Microenvironment-induced restoration of cohesive growth associated with focal activation of P-cadherin expression in lobular breast carcinoma metastatic to the colon.

Invasive lobular carcinoma (ILC) is a special breast cancer type characterized by noncohesive growth and E-cadherin loss. Focal activation of P-cadherin expression in tumor cells that are deficient for E-cadherin occurs in a subset of ILCs. Switching from an E-cadherin deficient to P-cadherin proficient status (EPS) partially restores cell-cell adhesion leading to the formation of cohesive tubular elements. It is unknown what conditions control EPS. Here, we report on EPS in ILC metastases in the large bowel. We reviewed endoscopic colon biopsies and colectomy specimens from a 52-year-old female (index patient) and of 18 additional patients (reference series) diagnosed with metastatic ILC in the colon. EPS was assessed by immunohistochemistry for E-cadherin and P-cadherin. CDH1/E-cadherin mutations were determined by next-generation sequencing. The index patient's colectomy showed transmural metastatic ILC harboring a CDH1/E-cadherin p.Q610* mutation. ILC cells displayed different growth patterns in different anatomic layers of the colon wall. In the tunica muscularis propria and the tela submucosa, ILC cells featured noncohesive growth and were E-cadherin-negative and P-cadherin-negative. However, ILC cells invading the mucosa formed cohesive tubular elements in the intercryptal stroma of the lamina propria mucosae. Inter-cryptal ILC cells switched to a P-cadherin-positive phenotype in this microenvironmental niche. In the reference series, colon mucosa infiltration was evident in 13 of 18 patients, one of which showed intercryptal EPS and conversion to cohesive growth as described in the index patient. The large bowel is a common metastatic site in ILC. In endoscopic colon biopsies, the typical noncohesive growth of ILC may be concealed by microenvironment-induced EPS and conversion to cohesive growth.

Immunohistochemical marker profiles for the differentiation of collagenous spherulosis from adenoid cystic carcinoma of the breast.

Collagenous spherulosis (CS) is a rare breast lesion of unknown histogenesis. Adenoid cystic carcinoma (ACC) is a rare basal-like breast carcinoma with low histological grade. CS is a benign lesion but resembles ACC. Both lesions show a similar histomorphology and feature bilineage differentiation. This study compared immunohistochemical markers in CS and ACC. We compiled n = 13 CS cases and n = 18 mammary ACCs. Fourteen marker proteins (ER, PR, HER2, GATA3, CK7, E-cadherin, CD117, CK5/14, p40, p63, SMA, CD10, calponin, P-cadherin) were evaluated by immunohistochemistry (IHC). MYB rearrangement, a common alteration in ACC, was assessed by fluorescence in situ hybridization. Patient age ranged between 40-60 years for CS lesions and 30-90 years for ACCs. 7/13 (54%) CS cases harbored a lobular carcinoma in situ (LCIS) in the luminal component. One CS/LCIS lesion occurred in a carrier of a pathogenic germline variant in CDH1/E-cadherin. MYB rearrangement was detected in 0/11 (0%) CS and 6/16 (37%) ACC cases (P = 0.054). CS was associated with expression of ER in the luminal component (P < 0.001), E-cadherin loss in the luminal component (P = 0.045), and expression of CD10 and calponin in the basal component (P < 0.001). Furthermore, CS was associated with GATA3 expression in the luminal component (12/13 [92%] versus 5/18 [27%], P < 0.001). In summary, IHC for GATA3 and E-cadherin may contribute to the differential diagnosis between CS and ACC, although these markers are not exclusively expressed in either lesion. Histologic evaluation has to take into account that CS is frequently colonized by LCIS, requiring thorough correlation of histomorphology and immunohistochemical features.

Optimized Modeling of Metastatic Triple-Negative Invasive Lobular Breast Carcinoma.

