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Patients with pathological nipple discharge (PND) often undergo local surgical procedures because standard radiologic imaging fails to identify the underlying cause. MicroRNA (MiRNA) expression analysis of nipple fluid holds potential for distinguishing between breast diseases. This study aimed to compare miRNA expression levels between nipple fluids from patients with PND to identify possible relevant miRNAs that could differentiate between intraductal papillomas and no abnormalities in the breast tissue. Nipple fluid samples from patients with PND without radiological and pathological suspicion for malignancy who underwent a ductoscopy procedure were analyzed. We used univariate and multivariate regression analyses to identify nipple fluid miRNAs differing between pathologically confirmed papillomas and breast tissue without abnormalities. A total of 27 nipple fluid samples from patients with PND were included for miRNA expression analysis. Out of the 22 miRNAs examined, only miR-145-5p was significantly differentially expressed (upregulated) in nipple fluid from patients with an intraductal papilloma compared to patients showing no breast abnormalities (OR 4.76, p = 0.046), with a diagnostic accuracy of 92%. miR-145-5p expression in nipple fluid differs for intraductal papillomas and breast tissue without abnormalities and, therefore, has potential as a diagnostic marker to signal presence of papillomas in PND patients. However, further refinement and validation in clinical trials are necessary to establish its clinical applicability.

Lineage tracing in mice indicates that LGR5 is an adult stem cell marker in multiple organs, such as the intestine, stomach, hair follicles, ovary, and mammary glands. Despite many studies exploring the presence of LGR5 cells in human tissues, little is known about its expression profile in either human mammary tissue or pathological lesions. In this study we aim to investigate LGR5 expression in normal, benign, and malignant lesions of the human breast using RNA in situ hybridization. LGR5 expression has not been observed in normal lactiferous ducts and terminal duct lobular units, whereas LGR5-positive cells have been specifically observed in the basal myoepithelium of ducts in the regenerative tissues, ductal carcinoma in situ, and in ducts surrounded by invasive cancer cells. These findings suggest LGR5 marks facultative stem cells that are involved in post injury regeneration instead of homeostatic stem cells. LGR5 positivity was found in 3% (9 of 278 cases) of invasive breast cancers (BC), and it showed positive associations with higher histologic grades (P = 0.001) and T stages (P < 0.001), while having negative correlations with estrogen receptor (P < 0.001) and progesterone receptor (P < 0.001) expression. Remarkably, all LGR5-positive BC, except one, belong to triple-negative BC (TNBC), representing 24% (9 of 38 cases) of all of them. LGR5 histoscores have no correlations with EGFR, CK5/6, Ki-67, or P53 expression. Additionally, no β-catenin nuclear localization was observed in LGR5-positive BC, indicating that canonical Wnt pathway activation is less likely involved in LGR5 expression in BC. Our results demonstrate that LGR5 expression is induced in regenerative conditions in the myoepithelium of human mammary ducts and that its expression is only observed in TNBC subtype among all invasive BC. Further studies regarding the functional and prognostic impact of LGR5 in TNBC are warranted., (© 2021. The Author(s).)

The pathologic feature of intraductal papillomas is defined as a papillary structure composed of a fibrovascular stromal core lined by luminal epithelial cells and myoepithelial cells. We used droplet digital PCR for the mutational analysis of AKT1 (E17K) and PIK3CA (H1047R, E542K, and E545K) in 60 papillomas. AKT1 and PIK3CA mutations were detected in 12 (20%) and 17 (28%) of the papillomas, respectively. In five tumors harboring mutations, mutational analysis of AKT1 or PIK3CA was performed separately using luminal epithelial cells and myoepithelial cells sorted using anti-cytokeratin 19 antibody and anti-α smooth muscle actin antibody. The two types of cells from a given papilloma had the identical mutation. Three patients with the PIK3CA mutation-positive papilloma developed breast cancers at the resection site of the papilloma, but none of these subsequent breast cancers had the PIK3CA mutation. These results indicate that a papilloma stems from a bipotent progenitor cell that contains the AKT1 or PIK3CA mutation and proliferates and differentiates to form the papilloma. Papilloma can be a risk factor for developing breast cancer but is unlikely to be its obligate precursor., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

