Targetable alterations in invasive pleomorphic lobular carcinoma of the breast.

BACKGROUND: Invasive pleomorphic lobular carcinoma (PLC) of the breast is a subtype of invasive lobular cancer which compromises approximately 1% of all epithelial breast malignancies and is characterized by higher nuclear pleomorphism and poorer prognosis than classic invasive lobular cancer (ILC). Since PLC is more aggressive than classical ILC, we examined the underlying molecular alterations in this subtype of breast cancer to understand the possible benefit from targeted therapies. METHODS: In this study, we investigate the clinical characteristics and molecular alterations in 16 PLC from our institution. Additionally, we examined the clinical and genomic features in 31 PLC from the Cancer Genome Atlas (TCGA). RESULTS: Overall, our analysis of PLC found that 28% had activating ERBB2 mutations, 21% had ERBB2 amplification, and 49% activating PIK3CA mutations. Among cases from our institution, we found 19% with activating ERBB2 mutations, 25% had ERBB2 amplification, and 38% with activating PIK3CA mutations. In data from TCGA, 32% had activating ERBB2 mutations, 19% had ERBB2 amplification, and 55% had activating PIK3CA mutations. While classic ILC in TCGA had similar percentages of PIK3CA alterations compared to PLC, activating ERBB2 alterations were exceedingly rare, with no activating ERBB2 mutations and only one case with ERBB2 amplification. Interestingly, in further examining TCGA data which included FGFR1 and PTEN, 94% of PLC had alterations in ERBB2, FGFR1, or the PI3K pathway. CONCLUSIONS: Our results show a high frequency of ERBB2 and PIK3CA alterations in PLC and suggest all PLC should be tested for potential therapeutic targeting.

Multiple glomus tumors presenting as an aesthetic abnormality.

UNLABELLED: Glomus tumors are benign soft-tissue neoplasms. Commonly found in the digits, glomus tumors can rarely arise in extradigital locations and may result in misdiagnosis or delay in diagnosis. Plastic surgeons should therefore include glomus tumors in the differential diagnosis of patients who present with painful, red- or blue-colored extradigital lesions. The authors present a rare case of extradigital glomangioma tumors of the right and left thigh in a 35-year-old woman. This case report describes an atypical presentation of multiple glomus tumors and reviews diagnostic and treatment modalities. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Dual radiolabeling as a technique to track nanocarriers: the case of gold nanoparticles.

Gold nanoparticles (AuNPs) have shown great potential for use in nanomedicine and nanotechnologies due to their ease of synthesis and functionalization. However, their apparent biocompatibility and biodistribution is still a matter of intense debate due to the lack of clear safety data. To investigate the biodistribution of AuNPs, monodisperse 14-nm dual-radiolabeled [14C]citrate-coated [198Au]AuNPs were synthesized and their physico-chemical characteristics compared to those of non-radiolabeled AuNPs synthesized by the same method. The dual-radiolabeled AuNPs were administered to rats by oral or intravenous routes. After 24 h, the amounts of Au core and citrate surface coating were quantified using gamma spectroscopy for 198Au and liquid scintillation for the 14C. The Au core and citrate surface coating had different biodistribution profiles in the organs/tissues analyzed, and no oral absorption was observed. We conclude that the different components of the AuNPs system, in this case the Au core and citrate surface coating, did not remain intact, resulting in the different distribution profiles observed. A better understanding of the biodistribution profiles of other surface attachments or cargo of AuNPs in relation to the Au core is required to successfully use AuNPs as drug delivery vehicles.

Carbonic anhydrase 4 and crystallin α-B immunoreactivity may distinguish benign from malignant thyroid nodules in patients with indeterminate thyroid cytology.