Invasive lobular carcinoma (ILC) is a common breast cancer subtype that is often diagnosed at advanced stages and causes significant morbidity. Late-onset secondary tumor recurrence affects up to 30% of ILC patients, posing a therapeutic challenge if resistance to systemic therapy develops. Nonetheless, there is a lack of preclinical models for ILC, and the current models do not accurately reproduce the complete range of the disease. We created clinically relevant metastatic xenografts to address this gap by grafting the triple-negative IPH-926 cell line into mouse milk ducts. The resulting intraductal xenografts accurately recapitulate lobular carcinoma in situ (LCIS), invasive lobular carcinoma, and metastatic ILC in relevant organs. Using a panel of 15 clinical markers, we characterized the intratumoral heterogeneity of primary and metastatic lesions. Interestingly, intraductal IPH-926 xenografts express low but actionable HER2 and are not dependent on supplementation with the ovarian hormone estradiol for their growth. This model provides a valuable tool to test the efficiency of potential new ILC therapeutics, and it may help detect vulnerabilities within ILC that can be exploited for therapeutic targeting.

p53 Expression in Luminal Breast Cancer Correlates With TP53 Mutation and Primary Endocrine Resistance.

TP53 mutation is associated with primary endocrine resistance in luminal breast cancer (BC). Nuclear accumulation of p53, as determined by immunohistochemistry (IHC), is a surrogate marker for TP53 mutation. The immunohistochemical p53 index that defines a p53-positive status is not well established. This study determined the optimal p53 index cutoff to identify luminal BCs harboring TP53 mutations. In total, 364 luminal BCs from the West German Study Group ADAPT trial (NCT01779206) were analyzed for TP53 mutations by next-generation sequencing and for p53 expression by IHC (DO-7 antibody). P53 indices were determined by automated image analysis. All tumors were from patients treated with short-term preoperative endocrine therapy (pET; tamoxifen or aromatase inhibitor) before tumor resection. IHC evaluation included needle biopsies before therapy (baseline) and resections specimens after therapy (post-pET). Optimal p53 index cutoffs were defined with Youden statistics. TP53 mutations were detected in 16.3% of BC cases. The median p53 indices were significantly higher in TP53-mutated BCs compared to BCs harboring wild-type TP53 (baseline: 47.0% vs 6.4%, P < .001; post-pET: 50.1% vs 1.1%, P < .001). Short-term pET decreased p53 indices in BCs harboring wild-type TP53 (P < .001) but not in TP53-mutated BCs (P = .102). For baseline biopsies, the optimal p53 index cutoff was ≥34.6% (specificity 0.92, sensitivity 0.63, Youden index 0.54, accuracy: 0.87). For post-pET specimens, the optimal cutoff was ≥25.3% (specificity 0.95, sensitivity 0.65, Youden index 0.60, accuracy: 0.90). Using these cutoffs to define the p53 status, p53-positive BCs were >2-fold more common in pET nonresponders compared to pET responders (baseline: 37/162, 22.8% vs 18/162, 11.1%, P = .007; post-pET: 36/179, 20.1% vs 16/179, 8.9%, P = .004). In summary, IHC for p53 identifies TP53-mutated luminal BCs with high specificity and accuracy. Optimal cutoffs are ≥35% and ≥25% for treatment-naïve and endocrine-pretreated patients, respectively.

ERBB2 mutation is associated with sustained tumor cell proliferation after short-term preoperative endocrine therapy in early lobular breast cancer.

Invasive lobular breast cancer (ILC) is a special breast cancer (BC) subtype and is mostly hormone receptor (HR)-positive and ERBB2 non-amplified. Endocrine therapy restrains tumor proliferation and is the mainstay of lobular BC treatment. Mutation of ERBB2 has been associated with recurrent ILC. However, it is unknown whether ERBB2 mutation impacts on the otherwise exquisite responsiveness of early ILC to endocrine therapy. We have recently profiled n = 622 HR-positive early BCs from the ADAPT trial for mutations in candidate genes involved in endocrine resistance, including ERBB2. All patients were treated with short-term preoperative endocrine therapy (pET, tamoxifen or aromatase inhibitors) before tumor resection. Tumor proliferation after endocrine therapy (post-pET Ki67 index) was determined prospectively by standardized central pathology assessment supported by computer-assisted image analysis. Sustained or suppressed proliferation were defined as post-pET Ki67 ≥10% or <10%. Here, we report a subgroup analysis pertaining to ILCs in this cohort. ILCs accounted for 179/622 (28.8%) cases. ILCs were enriched in mutations in CDH1 (124/179, 69.3%, P < 0.0001) and ERBB2 (14/179, 7.8%, P < 0.0001), but showed fewer mutations in TP53 (7/179, 3.9%, P = 0.0048) and GATA3 (11/179, 6.1%, P < 0.0001). Considering all BCs irrespective of subtypes, ERBB2 mutation was not associated with proliferation. In ILCs, however, ERBB2 mutations were 3.5-fold more common in cases with sustained post-pET proliferation compared to cases with suppressed post-pET proliferation (10/75, 13.3% versus 4/104, 3.8%, P = 0.0248). Moreover, ERBB2 mutation was associated with high Oncotype DX recurrence scores (P = 0.0087). In summary, our findings support that ERBB2 mutation influences endocrine responsiveness in early lobular BC.

