Faculty Bibliography

Next-generation sequencing has enabled genetic and genomic characterization of melanoma to an unprecedent depth. However, the high mutational background plus the limited deep-coverage whole-genome sequencing performed on cutaneous melanoma samples, make difficult the identification of novel driver mutations. We sought to explore the somatic mutation portfolio in exonic and gene regulatory regions in human melanoma samples, for which we performed targeted sequencing of tumors and matched germline DNA samples from 89 melanoma patients, identifying known and novel recurrent mutations. Two recurrent mutations found in the RPS27 promoter associated with decreased RPS27 mRNA levels in vitro. Data mining and IHC analyses revealed a bimodal pattern of RPS27 expression in melanoma, with RPS27-low patients displaying worse prognosis. In vitro characterization of RPS27-high and -low melanoma cell lines, as well as loss-of-function experiments, demonstrated that high RPS27 status provides increased proliferative and invasive capacities, while low RPS27 confers survival advantage in low-attachment and resistance to therapy. Additionally, we demonstrate that 10 other cancer types harbor bimodal RPS27 expression and in those, similarly to melanoma, RPS27-low expression associates with worse clinical outcomes. RPS27 promoter mutation could thus represent a mechanism of gene expression modulation in melanoma patients, which may have prognostic and predictive implications.

Dermal invasion is a hallmark of malignant melanoma. Thought the molecular alterations driving the progression of primary melanoma to metastatic disease have been studied extensively, the early progression of non-invasive primary melanoma to an invasive state is poorly understood. To elucidate the mechanisms underlying the transition from radial to vertical growth, the first step in melanoma invasion, we developed a zebrafish melanoma model in which constitutive activation of ribosomal protein S6 kinase A1 (Rps6ka1; RSK1) drives tumor invasion. Transcriptomic analysis of ribosomal protein S6 kinase A1-activated tumors identified metabolic changes, including up-regulation of genes associated with oxidative phosphorylation. Vertical growth phase human melanoma cells show higher oxygen consumption and preferential utilization of glutamine compared to radial growth phase melanoma cells. Peroxisome proliferator activated receptor gamma (PPARG) coactivator 1 alpha (PPARGC1A), also known as PPARG coactivator-1α (PGC1α), has been proposed as a master regulator of tumor oxidative phosphorylation. In human primary melanoma specimens, PGC1α protein expression was found to be positively associated with increased tumor thickness and expression of the proliferative marker Ki-67 and the reactive oxygen species scavenger scavenger receptor class A member 3 . PGC1α depletion modulated cellular processes associated with primary melanoma growth and invasion, including oxidative stress. These results support a role for PGC1α in mediating glutamine-driven oxidative phosphorylation to facilitate the invasive growth of primary melanoma.

Background: Intraoperative evaluation of the nipple/subareolar tissue (N/SAT) has been used by surgeons to assess for occult nipple involvement by malignancy and guide the decision-making process for nipple preservation during nipple sparing mastectomies (NSM). The aim of our study is to evaluate significance and accuracy of frozen section (FS) results compared to final pathology/permanent sections.
Design(s): We retrospectively reviewed records of patients that underwent NSM with FS of the N/SAT from 2014 to 2018. Positive FS or final pathology results include atypical hyperplasia, in situ and invasive carcinoma.
Result(s): Over a 5-year period a total of 339 NSM cases utilized FS to evaluate the N/SAT. Of the total 339 cases, 85(25%) were prophylactic and 254(75%) were therapeutic mastectomies. All 85 prophylactic mastectomies were negative (benign) on FS and final diagnosis. Among 254 therapeutic mastectomies, 217(85.4%) showed negative (benign) intraoperative FS with concordant benign final pathology; 22(8.7%) showed positive intraoperative FS with concordant positive final pathology; 15(5.9%) were false negative (benign) on FS and positive on final permanent sections (figure 1). Positive results consisted of atypical ductal or lobular hyperplasia (5, 27.8%), in situ ductal or lobular carcinoma (11, 61.1%) and invasive carcinoma (1, 5.6%). One false positive case showed "atypical intraductal proliferation" at FS and was diagnosed as intraductal papilloma on final pathology; the nipple was not removed at the time of surgery. Of the 37 cases with positive final nipple pathology, 14(37.8%) had intraoperative resection of the nipple/areola complex (NAC), 9(24.3%) required an additional surgery for removal of NAC and 13(35.1%) had no additional procedures performed. Residual pathology was identified in 9(39.1%) of the resected NAC. In our patient cohort frozen section diagnosis has a sensitivity of 58.3%, specificity of 99.5%, positive predictive value (PPV) of 95.5% and negative predictive value (NPV) of 93.5%. (Figure presented)
Conclusion(s): Intraoperative evaluation of the N/SAT is highly accurate (93.7%) and specific (99.5%) test that prevented additional surgical intervention in 8.7% of therapeutic mastectomies. At the same time, surgeons should be aware of the low/moderate sensitivity of intraoperative FS, which may be explained by processing artifacts, sampling or cautious approach not to overcall the FS findings

