Luminal-Type Invasive Carcinoma in Association With Microglandular Adenosis/Atypical Microglandular Adenosis: A Case Report and Molecular Comparison

Microglandular adenosis (MGA) is a proliferative breast lesion composed of small, uniform glands lacking a myoepithelial cell layer while still invested by the basement membrane. The glands percolate haphazardly through the breast parenchyma rather than maintaining a lobular architecture, typical of other forms of adenosis.MGA is a benign lesion though atypical forms have been well described, often in close association with carcinoma. MGA, atypical MGA (AMGA), and the vast majority of MGA-associated carcinomas (MGACA) are negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) by immunohistochemistry. In light of these findings and early molecular studies, MGA is hypothesized to represent a clonal process and nonobligate precursor of basal-type breast carcinomas. We present the case of a 58-year-old woman and the first published molecular comparison of a luminal-type invasive ductal carcinoma with its associated MGA/AMGA. Analysis of small nucleotide variants (SNVs) revealed that 63% of the SNVs identified in the MGA were present in the AMGA while only 10% of them were present in the MGACA, suggesting a direct relationship between MGA and AMGA but not MGA and MGACA.


Introduction
Microglandular adenosis (MGA) is a rare proliferative breast lesion that has been reported in women over a wide age range (28 to 82 years old) with a mean age of diagnosis in the sixth decade of life [1,2]. No specific predisposing factors have been identified. MGA is often an incidental finding but may present as a mammographic density or calcifications [2][3][4][5][6]. MGA is often inconspicuous on gross examination though it has been described as a poorly defined nodularity or mass. A spectrum of morphologies of MGA has been described, from the usual MGA lacking atypia to atypical MGA (AMGA) to carcinoma arising in association with MGA (MGACA) [2]. On the basis of shared morphological, immunohistochemical, and molecular features, it has been suggested that MGA is a nonobligate precursor to triple-negative breast cancer with increasing architectural and morphologic complexity reflecting the acquisition of increased genetic alterations [7][8][9][10][11].
MGA, AMGA, and MGACA are genetically heterogeneous entities, with the most common nonsynonymous mutation reported to be shared when they co-occur being found within the TP53 gene [2,22,23]. Additionally, somatic mutations in genes involved in the P13K/Akt/mammalian target of rapamycin (mTOR) pathway (PAM) and the tyrosine kinase receptor signaling-related genes FGFR2 and ERBB3 have been identified in TP53-mutation negative cases [11]. The PAM pathway is crucial to cellular growth/synthesis; growth factors binding to PI3K leads to a series of phosphorylation steps ultimately activating AKT, the central mediator of the pathway, which in turn activates mTOR to stimulate growth and synthesis [24]. Alterations of the PAM pathway are implicated in many cancer types, including as many as 70% of breast cancers (most commonly ER-positive), showing pathway activation [24]. Alterations have also been identified in hormone receptornegative breast cancer. Mutations in PAM pathway genes, PTEN, PIK3CA, and INPP4B, were identified in TP53-mutation negative MGA/AMGA [11,24,25]. FGFR2 encodes fibroblast growth factor receptors in epithelial (FGFR2b) and mesenchymal (FGFR2c) cells with cytoplasmic tyrosine kinase domains involved in the mitogen-activated protein kinase, PI3K, protein kinase C, and calmodulin-calcineurin-NFAT signaling cascades [26]. In mouse models of FGFR2, activation enhances branch morphogenesis in mammary glands and predisposes to tumorigenesis by promoting epithelial-mesenchymal transition and negatively regulating BRCA1 [27]. Copy number variations are another form of genetic alterations found in 1 2 2 1 1 MGA/AMGA/MGACA and described previously in the literature [10,22,23,28]. According to one case series, a median of 20.3% of the genome in a pooled sample of MGA, AMGA, and MGACA harbor copy number variants with an increase in the median number [22]. Moreover, a stepwise increase in copy number variants from MGA to AMGA to MGACA has been reported [11,22]. Recurrent gains found in MGA/AMGA include those in 1q, 2q, 7p, 7q, and 8q while recurrent losses include those in 1p, 8p, 17p, 5q 14q, 16q, and 17q [11,22]. Additional recurrent changes reported in AMGA include gains of 6p and losses of 10q [22]. Amplifications of the MYC gene (8q24.21) and DEPTOR (8q24.12) have been reported as well [10,11]. A subset of MGAs and AMGAs lacking copy number variants has also been described [10,22].
Rare case reports of hormone receptor-positive MGACA are documented in the literature, however, none have undergone molecular characterization [16,17,28,29]. The current study presents a case report and the first molecular characterization of hormone receptor-positive invasive ductal carcinoma arising in the setting of MGA and AMGA.