Inter-observer agreement for the histological diagnosis of invasive lobular breast carcinoma.

Invasive lobular breast carcinoma (ILC) is the second most common breast carcinoma (BC) subtype and is mainly driven by loss of E-cadherin expression. Correct classification of BC as ILC is important for patient treatment. This study assessed the degree of agreement among pathologists for the diagnosis of ILC. Two sets of hormone receptor (HR)-positive/HER2-negative BCs were independently reviewed by participating pathologists. In set A (61 cases), participants were provided with hematoxylin/eosin (HE)-stained sections. In set B (62 cases), participants were provided with HE-stained sections and E-cadherin immunohistochemistry (IHC). Tumor characteristics were balanced. Participants classified specimens as non-lobular BC versus mixed BC versus ILC. Pairwise inter-observer agreement and agreement with a pre-defined reference diagnosis were determined with Cohen's kappa statistics. Subtype calls were correlated with molecular features, including CDH1/E-cadherin mutation status. Thirty-five pathologists completed both sets, providing 4,305 subtype calls. Pairwise inter-observer agreement was moderate in set A (median κ = 0.58, interquartile range [IQR]: 0.48-0.66) and substantial in set B (median κ = 0.75, IQR: 0.56-0.86, p < 0.001). Agreement with the reference diagnosis was substantial in set A (median κ = 0.67, IQR: 0.57-0.75) and almost perfect in set B (median κ = 0.86, IQR: 0.73-0.93, p < 0.001). The median frequency of CDH1/E-cadherin mutations in specimens classified as ILC was 65% in set A (IQR: 56-72%) and 73% in set B (IQR: 65-75%, p < 0.001). Cases with variable subtype calls included E-cadherin-positive ILCs harboring CDH1 missense mutations, and E-cadherin-negative ILCs with tubular elements and focal P-cadherin expression. ILCs with trabecular growth pattern were often misclassified as non-lobular BC in set A but not in set B. In conclusion, subtyping of BC as ILC achieves almost perfect agreement with a pre-defined reference standard, if assessment is supported by E-cadherin IHC. CDH1 missense mutations associated with preserved E-cadherin protein expression, E- to P-cadherin switching in ILC with tubular elements, and trabecular ILC were identified as potential sources of discordant classification.

TP53 mutations are associated with primary endocrine resistance in luminal early breast cancer.

BACKGROUND: Whereas the genomic landscape of endocrine-resistant breast cancer has been intensely characterized in previously treated cases with local or distant recurrence, comparably little is known about genomic alterations conveying primary non-responsiveness to endocrine treatment in luminal early breast cancer. METHODS: In this study, 622 estrogen receptor-expressing breast cancer cases treated with short-term preoperative endocrine therapy (pET) from the WSG-ADAPT trial (NCT01779206) were analyzed for genetic alterations associated with impaired endocrine proliferative response (EPR) to 3-week pET with tamoxifen or aromatase inhibitors. EPR was categorized as optimal (post-pET Ki67 <10%) versus slightly, moderately, and severely impaired (post-pET Ki67 10%-19%, 20%-34%, and ≥35%, respectively). Recently described gene mutations frequently found in previously treated advanced breast cancer were analyzed (ARID1A, BRAF, ERBB2, ESR1, GATA3, HRAS, KRAS, NRAS, PIK3CA, and TP53) by next-generation sequencing. Amplifications of CCND1, FGFR1, ERBB2, and PAK1 were determined by digital PCR or fluorescence in situ hybridization. RESULTS: ERBB2 amplification (p = 0.0015) and mutations of TP53 (p < 0.0001) were significantly associated with impaired EPR. Impaired EPR in TP53-mutated breast cancer cases was independent from the Oncotype DX Recurrence Score group and was seen both with tamoxifen- and aromatase inhibitor-based pET (p = 0.0005 each). CONCLUSION: We conclude that impaired EPR to pET is suitable to identify cases with primary endocrine resistance in early luminal breast cancer and that TP53-mutated luminal cancers might not be sufficiently treated by endocrine therapy alone.

