Tumor-Infiltrating Lymphocytes and Immune Response in HER2-Positive Breast Cancer.

Human epidermal growth factor receptor 2-positive (HER2-positive) breast cancer accounts for 15 to 25% of breast cancer cases. Although therapies based on the use of monoclonal anti-HER2 antibodies present clinical benefit for a subtype of patients with HER2-positive breast cancer, more than 50% of them are unresponsive to targeted therapies or they eventually relapse. In recent years, reactivation of the adaptive immune system in patients with solid tumors has emerged as a therapeutic option with great potential for clinical benefit. Since the approval of the first treatment directed against HER2 as a therapeutic target, the range of clinical options has expanded greatly, and, in this sense, cellular immunotherapy with T cells relies on the cytotoxicity generated by these cells, which ultimately leads to antitumor activity. Lymphocytic infiltration of tumors encompasses a heterogeneous population of immune cells within the tumor microenvironment that exhibits distinct patterns of immune activation and exhaustion. The prevalence and prognostic value of tumor-infiltrating lymphocyte (TIL) counts are associated with a favorable prognosis in HER2-positive breast cancers. This review discusses emerging findings that contribute to a better understanding of the role of immune infiltrates in HER2-positive breast cancer. In addition, it summarizes the most recent results in HER2-positive breast cancer immunotherapy and anticipates which therapeutic strategies could be applied in the immediate future.

Role of total tumour load of sentinel lymph node on survival in early breast cancer patients.

BACKGROUND: Axillary staging (pN) is considered one of the most important prognostic factors in breast cancer patients. However, the Z0011 study data drastically reduced the number of surgical axillary dissections in a selected group of patients, limiting the prognostic information relating to axillary involvement to the sentinel lymph node (SLN). It is known that there is a relationship between SLN total tumour load (TTL) and axillary involvement. The objective of this study is to analyse the relationship between the TTL and outcomes in patients with early stage breast cancer. PATIENTS AND METHODS: clinicopathological and follow-up data were collected from 950 patients with breast cancer between 2009 and 2010 on whom SLN analysis was conducted by molecular methods (One Step Nucleic Acid Amplification, Sysmex, Kobe, Japan). RESULTS: TTL (defined as the total number of CK19 mRNA copies in all positive SLN) correlates with disease free survival (HR, 1.08; p = 0.000004), with local recurrence disease free survival (HR = 1.07; p = 0.0014) and overall survival (HR: 1.08, p = 0.0032), clearly defining a low-risk group (TTL <2.5 × 104 CK19 mRNA copies/µL) versus a high-risk group (>2.5 × 104 CK 19 mRNA copies/µL). CONCLUSIONS: SLN TTL permits the differentiation between two patient groups in terms of DFS and OS, independently of axillary staging (pN), age and tumour characteristics (size, grade, lymphovascular invasion). This new data confirms the clinical value of low axillary involvement and could partially replace the information that staging of the entire axilla provides in patients on whom no axillary lymph node dissection is performed.

Deletion at 6q24.2-26 predicts longer survival of high-grade serous epithelial ovarian cancer patients.

Standard treatments for advanced high-grade serous ovarian carcinomas (HGSOCs) show significant side-effects and provide only short-term survival benefits due to disease recurrence. Thus, identification of novel prognostic and predictive biomarkers is urgently needed. We have used 42 paraffin-embedded HGSOCs, to evaluate the utility of DNA copy number alterations, as potential predictors of clinical outcome. Copy number-based unsupervised clustering stratified HGSOCs into two clusters of different immunohistopathological features and survival outcome (HR = 0.15, 95%CI = 0.03-0.81; Padj = 0.03). We found that loss at 6q24.2-26 was significantly associated with the cluster of longer survival independently from other confounding factors (HR = 0.06, 95%CI = 0.01-0.43, Padj = 0.005). The prognostic value of this deletion was validated in two independent series, one consisting of 36 HGSOCs analyzed by fluorescent in situ hybridization (P = 0.04) and another comprised of 411 HGSOCs from the Cancer Genome Atlas study (TCGA) (HR = 0.67, 95%CI = 0.48-0.93, Padj = 0.019). In addition, we confirmed the association of low expression of the genes from the region with longer survival in 799 HGSOCs (HR = 0.74, 95%CI = 0.61-0.90, log-rank P = 0.002) and 675 high-FIGO stage HGSOCs (HR = 0.76, 95%CI = 0.61-0.96, log-rank P = 0.02) available from the online tool KM-plotter. Finally, by integrating copy number, RNAseq and survival data of 296 HGSOCs from TCGA we propose a few candidate genes that can potentially explain the association. Altogether our findings indicate that the 6q24.2-26 deletion is an independent marker of favorable outcome in HGSOCs with potential clinical value as it can be analyzed by FISH on tumor sections and guide the selection of patients towards more conservative therapeutic strategies in order to reduce side-effects and improve quality of life.

