±5.2 (range 7-23). In all cases, histopathological analysis of ML, 125I seed and/or marker within, correctly predicted axillary status after neoadjuvant chemotherapy. In all patients the 125I radioactive seed was recovered. CONCLUSIONS: Placing of 125I seeds is a feasible technique for intraoperative location of axillary positive node combined with SLN. The histopathological result of ML allows the axillary status to be determined after neoadjuvant chemotherapy.

HER2 assessment by bright-field dual in situ hybridization in cell blocks of recurrent and metastatic breast carcinoma.

BACKGROUND: Breast cancer recurrences or metastases often are diagnosed using cytology material. Cell blocks (CBs) with adequate cellularity are crucial for the determination of accurate hormonal and human epidermal growth factor receptor 2 (HER2) status and to guide treatment. In the current study, the authors evaluated the concordance of HER2 status between bright-field dual in situ hybridization (DISH), fluorescence in situ hybridization (FISH), and HER2 immunohistochemistry (IHC) performed on formalin-fixed CBs of recurrent and metastatic breast cancers. METHODS: The authors searched for patients who had breast carcinoma recurrences or metastases diagnosed between 2010 and 2018 by fine-needle aspiration or by the drainage of body cavity fluids with HER2 IHC and/or FISH performed on formalin-fixed CBs. Cases with adequate tumor cellularity (>50 cells) were selected. HER2 DISH was performed on all CBs. HER2 status of the primary breast carcinoma was recorded. RESULTS: Formalin-fixed CBs were identified from 30 patients with breast cancer recurrences and metastases in axillary lymph nodes (LNs) (5 patients), mediastinal LNs (8 patients), internal mammary LNs (1 patient), supraclavicular LNs (2 patients), portocaval LNs (1 patient), chest wall (3 patients), pleural fluid (3 patients), bone (4 patients), liver (2 patients), and lung (1 patient). All cases had HER2 IHC performed at the study institution and were scored by breast pathologists according to the American Society of Clinical Oncology/College of American Pathologists guidelines. The HER2 DISH results demonstrated 100% concordance (30 of 30 cases) with the concurrent IHC and/or FISH. CONCLUSIONS: All methods of HER2 evaluation were found to accurately identify the amplification status. DISH can be used in tandem with IHC as a reflex assay instead of FISH and is an efficient and reliable method with which to determine HER2 amplification in formalin-fixed CBs.

Breast Cancer Subtype Classification Using 4-Plex Droplet Digital PCR.

BACKGROUND: Infiltrating ductal carcinoma (IDCA) is the most common form of invasive breast cancer. Immunohistochemistry (IHC) is widely used to analyze estrogen receptor 1 (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) that can help classify the tumor to guide the medical treatment. IHC examinations require experienced pathologists to provide interpretations that are subjective, thereby lowering the reproducibility of IHC-based diagnosis. In this study, we developed a 4-plex droplet digital PCR (ddPCR) for the simultaneous and quantitative analyses of estrogen receptor 1 (ESR1), progesterone receptor (PGR), erb-b2 receptor tyrosine kinase 2 (ERBB2), and pumilio RNA binding family member 1 (PUM1) expression levels in formalin-fixed paraffin-embedded (FFPE) samples. METHODS: We evaluated the sensitivity, reproducibility, and linear dynamic range of 4-plex ddPCR. We applied this method to analyze 95 FFPE samples from patients with breast IDCA and assessed the agreement rates between ddPCR and IHC to evaluate its potential in classifying breast cancer subtypes. RESULTS: The limits of quantification (LOQ) were 25, 50, 50, and 50 copies per reaction for ERBB2, ESR1, PGR, and PUM1, respectively. The dynamic ranges of ESR1, PGR, and PUM1 extended over 50-1600 copies per reaction and those of ERBB2 from 25 to 1600 copies per reaction. The concordance correlation coefficients between 4-plex ddPCR and IHC were 96.8%, 91.5%, and 85.1% for ERBB2, ESR1, and PGR, respectively. Receiver operating characteristic curve area under the curve values of 0.991, 0.977, and 0.920 were generated for ERBB2, ESR1, and PGR, respectively. CONCLUSIONS: Evaluation of breast cancer biomarker status by 4-plex ddPCR was highly concordant with IHC in this study.

Quantitative label-free mass spectrometry using contralateral and adjacent breast tissues reveal differentially expressed proteins and their predicted impacts on pathways and cellular functions in breast cancer.

Proteins play an essential role in the biological processes associated with cancer. Their altered expression levels can deregulate critical cellular pathways and interactive networks. In this study, the mass spectrometry-based label-free quantification followed by functional annotation was performed to investigate the most significant deregulated proteins among tissues of primary breast tumor (PT) and axillary metastatic lymph node (LN) and corresponding non-tumor tissues contralateral (NCT) and adjacent (ANT) from patients diagnosed with invasive ductal carcinoma. A total of 462 proteins was observed as differentially expressed (DEPs) among the groups analyzed. A high level of similarity was observed in the proteome profile of both non-tumor breast tissues and DEPs (n = 12) were mainly predicted in the RNA metabolism. The DEPs among the malignant and non-tumor breast tissues [n = 396 (PTxNCT) and n = 410 (LNxNCT)] were related to pathways of the LXR/RXR, NO, eNOS, eIF2 and sirtuins, tumor-related functions, fatty acid metabolism and oxidative stress. Remarkable similarity was observed between both malignant tissues, which the DEPs were related to metastatic capabilities. Altogether, our findings revealed differential proteomic profiles that affected cancer associated and interconnected signaling processes. Validation studies are recommended to demonstrate the potential of individual proteins and/or pathways as biological markers in breast cancer. SIGNIFICANCE: The proteomic analysis of this study revealed high similarity in the proteomic profile of the contralateral and adjacent non-tumor breast tissues. Significant differences were identified among the proteome of the malignant and non-tumor tissue groups of the same patients, providing relevant insights into the hallmarks, signaling pathways, biological functions, and interactive protein networks that act during tumorigenesis and breast cancer progression. These proteins are suggested as targets of relevant interest to be explored as potential biological markers related to tumor development and metastatic progression in the breast cancer disease.