Chromosome 2q gain and epigenetic silencing of GATA3 in microglandular adenosis of the breast.

Microglandular adenosis (MGA) represents a rare neoplasm of the mammary gland, which in a subset of cases may be associated with triple-negative breast cancer (BC). The biology of MGA is poorly understood. In this study, eight MGA cases (n = 4 with and n = 4 without associated BC) were subjected to a comprehensive characterization using immunohistochemistry, genome-wide DNA copy number (CN) profiling, fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and DNA methylation profiling using 850 K arrays and bisulfite pyrosequencing. Median patient age was 61 years (range 57-76 years). MGA lesions were estrogen receptor (ER)-negative, progesterone receptor-negative, HER2-negative, and S100-positive. DNA CN alterations (CNAs) were complex or limited to few gains and losses. CN gain on chromosome 2q was the most common CNA and was validated by FISH in five of eight cases. NGS demonstrated an average of two mutations per case (range 0-5) affecting 10 different genes (ARID1A, ATM, CTNNB1, FBXW7, FGFR2, MET, PIK3CA, PMS2, PTEN, and TP53). CNAs and mutations were similar in MGA and adjacent BC, indicating clonal relatedness. DNA methylation profiling identified aberrant hypermethylation of CpG sites within GATA3, a key transcription factor required for luminal differentiation. Immunohistochemistry showed regular GATA3 protein expression in the normal mammary epithelium and in ER-positive BC. Conversely, GATA3 was reduced or lost in all MGA cases tested (8/8). In conclusion, MGA is characterized by common CN gain on chromosome 2q and loss of GATA3. Epigenetic inactivation of GATA3 may provide a new clue to the peculiar biology of this rare neoplasia.

Differential impact of prognostic parameters in hormone receptor-positive lobular breast cancer.

BACKGROUND: Invasive lobular breast cancer (BC) is the second most common BC subtype. Prognostic parameters (tumor classification, lymph node status, histologic grade, Oncotype DX recurrence score [RS], progesterone receptor status, and Ki67 index) were retrospectively studied in a large, prospective clinical trial encompassing 2585 patients who had hormone receptor-positive early BC (the West German Study Group PlanB trial). METHODS: BCs were centrally reviewed and classified as lobular (n = 353; 14%) or nonlobular (n = 2232; 86%). The median follow-up was 60 months. Five-year disease-free survival (DFS) estimates were obtained using the Kaplan-Meier method. Prognostic parameters were evaluated using Cox proportional hazard models. RESULTS: Lobular BC was associated with higher tumor classification, higher lymph node status, lower histologic grade, lower Ki67 index, and low or intermediate RS. The prevalence of high RS (RS range, 26-100) was 3-fold lower in patients who had lobular BC compared with those who had nonlobular BC (8% vs 24%; P < .001). However, 5-year DFS estimates for lobular and nonlobular BC were similar (92.1% and 92.3%, respectively; P = .673). In multivariate analyses, prognostic parameters for DFS in lobular BC included grade 3 (hazard ratio, 5.06; 95% CI, 1.91-13.39) and a pathologic lymph node status (pN) of pN3 (hazard ratio, 12.16; 95% CI, 3.87-38.24), but not RS. By contrast, prognostic parameters in nonlobular BC included grade 3 (hazard ratio, 1.65; 95% CI, 1.11-2.44), pN3 (hazard ratio, 3.68; 95% CI, 1.60-8.46), and high RS (hazard ratio, 2.49; 95% CI, 1.69-3.68). CONCLUSIONS: Lobular BC is associated with low and intermediate RS, although 5-year DFS is similar to that of nonlobular BC. The effect of the RS in lobular BC appears to be distinct from that in nonlobular BC. For risk assessment, the RS needs to be complemented by clinicopathologic parameters for therapy decision making.

E-cadherin to P-cadherin switching in lobular breast cancer with tubular elements.