BACKGROUND: Thyroid nodules are present in 19%-67% of the population and carry a 5%-10% risk of malignancy. Unfortunately, fine-needle aspiration biopsies are indeterminate in 20%-30% of patients, often necessitating thyroid surgery for diagnosis. Numerous DNA microarray studies including a recently commercialized molecular classifier have helped to better distinguish benign from malignant thyroid nodules. Unfortunately, these assays often require probes for >100 genes, are expensive, and only available at a few laboratories. We sought to validate these DNA microarray assays at the protein level and determine whether simple and widely available immunohistochemical biomarkers alone could distinguish benign from malignant thyroid nodules. METHODS: A tissue microarray (TMA) composed of 26 follicular thyroid carcinomas (FTCs) and 53 follicular adenomas (FAs) from patients with indeterminate thyroid nodules was stained with 17 immunohistochemical biomarkers selected based on prior DNA microarray studies. Antibodies used included galectin 3, growth and differentiation factor 15, protein convertase 2, cluster of differentiation 44 (CD44), glutamic oxaloacetic transaminase 1 (GOT1), trefoil factor 3 (TFF3), Friedreich Ataxia gene (X123), fibroblast growth factor 13 (FGF13), carbonic anhydrase 4 (CA4), crystallin alpha-B (CRYAB), peptidylprolyl isomerase F (PPIF), asparagine synthase (ASNS), sodium channel, non-voltage gated, 1 alpha subunit (SCNN1A), frizzled homolog 1 (FZD1), tyrosine related protein 1 (TYRP1), E cadherin, type 1 (ECAD), and thyroid hormone receptor associated protein 220 (TRAP220). Of note, two of these biomarkers (GOT1 and CD44) are now used in the Afirma classifier assay. We chose to compare specifically FTC versus FA rather than include all histologic categories to create a more uniform immunohistochemical comparison. In addition, we have found that most papillary thyroid carcinoma could often be reasonably distinguished from benign disease by morphological cytology findings alone. RESULTS: Increased immunoreactivity of CRYAB was associated with thyroid malignancy (c-statistic, 0.644; negative predictive value [NPV], 0.90) and loss of immunoreactivity of CA4 was also associated with malignancy (c-statistic, 0.715; NPV, 0.90) in indeterminate thyroid specimens. The combination of CA4 and CRYAB for discriminating FTC from FA resulted in a better c-statistic of 0.75, sensitivity of 0.76, specificity of 0.59, positive predictive value (PPV) of 0.32, and NPV of 0.91. When comparing widely angioinvasive FTC from FA, the resultant c-statistic improved to 0.84, sensitivity of 0.75, specificity of 0.76, PPV of 0.11, and NPV of 0.99. CONCLUSIONS: Loss of CA4 and increase in CRYAB immunoreactivity distinguish FTC from FA in indeterminate thyroid nodules on a thyroid TMA with an NPV of 91%. Further studies in preoperative patient fine needle aspiration (FNAs) are needed to validate these results.

Unexpected encounter with painless endometriosis during abdominoplasty.

UNLABELLED: Subcutaneous endometriomas are cystic lesions that are rarely described. During the course of abdominoplasty surgery, a "surprise" encounter with a cyst containing brown material brought up the possibility that an atypical incarcerated hernia was entered. Dissection revealed no connection between the cyst and the rectus fascia and subsequent pathologic evaluation revealed an endometrioma. This report describes the unusual encounter with a painless endometrioma during abdominoplasty. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Bis(diphenyl-p-tolyl-phosphane-κP)(2-hy-droxy-3,5,7-bromo-cyclo-hepta-2,4,6-trienonato-κ(2)O,O')copper(I).

The Cu(I) atom in the title compund, [Cu(C(7)H(2)Br(3)O(2))(C(19)H(17)P)(2)], is located on a twofold rotation axis; the 3,5,7-tribromo-tropolonate anion coordinates as a bidentate ligand with a bite angle of 76.42 (9)°. An intra-molecular C-H⋯O inter-action occurs. Within the crystal, extensive weak C-H⋯π inter-actions contribute to the herringbone pattern observed in the packing of the mol-ecules.

Loss of the BRCA1-PALB2 interaction accelerates p53-associated tumor development in mice.