More than 2% of adults harbor a pancreatic cyst, a subset of which progresses to invasive lesions with lethal consequences. To assess the genomic landscapes of neoplastic cysts of the pancreas, we determined the exomic sequences of DNA from the neoplastic epithelium of eight surgically resected cysts of each of the major neoplastic cyst types: serous cystadenomas (SCAs), intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs), and solid pseudopapillary neoplasms (SPNs). SPNs are low-grade malignancies, and IPMNs and MCNs, but not SCAs, have the capacity to progress to cancer. We found that SCAs, IPMNs, MCNs, and SPNs contained 10 ± 4.6, 27 ± 12, 16 ± 7.6, and 2.9 ± 2.1 somatic mutations per tumor, respectively. Among the mutations identified, E3 ubiquitin ligase components were of particular note. Four of the eight SCAs contained mutations of the von Hippel-Lindau gene (VHL), a key component of the VHL ubiquitin ligase complex that has previously been associated with renal cell carcinomas, SCAs, and other neoplasms. Six of the eight IPMNs and three of the eight MCNs harbored mutations of RNF43, a gene coding for a protein with intrinsic E3 ubiquitin ligase activity that has not previously been found to be genetically altered in any human cancer. The preponderance of inactivating mutations in RNF43 unequivocally establish it as a suppressor of both IPMNs and MCNs. SPNs contained remarkably few genetic alterations but always contained mutations of CTNNB1, previously demonstrated to inhibit degradation of the encoded protein (β-catenin) by E3 ubiquitin ligases. These results highlight the essential role of ubiquitin ligases in these neoplasms and have important implications for the diagnosis and treatment of patients with cystic tumors.

Atypical proliferative lesions of the breast, such as atypical ductal hyperplasia and atypical papilloma, are considered to be precursors of breast carcinomas and have frequently been shown to have loss of heterozygosity (LOH) on chromosome 16q at the DNA level. We evaluated whether an atypical proliferative lesion and a carcinoma that subsequently occurred in the same area of the ipsilateral breast were of identical clonal origin in seven patients. Using DNA isolated from microdissected archival tissue of epithelial components of both the biopsy specimen of the atypical proliferative lesion and the mastectomy specimen of the carcinoma, the pattern of LOH on 16q was compared between these two lesions using polymerase chain reaction -microsatellite LOH analysis. As a control, LOH on 16q was examined in 13 cases of usual ductal hyperplasia, 10 usual papillomas, and 6 atypical ductal hyperplasias. In the seven cases, LOH on 16q was detected in three of the six atypical proliferative lesions and in five of the seven carcinomas, but the allele with LOH or a deleted region always differed between the two. LOH was detected in both atypical proliferative lesions and carcinomas in one case, only in the atypical proliferative lesion in two cases, and only in carcinomas in three cases. In the controls, LOH on 16q was absent in usual ductal hyperplasias or usual papillomas but was detected in two of six atypical ductal hyperplasias. Although atypical proliferative lesions were frequently confirmed to be of clonal nature with LOH on 16q, these lesions and carcinomas were considered to be clones, probably originated from a field with these clones.

The expression of fibronectin (FN) isoforms including extra domain A (EDA) and extra domain B (EDB) segments, was investigated in 36 invasive ductal carcinomas and 13 benign tumors of human breast tissues by in situ hybridization using probes specific to alternative splicing sites. Signals for the constant region of FN mRNA in cancer cells were found in 53% of the invasive ductal carcinomas. The EDA+ and EDB+ mRNA signals were found in 47% and 33%, respectively. Stromal cells expressing FN, EDA+ and EDB+ mRNA signals were present in 100%, 69% and 14% of cases, respectively. Expression of FN mRNAs by cancer cells was most frequent in intraductal lesions or large cancer nests, and that by stromal cells was associated with desmoplastic areas. In representative cases, proportions of FN mRNA-positive cancer cells expressing EDA and EDB segments were 45% and 39%, respectively, signals for both being frequently found in the same cells. EDA+ and EDB+ mRNA were labeled in 25% and 6% of the FN mRNA-positive stromal cells, a large proportion thus being EDA-/EDB- FN. In conclusion, the splicing pattern of FN pre-mRNA is dependent on the cell type and histology of breast cancer tissues. The observed lack of expression in fibroadenomas and other benign conditions suggests a link with tumor progression.