Loss of E-cadherin expression due to mutation of the CDH1 gene is a characteristic feature of invasive lobular breast cancer (ILBC). Beta-catenin, which binds to the cytoplasmic domain of E-cadherin, is simultaneously downregulated, reflecting disassembly of adherens junctions (AJs) and loss of cell adhesion. E-cadherin to P-cadherin expression switching can rescue AJs and cell adhesion. However, P-cadherin has not been implicated in ILBC, so far. We aimed to characterize 13 ILBCs with exceptional histomorphology, which we termed ILBCs with tubular elements. The CDH1 mutational status was determined by next generation sequencing and whole-genome copy number (CN) profiling. Expression of cadherins was assessed by immunohistochemistry. ILBCs with tubular elements were ER-positive (13/13) and HER2-negative (13/13) and harbored deleterious CDH1 mutations (11/13) accompanied by loss of heterozygosity due to deletion of chromosome 16q22.1 (9/11). E-cadherin expression was lost or reduced in noncohesive tumor cells and in admixed tubular elements (13/13). Beta-catenin expression was lost in noncohesive tumor cells, but was retained in tubular elements (11/13), indicating focal rescue of AJ formation. N-cadherin and R-cadherin were always negative (0/13). Strikingly, P-cadherin was commonly positive (12/13) and immunoreactivity was accentuated in tubular elements. Adjacent lobular carcinoma in situ (LCIS) was always P-cadherin-negative (0/7). In a reference cohort of LCIS specimens, P-cadherin was constantly not expressed (0/25). In a reference cohort of invasive mammary carcinomas, P-cadherin-positive cases (36/268, 13%) were associated with triple-negative nonlobular breast cancer (P < 0.001). Compared with ILBCs from the reference cohort, P-cadherin expression was more common in ILBCs with tubular elements (12/13 versus 7/84, P < 0.001). In summary, E-cadherin to P-cadherin switching occurs in a subset of ILBCs. P-cadherin is the molecular determinant of a mixed-appearing histomorphology in ILBCs with tubular elements.

ERBB2 mutation frequency in lobular breast cancer with pleomorphic histology or high-risk characteristics by molecular expression profiling.

HER2-positive breast cancer is defined by amplification or overexpression of the HER2/ERBB2 oncogene and accounts for about 15% of breast cancer cases. Somatic mutation of ERBB2 is an alternative mechanism, by which activation of HER2 signaling can occur. ERBB2 mutation has been associated with invasive lobular breast cancer (ILBC). This study investigates the frequency and phenotype of ILBC harboring mutated ERBB2. The ERBB2 mutation status was determined by next generation sequencing and/or pyrosequencing in n = 106 ILBCs, including n = 86 primary or locally recurrent tumors and n = 20 metastases from visceral organs, soft tissue, or skin. Immunohistochemical characteristics were determined using tissue microarrays. This series was enriched for ILBCs with pleomorphic histology and/or high-risk expression profiles (Oncotype DX, recurrence score RS > 25). Nearly all specimens were E-cadherin-negative (99%), estrogen receptor (ER)-positive (92%), and lacked ERBB2 overexpression (96%). ERBB2 mutations (p.V777L, p.L755S, p.S310F) were identified in 5/106 (5%) cases. ERBB2-mutated cases included 2/86 (2%) primary tumors and 3/20 (15%) metastases (P = 0.045). ERBB2-mutated cases were associated with loss of ER (2/7, 29%, P = 0.035) and histological grade 3 (4/34, 12%, P = 0.023), but not with solid growth (3/31, 10%, P = 0.148) or pleomorphic histology (2/27, 7%, P = 0.599). No ERBB2 mutation was detected in ILBCs with RS > 25 (0/22, 0%). In 10 patients with multiple matched specimens (n = 25), the ERBB2 mutational status was always concordant. In summary, a small subset of ILBCs harbors potentially actionable ERBB2 mutations. In ERBB2-mutated ILBCs, no association with pleomorphic histology was found.

Lobular carcinoma in situ and invasive lobular breast cancer are characterized by enhanced expression of transcription factor AP-2ß.