Background: Encapsulated papillary carcinomas (EPC) of the breast is a variant of papillary carcinoma that are confined to a cystic space, surrounded by a fibrous capsule and lack the myoepithelial coat. Despite the latter finding, it is recommended that EPC be staged as pTis due to its indolent course. Concurrent frank invasive carcinomas are staged commensurate with their size. We sought to investigate the molecular pathways differentially expressed in pure EPC and EPC with frank invasion at the genomic and transcriptomic level. In addition, we compared EPC with its corresponding invasive ductal carcinoma (IDC) at the transcriptomic level.
Design(s): We selected 3 cases of pure EPC (C1-C3) and 3 cases of EPC (C4e-C6e) with corresponding IDC (C4i-C6i).We performed whole transcriptome analysis on laser-capture microdissected samples from formalin-fixed, paraffin-embedded tissue. We used CloneTech Mammalian stranded pico kit for sequencing RNA. KEGG pathway analysis and Gene Ontology (GO) analysis was performed using the cluster Profiler R package (v3.0.0) and Database for Annotation, Visualization and Integrated Discovery. DNA analysis was performed using our in-house next generation sequencing hybrid capture covering 580 genes on C1-C3 and C4e-C6e.
Result(s): There were 5 female and 1 male patients. The mean age was 73 years (range 62-90). All cases were hormone receptor positive. C4e-C6e showed upregulation of NTRK2 and MAGI2 (lg2FC= 3.14 and lg2FC = 3.0 fold, respectively) and downregulation of PRKACB (lg2FC= -4.4) compared to C1-C3 on RNAseq. C4i-C6i showed upregulation of collagen-related genes (COL10A1, COL11A1, COL14A1, COL16A1, COL1A1, COL3A1, COL8A1) (lg2FC range: 6.28 fold change) and ADAM12/ADAMTS2 (lg2FC=6.2 and lg2FC= 6.9 fold change) compared to C4e-C6e (FDR =0.014). Pathway analysis showed upregulation of collagen fibril organization and extracellular matrix organization pathways in C4i-C6i compared to C4e-C6e and upregulation of kinase activity pathway (GO: 0016301) in C4e-C6e compared to C1-C3.Recurrent PIK3CA hotspot non-synonymous mutation was identified in C3, C4e, C5e and C6e (c.G1633A in C5 and c.A3140G in C3, C4 and C6).
Conclusion(s): Our findings suggest that kinase and matrix metalloproteinase pathways contribute to EPC with invasion compared to pure EPC cases. Furthermore, enrichment of collagen-related genes in IDCs compared to their corresponding EPC suggest a synergistic potential with the aforementioned pathways. Mechanistic studies are warranted to validate the findings