The BRCA1-PALB2-BRCA2 axis, or the BRCA pathway, plays key roles in genome stability maintenance and suppression of breast and several other cancers. Due to frequent p53 mutations in human BRCA1 breast cancers and mouse mammary tumors from Brca1, Brca2 and Palb2 conditional knockout models, it is often thought that p53 inactivation accelerates BRCA1/2 and PALB2-associated tumorigenesis. Here, we studied tumor development in mice with a mutation in Palb2 that disengages the PALB2-BRCA1 interaction in different Trp53 backgrounds. Rather than mammary tumors, Palb2 and Trp53 compound mutant mice developed, with greatly reduced latencies, lymphomas and sarcomas that are typically associated with germline Trp53 inactivation. Whole exome sequencing failed to identify any significant differences in genomic features between the same tumor types of Trp53 single mutant and Palb2;Trp53 compound mutant mice. These results suggest that loss of the BRCA pathway accelerates p53-associated tumor development, possibly without altering the fundamental tumorigenic processes.

A genetic variant in a PP2A regulatory subunit encoded by the PPP2R2B gene associates with altered breast cancer risk and recurrence.

A recent candidate gene association study identified a single nucleotide polymorphism (SNP) in the PPP2R2B gene (rs319217, A/G) that manifests allelic differences in the cellular responses to treatment with chemotherapeutic agents (Vazquez et al., Nat Rev Drug Discov 2008;7:979-87). This gene encodes a regulatory subunit of protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases implicated in the negative control of cell growth and division. Given the tumor suppressor activities of PP2A, here we evaluate whether this genetic variant associates with the age of diagnosis and recurrence of breast cancer in women. To investigate the linkage disequilibrium in the vicinity of this SNP, PPP2R2B haplotypes were analyzed using HapMap data for 90 Caucasians. It is found that the A variant of rs319217 tags a haplotype that appears tobe under positive selection in the Caucasian population, implying that this SNP is functional. Subsequently, associations with cellular responses were investigated using data reported by the NCI anticancer drug screen and associations with breast cancer clinical variables were analyzed in a cohort of 819 Caucasian women. The A allele associates with a better response of tumor derived cell lines, lower risk of breast cancer recurrence, later time to recurrence, and later age of diagnosis of breast cancer in Caucasian women. Taken together these results indicate that the A variant of the rs319217 SNP is a marker of better prognosis in breast cancer.

Genetic interactions among Brca1, Brca2, Palb2, and Trp53 in mammary tumor development.

Inherited mutations in BRCA1, BRCA2, and PALB2 cause a high risk of breast cancer. Here, we conducted parallel conditional knockout (CKO) of Brca1, Palb2, and Brca2, individually and in combination, along with one copy of Trp53, in the mammary gland of nulliparous female mice. We observed a functional equivalence of the three genes in their basic tumor-suppressive activity, a linear epistasis of Palb2 and Brca2, but complementary roles of Brca1 and Palb2 in mammary tumor suppression, as combined ablation of either Palb2 or Brca2 with Brca1 led to delayed tumor formation. Whole-exome sequencing (WES) revealed both similarities and differences between Brca1 and Palb2 or Brca2 null tumors. Analyses of mouse mammary glands and cultured human cells showed that combined loss of BRCA1 and PALB2 led to high levels of reactive oxygen species (ROS) and increased apoptosis, implicating oxidative stress in the delayed tumor development in Brca1;Palb2 double CKO mice. The functional complementarity between BRCA1 and PALB2/BRCA2 and the role of ROS in tumorigenesis require further investigation.

A 2D mechanistic model of breast ductal carcinoma in situ (DCIS) morphology and progression.

Ductal carcinoma in situ (DCIS) of the breast is a non-invasive tumor in which cells proliferate abnormally, but remain confined within a duct. Although four distinguishable DCIS morphologies are recognized, the mechanisms that generate these different morphological classes remain unclear, and consequently the prognostic strength of DCIS classification is not strong. To improve the understanding of the relation between morphology and time course, we have developed a 2D in silico particle model of the growth of DCIS within a single breast duct. This model considers mechanical effects such as cellular adhesion and intra-ductal pressure, and biological features including proliferation, apoptosis, necrosis, and cell polarity. Using this model, we find that different regions of parameter space generate distinct morphological subtypes of DCIS, so elucidating the relation between morphology and time course. Furthermore, we find that tumors with similar architectures may in fact be produced through different mechanisms, and we propose future work to further disentangle the mechanisms involved in DCIS progression.