Transcription factor AP-2ß (TFAP2B) regulates embryonic organ development and is overexpressed in alveolar rhabdomyosarcoma, a rare childhood malignancy. Gene expression profiling has implicated AP-2ß in breast cancer (BC). This study characterizes AP-2ß expression in the mammary gland and in BC. AP-2ß protein expression was assessed in the normal mammary gland epithelium, in various reactive, metaplastic and pre-invasive neoplastic lesions and in two clinical BC cohorts comprising >2000 patients. BCs from various genetically engineered mouse (GEM) models were also evaluated. Human BC cell lines served as functional models to study siRNA-mediated inhibition of AP-2ß. The normal mammary gland epithelium showed scattered AP-2ß-positive cells in the luminal cell layer. Various reactive and pre-invasive neoplastic lesions, including apocrine metaplasia, usual ductal hyperplasia and lobular carcinoma in situ (LCIS) showed enhanced AP-2ß expression. Cases of ductal carcinoma in situ (DCIS) were more often AP-2ß-negative (P<0.001). In invasive BC cohorts, AP-2ß-positivity was associated with the lobular BC subtype (P<0.001), loss of E-cadherin (P<0.001), a positive estrogen receptor (ER) status (P<0.001), low Ki67 (P<0.001), low/intermediate Oncotype DX recurrence scores (P<0.001), and prolonged event-free survival (P=0.003). BCs from GEM models were all AP-2ß-negative. In human BC cell lines, AP-2ß expression was independent from ER-signaling. SiRNA-mediated inhibition of AP-2ß diminished proliferation of lobular BC cell lines in vitro. In summary, AP-2ß is a new mammary epithelial differentiation marker. Its expression is preferentially retained and enhanced in LCIS and invasive lobular BC and has prognostic implications. Our findings indicate that AP-2ß controls tumor cell proliferation in this slow-growing BC subtype.

The region-of-interest size impacts on Ki67 quantification by computer-assisted image analysis in breast cancer.

Therapeutic decision-making in breast cancer depends on histopathologic biomarkers and is influenced by the Ki67 proliferation index. Computer-assisted image analysis (CAIA) promises to improve Ki67 quantification. Several commercial applications have been developed for semiautomated CAIA-based Ki67 quantification, many of which rely on measurements in user-defined regions of interest (ROIs). Because of intratumoral proliferative heterogeneity, definition of the ROI is an important step in the analytical procedure. This study explores the ROI size impacts on Ki67 quantification. Whole-slide sections of 100 breast cancers were immunostained with the anti-Ki67 antibody 30-9 and were analyzed on the iScan Coreo digital pathology platform using a Food and Drug Administration-cleared Ki67 quantification software version v5.3 (Virtuoso; Ventana, Tucson, TX). For each case, the Ki67 labeling index (LI) was determined in multiple ROIs of gradually increasing size centered around a high-proliferation area. The spatial Ki67 decline was modeled with nonlinear regression. Depending on the ROI size, the median Ki67 LI varied between 55% and 15%. The proportion of tumors classified as Ki67 low according to the St Gallen 2013/2015 cutoff increased from 2% to 56%, as the ROI size increased from 50 to 10,000 cells captured. The interrater reliability of conventional Ki67 assessment versus CAIA-based Ki67 quantification was also dependent on the ROI size and varied between slight and almost perfect agreement (Cohen κ = 0.06-0.85). In conclusion, the ROI size is a critically important parameter for semiautomated Ki67 quantification by CAIA. Ki67 LIs determined on platforms like iScan Coreo/Virtuoso require an ROI size adjustment, for which we offer a downloadable data transformation tool.

High frequency of lobular breast cancer in distant metastases to the orbit.

Metastasis to the periocular soft tissue of the orbit is a rare manifestation of metastatic cancer. Infiltrating lobular breast cancer (ILBC) is a special breast cancer subtype, which accounts for 10-15% of all mammary carcinomas and for ~1% of all malignancies. Here, we report on a high frequency of lobular breast cancer in patients with orbital metastases identified in an original series of metastatic tumor specimens and by a systematic literature review. A series of 14 orbital metastases was compiled from formalin-fixed paraffin-embedded archival tissues. All cases were subjected to histological re-review and detailed immunophenotypical characterization. In addition, we performed a meta-analysis of 68 previously published case reports describing orbital metastases, with special reference to breast cancer subtypes. Based on clinical history, histomorphology, immunophenotype, and/or comparison with matched primary tumors, orbital metastases were derived from breast cancer in 8/14 cases, seven of which were classified as metastatic lobular breast cancer. Other entities included non-small cell lung cancer (4/14), infiltrating ductal breast cancer (1/14), prostate cancer (1/14) and adenocarcinoma of the esophagus (1/14). In line with this original series of orbital metastases, lobular breast cancer was the most common malignancy in 72 patients with orbital metastases described in 68 independent case reports. In conclusion, lobular breast cancer represents the cancer subtype with the highest prevalence among orbital metastases. The high frequency of ILBC in orbital metastases illustrates the special metastatic behavior of this tumor entity and may have implications for the understanding of the organotropism of metastatic lobular breast cancer.