Case Presentation
A 58-year-old nulliparous female presented to our institution with a new, palpable left breast mass, first noticed four days prior. Her medical history was significant for menarche at age 15, menopause at age 50, a 14-year history of birth control use, and a family history of breast cancer in a maternal aunt (62 years old at the time of diagnosis). At the time of presentation, the patient denied breast pain, dimpling, retraction, or discharge. Physical examination was notable for a 4 x 3 cm palpable left breast mass at the 12:00 position, approximately 2 cm from the nipple involving both upper quadrants with unremarkable overlying skin. A diagnostic mammogram revealed a 1.3 cm oval mass in the left breast with obscured margins favored to be benign (BI-RADS (Breast Imaging Reporting & Data System) Category 3). On follow-up seven months later, a 2.3 x 1.2 x 2.5 cm irregular, hypodense mass with spiculated margins located 2 cm from the nipple in the 12:00 position classified as BI-RADS Category 5 was identified on a diagnostic mammogram ( Figure 1). No axillary or internal mammary chain lymphadenopathy was appreciated on imaging. The patient underwent an ultrasound-guided needle core biopsy that revealed moderately differentiated luminal-type invasive ductal carcinoma and a background of MGA. Notably, cases of hormone receptor-positive invasive ductal carcinoma arising in the setting of MGA are exceedingly rare, limited to a handful of case reports in the literature. She was clinically staged as a cT2, cN0, M0 and was referred for breast-conserving therapy. The patient underwent a left lumpectomy with sentinel lymph node biopsy with clear margins for her invasive disease, but extensive AMGA was present at the superior-medial margin. She was subsequently treated with adjuvant radiation therapy and maintenance anastrozole. The patient remains radiographically free of disease at the time of this publication, 18 months post-surgery.

Gross examination
The biopsy specimen consisted of a 2.1 x 1.2 x 0.3 cm aggregate of unremarkable white-tan soft tissue. The lumpectomy specimen demonstrated a single 3.5 x 2.1 x 1.6 cm, firm, white-tan mass with ill-defined, irregular borders that appeared to extend to the anterior, medial, lateral, superior, and inferior margins grossly. An additional shave of the superior-medial margin was submitted at the time of surgery, which appeared to have a grossly unremarkable cut surface. Two grossly unremarkable lymph nodes measuring 1.5 x 0.7 x 0.5 cm and 1.2 x 1 x 0.8 cm were recovered from the sentinel lymph node biopsy specimen.

Histology results
Microscopic evaluation of the biopsy specimen revealed moderately differentiated invasive ductal carcinoma measuring in a background of microglandular adenosis ( Figure 2). The microglandular adenosis was characterized by well-formed uniform glands with eosinophilic luminal secretions scattered throughout the fibrous and fatty stroma. A preliminary Nottingham histologic score of 2 was assigned to the invasive component (3/3 tubule formation, 2/3 nuclear pleomorphism, 2/3 mitotic activity; total score=7/9). No lymphovascular invasion was identified on routine sections.

FIGURE 2: Ultrasound-guided core needle biopsy demonstrated moderately differentiated invasive ductal carcinoma (2a) in a background of small uniform glands lined by bland cuboidal cells with open lumina and intraluminal eosinophilic secretions scattered in the fibrous and fatty stroma consistent with MGA (2b). Immunohistochemistry was performed on representative sections and further characterized the invasive component as ER-positive (2c), PRpositive (2d), HER2-negative
A-B: H&E stain, 10x objective lens. B-C: 10x objective lens Microscopic evaluation of the excision specimen showed a 3.5 cm, moderately differentiated invasive ductal carcinoma with a final Nottingham histologic grade of 2 (3/3 tubule formation, 2/3 nuclear pleomorphism, 2/3 mitotic activity; total score=7/9) ( Figure 3). Directly adjacent to the invasive carcinoma were areas of MGA with classic morphology consistent with the biopsy specimen, as well as MGA with atypical features, including nuclear stratification, nuclear atypia, prominent nucleoli, increased mitoses, compressed lumina, elongated/fused glandular units, cribriform glands, and single-cell infiltration, consistent with AMGA. The final pTNM AJCC 8th Edition Pathologic Stage Classification assigned was pT2 pN1 mi(sn).  Areas of MGA and AMGA demonstrated typical strong, diffuse S100 positivity and negative for ER and PR ( Figure 3). Further, the MGA and AMGA areas were negative for myoepithelial cells by p63 and calponin. IHC for p53 was wild-type (patchy, weak staining) within the MGA, AMGA, and MGACA components. One of two submitted sentinel lymph nodes was involved in micrometastatic carcinoma, highlighted by IHC for pancytokeratin.
These histological and immunohistochemical findings are diagnostic of a rare case of luminal-type invasive ductal carcinoma arising in the setting of MGA/AMGA. To date, instances of hormone receptor-positive MGACA are limited to a handful of case reports in the literature ( Table 1).