Transcriptional characteristics of familial non-BRCA1/BRCA2 breast tumors.

To better understand the alterations present in the group of the so-called BRCAX tumors, we have used a cDNA microarray containing genes related to tumorigenesis and analyzed a series of 49 tumors consisting of 13 BRCA1, 14 BRCAX and 22 sporadic. We have confirmed that the BRCAX tumors are heterogeneous and can be divided in at least two main subgroups, so-called A and B, transcriptionally distinguishable and with different altered pathways within each of the groups. We have found that BRCAX-A and B subgroups, can be classified as Luminal A and Luminal B, respectively, taking into account the intrinsic phenotypes defined for the sporadic breast tumors. We have found that, at the somatic level, the BRCAX-B tumors are identical to their sporadic Luminal B counterparts, whereas BRCAX-A, despite having a Luminal A phenotype, shows additional genomic alterations. We have found 21 deregulated genes in the BRCAX-A group that we have called "the BRCAX susceptibility pathway" and suggested it as a candidate to search for new genes involved in the inherited susceptibility underlying the disease in this group.

Immunohistochemistry for the differential diagnosis of renal tumors with oncocytic features.

OBJECTIVE: Histologic analysis of renal tumors usually enables accurate diagnosis, but some tumors may show overlapping histopathologic features, which makes classification difficult. Diagnosis can be extremely challenging in tumors showing oncocytic change, mainly for oncocytomas, chromophobe carcinoma, papillary carcinomas with eosinophilic cells (type 2), and conventional renal cell carcinomas with eosinophilic oncocytic-like cells. The purpose of the present study is to analyze whether the patterns of immunohistochemical expression can help differential diagnosis of these tumors. MATERIAL AND METHODS: A thorough review of the files of the Department of Surgical Pathology, Fundación Jiménez Díaz, Madrid, Spain has retrieved 308 records of renal cell carcinomas in the last 12 years, with 15 renal oncocytomas (RO), 28 papillary carcinomas (PRCC), and 13 chromophobe carcinomas (CHRCC). We have performed immunohistochemistry on representative paraffin blocks from 9 oncocytomas, 11 chromophobe carcinomas, 10 papillary carcinomas, and 11 conventional cell carcinomas with oncocytic cells, from which we had suitable material. The immunohistochemical panel included CD117, α-methylacyl-coenzyme A racemase, CD10, p53, progesterone receptors, and cytokeratins 7 and 20. RESULTS: Our results show a considerable overlap between immunohistochemical expression in these tumors. Although immunohistochemistry can be helpful in some difficult cases, no markers allow a clear-cut distinction between these tumors, and diagnosis must still rely on morphologic features and histochemical techniques, as well as on molecular techniques when available.

Immunohistochemical classification of non-BRCA1/2 tumors identifies different groups that demonstrate the heterogeneity of BRCAX families.