Is upregulation of BCL2 a determinant of tumor development driven by inactivation of CDH1/E-cadherin?

Inactivation of CDH1, encoding E-cadherin, promotes cancer initiation and progression. According to a newly proposed molecular mechanism, loss of E-cadherin triggers an upregulation of the anti-apoptotic oncoprotein BCL2. Conversely, reconstitution of E-cadherin counteracts overexpression of BCL2. This reciprocal regulation is thought to be critical for early tumor development. We determined the relevance of this new concept in human infiltrating lobular breast cancer (ILBC), the prime tumor entity associated with CDH1 inactivation. BCL2 expression was examined in human ILBC cell lines (IPH-926, MDA-MB-134, SUM-44) harboring deleterious CDH1 mutations. To test for an intact regulatory axis between E-cadherin and BCL2, wild-type E-cadherin was reconstituted in ILBC cells by ectopic expression. Moreover, BCL2 and E-cadherin were evaluated in primary invasive breast cancers and in synchronous lobular carcinomas in situ (LCIS). MDA-MB-134 and IPH-926 showed little or no BCL2 expression, while SUM-44 ILBC cells were BCL2-positive. Reconstitution of E-cadherin failed to impact on BCL2 expression in all cell lines tested. Primary ILBCs were almost uniformly E-cadherin-negative (97%) and were frequently BCL2-negative (46%). When compared with an appropriate control group, ILBCs showed a trend towards an increased frequency of BCL2-negative cases (P = 0.064). In terminal duct-lobular units affected by LCIS, the E-cadherin-negative neoplastic component showed a similar or a reduced BCL2-immunoreactivity, when compared with the adjacent epithelium. In conclusion, upregulation of BCL2 is not involved in lobular breast carcinogenesis and is unlikely to represent an important determinant of tumor development driven by CDH1 inactivation.

Oncogenic PIK3CA mutations in lobular breast cancer progression.

Infiltrating lobular breast cancer (ILBC) is a tumor-biologically distinct breast cancer subtype. A high frequency of oncogenic PIK3CA mutations has been reported in ILBC, which may allow for targeted therapy with newly developed PI3K inhibitors. This is of particular clinical relevance for ILBC patients, who have failed to respond to current treatment regimes and suffer from tumor recurrence or dissemination. In anticipation of this therapeutic strategy, we investigated PIK3CA mutations in ILBC with special reference to late stage tumor progression. A total of 88 ILBCs from 73 patients, including primary tumors (PTs, n = 43), ipsilateral locally recurrent tumors (LRTs, n = 15), and distant organ metastases (DOMs, n = 30), were compiled on tissue microarrays. Established ILBC marker proteins were evaluated by immunohistochemistry and PIK3CA hot spot mutations in exons 9 and 20 by direct sequencing. Matched PT/LRT, PT/DOM, and DOM/DOM cases were characterized on a patient-by-patient basis. Following correction for redundant patient representations, mutation frequencies were compared in PTs versus LRTs or DOMs. Nearly all specimens were E-cadherin-negative (99%), estrogen receptor (ER)-positive (91%), and lacked basal epithelial markers (100%), demonstrating correct ILBC classification. PIK3CA mutations were detected in 32/88 (36%) specimens. The mutation rate was similar in PTs (33%) and DOMs (26%, P = 0.769), but approximately two-fold increased in LRTs (69%, P = 0.022). Consistently, matched PT/LRT and LRT/DOM cases showed additional PIK3CA mutations in LRTs. Intriguingly, these findings imply that PIK3CA mutations are positively selected for during ILBC progression to local recurrence but not distant metastasis, which may have clinical implications for PI3K inhibitor-based therapy.

IPH-926 lobular breast cancer cells harbor a p53 mutant with temperature-sensitive functional activity and allow for profiling of p53-responsive genes.