Sequencing results
Twenty

Conclusions
To date, cases of hormone receptor-positive invasive carcinoma arising in association with MGA and/or AMGA are limited to the few case reports described in the literature, as shown in Table 1, which includes salient clinicopathologic data and hormone receptor status. In these cases, it is unclear if MGA is, in fact, a precursor lesion to the synchronous invasive component or is merely a coexisting, unrelated process.
Our SNV analysis indicates an increasing mutational burden with progression from MGA to AMGA to MGACA. The majority (63%) of the SNVs identified in the MGA were retained in the AMGA. Relatively few SNVs are shared by either the MGA (11%) or the AMGA (10%) with the adjacent carcinoma. The considerable overlap between the MGA and AMGA supports the conclusion that the former is a precursor to the latter. This is also consistent with the literature positing a progression from MGA to AMGA through the accumulation of mutations. In this particular case, there is a lack of evidence that either the MGA or the AMGA served as a precursor to the luminal-type MGACA, as the molecular results show a lack of clonal evolution between the entities. A notable finding, however, included the identification of unique oncogenic driver mutations isolated within the AMGA (FGFR2 Ser24Trp) and MGACA (AKT1 Glu17Lys). FGFR2 Ser24Trp is associated with endometrial, breast, ovarian, and cervical cancers (COSMIC database accessed 15 Nov 22). AKT1 Glu17Lys is strongly associated with breast cancer and cited in 374 cases. Interestingly, the FGFR2 Ser24Trp variant has also been previously identified in TP53-mutation negative MGA/AMGA. Mouse studies have demonstrated that this mutation promotes breast cancer by activating the FGFR2/STAT3/MAPK pathway and stimulating the epithelial-to-mesenchymal transition. This missense mutation has also been described within the context of uterine cancer and is one of several oncogenic mutations clustered around the third Ig-like extracellular domain of FGFR2, where they are thought to alter ligand-receptor specificity. The finding of the FGFR2 Ser24Trp variant, a previously characterized oncogenic driver mutation, in this case of MAGA is consistent with AMGA as a precursor lesion for carcinoma, though not in this particular case. AKT1 E17K has been identified in a variety of cancer types, including an estimated 6.7% of ER-positive breast cancers, where it leads to constitutive membrane localization and activation, stimulating survival and proliferation. Taken together, these findings suggest that MGA is the precursor of the AMGA but not of the MGACA in this case, even though they are spatially and temporally related. In the present case, our next-generation sequencing was limited by formalin fixation artifact, precluding copy number variant evaluation, however, future reports would benefit from characterizing copy number variants in hormone receptor-positive MGACA. Documentation of additional cases incorporating molecular information will better elucidate the relationship suggested by this unique case.

Case Selection, Histochemistry, and Immunohistochemistry
We

Macrodissection and Genomic DNA Extraction
Genomic DNA was extracted from the formalin-fixed, paraffin-embedded normal breast tissue and breast biopsies with representative sections of histologically and immunohistochemically confirmed MGA, AMGA, and hormone receptor-positive invasive ductal carcinoma (3-4,   All single nucleotide variants identified by whole exome sequencing with associated gene, hgsvg, coding change, protein change, and variant allele frequency identified in microglandular adenosis (MGA), atypical microglandular adenosis (AMGA), and microglandular associated carcinoma (MGACA)

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. N/A issued approval N/A. N/A -Granted non-research designation by the Brooke Army Medical Center IRB. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: DISCLAIMER: The views expressed herein are those of the author(s) and do not reflect the official policy or position of Brooke Army Medical Center, the Department of Defense, or any agencies under the U.S. Government. This work was prepared as part of our official duties. Title 17, USC, Section105 provides that "Copyright protection under this title is not available for any work of the United States Government." Title 17, USC, Section 101defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person's official duties. .