Background: Diagnosis of microinvasion (MI) in breast core needle biopsy (CNB) can be challenging particularly in a background of carcinoma in situ (CIS) involving sclerosing lesion with periductal fibrosis and lymphocytic infiltrate. Immunohistochemical stains (IHC) for myoepithelial cells aid in confirming MI. Surgical management of MI deviates from CIS as the former includes sentinel lymph node biopsy (SLNB) while the latter typically includes SLNB only when total mastectomy (TM) is planned. We investigated the utility of IHC in diagnosing MI in our CNBs and its impact on final histopathology on surgical excision.
Design(s): We conducted a search for cases of CIS with foci suspicious for MI, in which IHC for calponin and p63 was used to confirm MI (defined as invasive carcinoma <=1 mm) between January 2010 and June 2019. CIS included ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS). MI cases diagnosed based on routine histology were also collected for the same time period. Only cases with follow up excision data were included. Cases with synchronous invasive carcinoma were excluded. Clinicopathologic data including age, size, laterality, resection type, SLNB status and biomarker profiles were compared. Graphpad Prism software was used for statistical analysis.
Result(s): We identified 106 cases of CIS (102 DCIS, 4 LCIS), where IHC was used to confirm MI (MI-IHC hereafter). Mean age was 58 years. Of the 106 cases MI-IHC was identified in 24 cases (23%). See table. All 24 MI-IHC cases had SLNB (100%). Of the 82 CIS cases, 39 had SLNB (48%). Relative risk of finding invasive carcinoma/MI on resection in MI-IHC was 1.8 (p=0.03) compared to CIS. There was no correlation between the biomarker profile with the resection outcome in either CIS (p=0.5, Fisher's exact test) or MI-IHC cases (p=3.4, Chi-square test). We identified 7 cases of MI, diagnosed on routine histology without IHC, of which 5 (71%) had invasive carcinoma/MI and 2 (29%) had CIS or no residual carcinoma on resection. Mean size of invasive carcinoma and CIS on resection in this group was 11 mm and 25 mm, respectively. The resection outcome between MI-IHC and MI based on routine histology was not significant (p=0.6). (Table presented)
Conclusion(s): IHC helped diagnose MI in CNB for CIS in 23% of cases. Compared to CIS, the diagnosis of MI-IHC carried a relative risk of 1.8 in finding invasive carcinoma/MI on resection. There was no difference in the significance of the method used for the diagnosis of MI

Background: The American Joint Committee on Cancer (AJCC) recommends the use of the largest tumor size for pathologic staging of multifocal breast cancer (MBC). However, the recently updated AJCC 8th edition now takes biologic factors such as grade, biomarker profile, and genomic panels into account for breast cancer prognostication. We sought to study MBCs at our institution and track their path to metastasis in an attempt to gain further insight into variables of aggressiveness other than traditional size.
Design(s): We searched for MBC cases within our database from 2010 through 2017. Age, procedure type, laterality, size, histologic grade (G), presence of metastasis (axillary or distant) and biomarker profile of the primary and metastases were recorded. The biomarker profiles were classified as hormone receptor positive (HR+), human epidermal growth factor 2 positive (Her2+) or triple negative (TN), and deemed concordant (C) if similar or discordant (D) if dissimilar.
Result(s): Of 473 MBCs, 260 had at least two biomarker profiles reported. The majority of the latter patients underwent total mastectomy (67%) while 33% had partial mastectomy. Of 554 total tumor foci, 462 (93% C, 7% D) were HR+ (17% G1, 62% G2, 21% G3), 65 (66% C, 34% D) were Her2+ (34% G2, 66% G3) and 27 (74% C, 26% D) were TN (19% G2, 81% G3). There were 233 (90%) concordant cases and 27 (10%) discordant. Of the 260 cases, 12 (4.6%) discordant cases had tumor foci with the same morphology and G. Twelve of the discordant cases had axillary and/or distant metastases, 4 of which with a biomarker profile available for the metastatic tumor (Table). The metastatic biomarker profiles of these 4 cases indicate that, of the two primary foci, the focus with the largest tumor size, equal or higher G, and Her2+ status metastasized in 75%, 75% and 50% of cases, respectively. (Table presented)
Conclusion(s): Our data show that about 90% of patients with MBC can be expected to have the same biomarker profile in all tumor foci. However, 4.6% of cases with multiple biomarker profiles showed discordant profiles despite similar morphology and G. It can be concluded then, that a similar percentage of cases on which only one set of biomarkers were performed, actually had discordant profiles. These patients may have had additional treatment options than otherwise indicated by the single biomarker profile. In addition, our data shows that tumor G, size and biomarker profile all contribute to predicting tumor behavior, supporting the updated AJCC 8th edition