Profiling of p53-responsive genes has been carried out in different cellular models, most of which involved genetic modifications or cytotoxic stimulation. We report on the utilization of IPH-926 human lobular breast cancer cells for the profiling of p53-responsive genes using a novel approach without such modifications. We discovered that IPH-926 cells harbor a homozygous TP53 missense mutation encoding for a rare p53 mutant (E285K) with temperature-sensitive (ts) loss of function characteristics. This mutation had evolved as a late, secondary genetic event during the natural clonal evolution of the corresponding lobular carcinoma. In vitro temperature shifts reconstituted endogenous wild-type p53 activity in IPH-926, as evidenced by induction of p21(Waf1). Transcriptional alterations associated with restored p53 function were profiled using Affymetrix microarrays and a new strategy to gate out non-specific temperature effects. At the P=0.0005 significance level, 60 genes were differentially expressed following reconstitution of p53 activity. These genes included CDKN1A, MDM2 and PHLDA3, a recently described p53-inducible inhibitor of AKT. Similar transcriptional alterations were observed upon reconstitution of p53 activity in BT-474 cells, which also harbor ts-p53 E285K, and in ASPC1 cells transduced with ts-p53 A138V. Consistent with these models, low PHLDA3 expression was associated with nuclear p53 accumulation, indicative of deleterious TP53 mutations, in primary breast cancers. From a molecular point of view, IPH-926 thus provides a new tool to study transcriptional programs controlled by p53. From a tumor pathology perspective, IPH-926 also provides the first direct evidence of a p53-related clonal evolutionary pathway in lobular breast cancer progression.

Identification of differentially expressed microRNAs in human male breast cancer.

BACKGROUND: The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases. METHODS: The expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods.Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer. RESULTS: Unsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer. CONCLUSIONS: Male breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.

Down-regulation of the fetal stem cell factor SOX17 by H33342: a mechanism responsible for differential gene expression in breast cancer side population cells.

Human solid tumors contain rare cancer side population (SP) cells, which expel the fluorescent dye Hoechst 33342 (H33342) and display cancer stem cell characteristics. Transcriptional profiling of cancer SP cells isolated by H33342 fluorescence analysis is a newly emerging approach to discover cancer stem cell markers and aberrant differentiation pathways. Using Affymetrix expression microarrays and quantitative reverse transcription-PCR, we investigated differential gene expression between SP and non-SP (NSP) cells isolated from human mammary carcinoma cell lines. A total of 136 genes were up-regulated in breast cancer SP relative to NSP cells, one of which was the fetal stem cell factor and Wnt/beta-catenin signaling pathway target SOX17. Strikingly, we discovered that SOX17 was down-regulated by H33342 in a dose-dependent manner. In SP cells, which expel H33342, down-regulation of SOX17 was less pronounced than in NSP cells, which retain H33342. As a result of this, SOX17 displayed a 10-20-fold overexpression in cancer SP relative to NSP cells. Similar results were obtained for further stemness-related genes, namely EPC1 and SPRY1. These findings establish a previously unidentified gene-regulatory impact of H33342 as a novel mechanism responsible for differential gene expression in cancer SP cells. This has significant implications for the future interpretation of cancer SP cells.

Expression of KAI1/CD82 in distant metastases from estrogen receptor-negative breast cancer.

The tetraspanin protein superfamily member KAI1 suppresses tumor growth and metastasis in animal models and is downregulated in various human malignancies. In breast cancer, KAI1 is preferentially lost in estrogen receptor (ER)-positive tumors. Interestingly, most ER-negative primary breast cancers retain KAI1 expression. This study aimed to evaluate whether or not KAI1 is downregulated during progression to metastasis of these carcinomas. Expression of KAI1, ER, progesterone receptor, c-ErbB2, and Ki67 was analyzed in tissue microarrays comprising a large collection of distant organ metastases from human breast cancers (n = 92) by immunohistochemistry. Results were compared with a previously characterized set of primary breast tumors (n = 209). Immunoreactivity for KAI1 was observed in one-third of the metastases and was associated with lack of ER expression (P = 0.005). The high frequency of KAI1-positive cases in ER-negative primary tumors was maintained in ER-negative metastases. Expression of KAI1 was also observed in MDA-MB-468 and SK-BR-3, two ER-negative breast cancer cell lines of metastatic origin. Moreover, a reanalysis of independent microarray gene expression data indicated maintenance of KAI1 mRNA expression in metastases from ER-negative breast cancers. Furthermore, in a series of matched pairs of mammary carcinomas and metachronous distant metastases, all metastases from KAI1-positive/ER-negative primary tumors were KAI1-positive as well. Collectively, these findings demonstrate that the expression of KAI1 is maintained during progression to metastasis in a large proportion of ER-negative mammary carcinomas. This has significant implications for the use of KAI1 as a clinical marker and the understanding of the metastatic process in human breast cancer.