Around 25% of hereditary breast and ovarian cancer families have mutations in the BRCA1 and BRCA2 genes. The search for other genes has until now failed, probably because there is not one single BRCAX gene, but rather various genes that may each be responsible for a small number of breast cancer families and/or may interact according to a polygenic model. We have studied 50 tumors from probands belonging to non-BRCA1/2 breast cancer families (BRCAX), using 25 immunohistochemical markers. The objective was to classify these tumors and confirm that they are heterogeneous. Unsupervised cluster analysis showed the existence of the following two main groups of tumors: high-grade and estrogen receptor (ER)-negative tumors (50%), and low-grade and ER-positive tumors (50%). In addition we identified five subgroups, three among the high-grade and two among the low-grade groups; one overexpressing HER-2 (18%); one with a basal-like phenotype (14%); one with a normal breast-like phenotype (18%); a luminal A subgroup (36%), and a luminal B subgroup (14%). Hypermethylation of the BRCA1 gene was observed in 42% of the cases, spread across all five subgroups, but only 37% of those had loss of heterozygosity as well. These latter cases were all clustered in the high-grade group and the majority of them in the basal-like subgroup. Our results show that familial non-BRCA1/2 tumors are heterogeneous and suggest a polygenic model for explaining the majority of BRCAX families. In addition we have defined a subset of them that have somatic inactivation of the BRCA1 gene.

Immunohistochemical expression of DNA repair proteins in familial breast cancer differentiate BRCA2-associated tumors.

PURPOSE: Morphologic and immunohistochemical studies of familial breast cancers have identified specific characteristics associated with BRCA1 mutation-associated tumors when compared with BRCA2 and non-BRCA1/2 tumors, but have not identified differences between BRCA2 and non-BRCA1/2 tumors. Because BRCA1 and BRCA2 genes participate in the DNA repair pathway, we have performed an immunohistochemical study with markers related to this pathway to establish the profile of the three groups. MATERIALS AND METHODS: We have studied two tissue microarrays that include 103 familial and 104 sporadic breast tumors, with a panel of DNA repair markers including ATM, CHEK2, RAD51, RAD50, XRCC3, and proliferating cell nuclear antigen. RESULTS: We found more frequent expression of CHEK2 in BRCA1 and BRCA2 tumors than in non-BRCA1/2 and sporadic tumors. We found absence of nuclear expression and presence of cytoplasmic expression of RAD51 in BRCA2 tumors that differentiate them from other familial tumors. We validated these results with a new series of patient cases. The final study with 253 familial patient cases (74 BRCA1, 71 BRCA2, 108 non-BRCA1/2), and 288 sporadic patient cases, has allowed us to confirm our preliminary results. Because BRCA2 tumors present a specific immunohistochemical profile for RAD51 and CHEK2 markers that is different from non-BRCA1/2 tumors, we have built a multivariate model with these markers that distinguish both tumors with an estimated probability of at least 76%. CONCLUSION: Our results suggest that BRCA2 tumors demonstrate more cytoplasmic and less nuclear RAD51 staining, and increased CHEK2 staining. This pattern may distinguish BRCA2 from familial non-BRCA1/2 tumors.

Phenotypic characterization of BRCA1 and BRCA2 tumors based in a tissue microarray study with 37 immunohistochemical markers.

Familial breast cancers that are associated with BRCA1 or BRCA2 germline mutations differ in both their morphological and immunohistochemical characteristics. To further characterize the molecular difference between genotypes, the authors evaluated the expression of 37 immunohistochemical markers in a tissue microarray (TMA) containing cores from 20 BRCA1, 14 BRCA2, and 59 sporadic age-matched breast carcinomas. Markers analyzed included, amog others, common markers in breast cancer, such as hormone receptors, p53 and HER2, along with 15 molecules involved in cell cycle regulation, such as cyclins, cyclin dependent kinases (CDK) and CDK inhibitors (CDKI), apoptosis markers, such as BCL2 and active caspase 3, and two basal/myoepithelial markers (CK 5/6 and P-cadherin). In addition, we analyzed the amplification of CCND1, CCNE, HER2 and MYC by FISH. Unsupervised cluster data analysis of both hereditary and sporadic cases using the complete set of immunohistochemical markers demonstrated that most BRCA1-associated carcinomas grouped in a branch of ER-, HER2-negative tumors that expressed basal cell markers and/or p53 and had higher expression of activated caspase 3. The cell cycle proteins associated with these tumors were E2F6, cyclins A, B1 and E, SKP2 and Topo IIalpha. In contrast, most BRCA2-associated carcinomas grouped in a branch composed by ER/PR/BCL2-positive tumors with a higher expression of the cell cycle proteins cyclin D1, cyclin D3, p27, p16, p21, CDK4, CDK2 and CDK1. In conclusion, our study in hereditary breast cancer tumors analyzing 37 immunohistochemical markers, define the molecular differences between BRCA1 and BRCA2 tumors with respect to hormonal receptors, cell cycle, apoptosis and basal cell markers.