Multifocal breast cancer (MFBC), ductal type, has been hypothesized to arise by one of two mechanisms: either through intramammary/intralymphatic spread from a single index tumor (MBC-1), or as multiple independent tumors with each focus carrying its corresponding ductal carcinoma in-situ (MBC-2). In order to improve our understanding of MFBC pathogenesis, we employed laser capture microdissection coupled with whole-exome sequencing to study clonal origin in MFBC. We selected three cases of MBC-1 (C1 to C3) and MBC-2 (C4 to C6) and analyzed three foci from each case. MBC-1 cases were histologically similar and showed a strong predilection for satellite foci, vascular invasion and nodal metastasis when compared to MBC-2. Our bioinformatics approach provided strong evidence for clonal relationships in MBC-1, as demonstrated by distinct clusters of genes conserved across all tumor foci. Conversely, no gene clusters were shared across all the foci in MBC-2, suggesting multiple independent tumors. These findings provide further support for the two distinct pathogenetic mechanisms in MFBC.

Amyloidosis is characterized by extracellular deposition of insoluble protein fibrils in a beta-pleated sheet configuration. Breast amyloidosis is a rare entity which has previously been reported to present with localized involvement, or as a late manifestation of systemic amyloidosis. However, descriptions of the clinicopathologic features of localized breast amyloidosis remain limited. A retrospective search for breast amyloidosis diagnosed at our institution yielded 10 cases of breast amyloidosis. All patients were female, with a mean age of 69. Median follow-up for survival or progression was 13 months. Indications for breast or axilla biopsy included mammographic calcifications, mass, and axillary lymphadenopathy. Amyloid showed positive staining with Congo red in all cases, and amyloid typing revealed light chain lambda in 3 cases, amyloid transthyretin in 2 cases, light chain kappa in 1 case, and iatrogenic insulin-derived amyloidosis in 1 case. Amyloid occurred within axillary lymph nodes and alongside both benign and neoplastic breast tissue, including atypical ductal hyperplasia, lobular carcinoma in situ and ductal carcinoma in situ. Most cases were associated with predisposing clinical conditions, including autoimmune disease in 4 cases, B cell lymphomas in 2 cases, and diabetes mellitus treated with insulin in 1 case. In contrast to previously published case series, no patient had clinical evidence of systemic amyloidosis. Amyloidosis of the breast should be considered in the differential diagnosis of all mammographic calcifications and masses of the breast or axilla. When recognized correctly on biopsy, the diagnosis of amyloidosis can not only prevent further unnecessary surgical interventions due to radiology-pathology discordance, but initiate the necessary amyloidosis work-up. Although rare, an awareness of the clinicopathologic characteristics of this easily overlooked entity is of great importance for every practicing pathologist reviewing breast biopsies.

BACKGROUND:Growing evidence suggests that the tumor immune microenvironment influences breast cancer development and prognosis. Density of tumor-infiltrating lymphocytes (TILs) within invasive breast cancer is correlated with response to therapy, especially in triple-negative disease. The clinical relevance and outcomes of TILs within ductal carcinoma in situ (DCIS) are less understood. METHODS:Our institutional database of 668 patients with pure DCIS from 2010 to 2018 was queried. TILs were evaluated by International TILs Working Group guidelines. Percentage of TILs was assessed from the densest focus (hotspot) in one high-power field of stroma touching the basement membrane. Statistical methods included cluster analyses (to define sparse versus dense TILs), logistic, and Cox regression models. RESULTS:Sixty-nine patients with DCIS and TILs were evaluated, of whom 54 (78%) were treated by breast-conserving surgery. Thirteen (19%) patients had ipsilateral recurrence. Each recurrence (n = 13) was matched to four controls (n = 56) based on date of surgery. Median follow-up was 6.7 years. TILs were defined as sparse (< 45%) or dense (≥ 45%). Dense TILs were associated with younger age (p = 0.045), larger tumor size (p < 0.001), high nuclear grade (p = 0.010), comedo histology (p = 0.033), necrosis (p = 0.027), estrogen receptor (ER) negativity (p = 0.037), and ipsilateral recurrence (p = 0.001). Nine patients with dense TILs had mean time to recurrence of 73.5 months compared with four patients with sparse TILs with mean time to recurrence of 97.9 months (p = 0.003). CONCLUSIONS:Dense TILs were significantly associated with age, tumor size, nuclear grade, comedo histology, necrosis, and ER status and was a significant predictor of recurrence in patients with pure DCIS.