An autopsy case of double cancer is reported. The patient had undergone right hemicolectomy for colon carcinoma in 1982, and the second cancer was detected in the pancreas in April 1989, which was diagnosed as intraductal papillary adenocarcinoma that is known for its favorable prognosis after surgical resection. However, as the patient did not consent to the operation, she died in August 1994, five years after the diagnosis of the second cancer. Histopathological study revealed neither recurrence nor metastasis of colon carcinoma. The pancreatic carcinoma metastasized to the lung, liver, and peritoneum. DNAs were extracted from paraffin-embedded tissue for molecular pathological examinations. Different mutations were found at codon 12 of the K-ras gene by nucleotide sequencing analysis: one in the colon and the other in the pancreas and lung. Over-expression of p53 protein was also detected in the colon by immunostaining. Replication error was not observed in these three tumors suggesting that a factor(s) other than genetic instability was playing a role in the development of double cancer in this patient.

Loss of heterozygosity (LOH) on chromosome 16q was examined in DNA isolated from 11 intraductal papillomas and 12 intracystic papillary adenocarcinomas of the breast. Such LOH was detected in 8 (67%) out of 12 intracystic papillary adenocarcinomas, and in 7 (64%) of 11 of these adenocarcinomas of low grade atypia (Grade 1), whereas it was not detected in 11 intraductal papillomas. Therefore, inactivation of tumor-suppressor genes on chromosome 16q was suggested to be involved in acquisition of malignant phenotype rather than in tumorigenesis in mammary gland epithelial cell. Examination of LOH on 16q should be helpful for differential diagnosis of intracystic papillary tumors.

Clonality of solitary intraductal papillomas of the breast was analyzed using a method based on restriction fragment length polymorphism of the X-chromosome-linked phosphoglycerokinase (PGK) gene and on random inactivation of the gene by methylation. The application of polymerase chain reaction to this method enabled clonal analysis of such a small intraductal lesion as papilloma. Clonal analysis of DNA samples obtained from the nine solitary intraductal papillomas and adjacent normal breast tissues showed that all of the papillomas were monoclonal and all the normal breast tissues were polyclonal in origin. When DNA samples were obtained from four widely separated sites in the papillomas, clonal analysis showed that all were monoclonal and, in addition, the same allele of PGK gene was inactivated in each case. These results demonstrate that solitary intraductal papilloma arises as a single monoclonal tumor and extends along the ducts rather than occurring as multicentric monoclonal tumors and merging together subsequently. Immunohistochemical staining of smooth muscle alpha-actin, a marker protein of myoepithelial cells, revealed that solitary intraductal papilloma was composed of approximately equal mixtures of luminal epithelial and myoepithelial cells. Since solitary intraductal papillomas were shown to be monoclonal in origin, it was suggested that this disease originates from a common precursor that could differentiate into both luminal epithelial and myoepithelial cells.

Clonality of predominantly intraductal carcinoma (PIC) and precancerous lesions of the breast was analyzed by a method based on restriction fragment length polymorphism of the X-chromosome-linked phosphoglycerokinase gene and on random inactivation of the gene by methylation. The application of polymerase chain reaction to this method enabled clonal analysis of small lesions. In order to eliminate the contamination by normal stromal cells, intraductal components were microdissected from the frozen sections of PIC under a dissection microscope. Clonal analysis of the intraductal components from seven PICs revealed that all were monoclonal in origin. In three PICs with intraductal spreading of carcinoma cells over nearly a whole breast gland, the intraductal components were collected from eight widely separated sites in each case. Clonal analysis of these samples showed that every sample was monoclonal and the same allele of the phosphoglycerokinase gene was consistently inactivated in each case. These results suggest that PIC arises as a single monoclonal carcinoma and spreads through the ducts over the gland rather than having multicentric origins. Clonality of precancerous lesions such as atypical ductal hyperplasia and intraductal papilloma arising in the terminal ducts was also studied. Intraductal components were microdissected from the paraffin sections of these lesions and subjected to clonal analysis. Both atypical ductal hyperplasia and intraductal papilloma were found to be monoclonal in origin, suggesting that certain genetic changes had already occurred in the precancerous lesions. A further study is needed to elucidate these genetic changes, which would greatly help our understanding of the mechanism of carcinogenesis.