Immunohistochemical characteristics defined by tissue microarray of hereditary breast cancer not attributable to BRCA1 or BRCA2 mutations: differences from breast carcinomas arising in BRCA1 and BRCA2 mutation carriers.

PURPOSE: Most familial breast cancers are not associated with BRCA1 or BRCA2 germ-line mutations. Therefore, it is of major importance to define the morphological, immunohistochemical, and molecular features of this group of tumors to improve genetic testing and also gain further insight into the biological characteristics of tumors. EXPERIMENTAL DESIGN: We evaluated the morphological characteristics of 37 tumors arising in women without BRCA1 or BRCA2 mutations, 20 tumors from BRCA1 mutation carriers, and 18 from BRCA2 mutation carriers, all of which were from index patients from breast cancer families. In addition, a tissue microarray was constructed with all tumoral samples to evaluate the immunohistochemical expression of a wide panel of antibodies (11 antibodies) and the amplification of HER-2 and c-MYC genes by fluorescence in situ hybridization. An age-matched group with 50 sporadic breast cancers as controls for non-BRCA1/2 was also included. RESULTS: Non-BRCA1/2 infiltrating ductal carcinomas (IDCs) showed specific differences from BRCA1 tumors. They were of lower grade (1 and 2); more frequently estrogen receptor, progesterone receptor, BCL2 positive, and p53 negative; had a low proliferation rate (Ki-67 immunostaining < 5%); and did not express P-cadherin. With respect to BRCA2 IDCs and control group, non-BRCA1/2 tumors were of lower grade and had a lower proliferation rate. No cases of HER-2 amplification and/or overexpression were observed except in the control group ( approximately 20%). In contrast, c-MYC amplification was present in 18.2, 62.5, and 12.5% of BRCA1, BRCA2, and non-BRCA1/2 IDCs, respectively, and 31% in the control group. CONCLUSIONS: This study thus reveals distinct morphological and immunohistochemical features in non-BRCA1/2 and BRCA1 tumors, whereas BRCA2 tumors present characteristics intermediate between the two phenotypes. In addition, the study also demonstrates the usefulness of tissue microarray technology in the evaluation of the immunophenotypic features of hereditary breast cancer.

Loss of heterozygosity analysis at the BRCA loci in tumor samples from patients with familial breast cancer.

The BRCA1 and BRCA2 genes are responsible for a high proportion of familial breast cancer; germline mutations in these genes confer a lifetime risk of about 70% for developing breast cancer. Most of the described deleterious mutations are small deletions or insertions that originate a truncated protein; however, in many cases, they are amino acid changes whose significance is unknown. In these cases, there are some tests that can analyze the meaning of these variants, but most remain unclassified. The BRCA genes are tumor suppressors and it is believed that complete loss of the wild-type allele is a common mechanism of inactivation in tumors from patients carrying a germline deleterious mutation in these genes; if this is true, loss of heterozygosity (LOH) analysis in the tumor sample could help to distinguish if a rare variant is either a deleterious mutation or a common polymorphism. In the present study, we performed LOH analysis at the BRCA loci in 47 tumors from patients who belonged to high-risk breast cancer families and were carriers of any type of alteration in these genes. Our results suggest that (i) loss of the wild-type allele is the most common mechanism of inactivation in tumors from patients who carry a deleterious mutation in any of the genes, (ii) this loss is not common when we analyze familial tumors not associated with mutations in BRCA and (iii) LOH can be used to clarify variants of unknown significance in the BRCA genes.