Analysis and Comparison of Tissue-Marking Dye Detection via Light Microscopy, Telemicroscopy, and Virtual Microscopy.

Objectives: To examine the fidelity of ink color identification using light microscopy (LM), telemicroscopy (TM), and virtual microscopy (VM). Methods: Twenty H&E-stained frozen section slides, prepared after tissue inking with five stain combinations, were assessed by three pathologists using LM, TM, and VM. TM was performed using Mikroscan D2 slide scanner/LiveQ software with various objectives. VM was performed using Mikroscan D2 scanner/Qumulus software, specimens digitized at20×. Results: Sensitivity/specificity by LM was 100%/100% for all colors. TM showed high overall specificity but poor sensitivity, particularly red (54%). VM showed high specificity for all colors except black (69%) and, consequently, poor sensitivity for all colors except black (96%). Conclusions: TMD identification via telepathology showed loss of sensitivity/specificity vs LM and highlighted the need for caution when interpreting TMDs with digital technologies and the need for validation protocols.

Inhibition of growth and metastasis of mouse mammary carcinoma by selective inhibitor of transforming growth factor-beta type I receptor kinase in vivo.

PURPOSE: Transforming growth factor-beta (TGF-beta) suppresses tumor development by inhibiting cellular proliferation, inducing differentiation and apoptosis, and maintaining genomic integrity. However, once tumor cells escape from the tumor-suppressive effects of TGF-beta, they often constitutively overexpress and activate TGF-beta, which may promote tumor progression by enhancing invasion, metastasis, and angiogenesis and by suppressing antitumor immunity. The purpose of this study was to test this hypothesis using TGF-beta pathway antagonists. EXPERIMENTAL DESIGN: We examined the effects of selective TGF-beta type I receptor kinase inhibitors, SD-093 and SD-208, on two murine mammary carcinoma cell lines (R3T and 4T1) in vitro and in vivo. RESULTS: Both agents blocked TGF-beta-induced phosphorylation of the receptor-associated Smads, Smad2 and Smad3, in a dose-dependent manner, with IC50 between 20 and 80 nmol/L. TGF-beta failed to inhibit growth of these cell lines but stimulated epithelial-to-mesenchymal transdifferentiation, migration, and invasiveness into Matrigel in vitro. These effects were inhibited by SD-093, indicating that these processes are partly driven by TGF-beta. Treatment of syngeneic R3T or 4T1 tumor-bearing mice with orally given SD-208 inhibited primary tumor growth as well as the number and size of metastases. In contrast, SD-208 failed to inhibit R3T tumor growth or metastasis in athymic nude mice. Moreover, in vitro anti-4T1 cell cytotoxic T-cell responses of splenocytes from drug-treated animals were enhanced compared with cells from control animals. In addition, SD-208 treatment resulted in a decrease in tumor angiogenesis. CONCLUSION: TGF-beta type I receptor kinase inhibitors hold promise as novel therapeutic agents for metastatic breast cancer.

Massive gastrointestinal bleeding: an unusual case of asymptomatic extrarenal, visceral, fibromuscular dysplasia.

Extrarenal fibromuscular dysplasia causing gastro-intestinal bleeding without other manifestations and especially sparing renal vasculature is uncommon. The diagnosis of this entity is usually made by radiographic appearance and the treatment is controversial. To our knowledge only seven cases of visceral fibromuscular dysplasia as a primary manifestation of the disease have been described, symptoms range from abdominal pain to gangrene. This is the first case of visceral fibromuscular dysplasia presenting with otherwise asymptomatic gastrointestinal bleeding, without bowel necrosis or ischemic changes. We provide a review of the literature.

Primary synovial sarcoma of the kidney with rhabdoid features.

Synovial sarcoma is a soft tissue sarcoma with clearly defined histologic, immunophenotypic, and molecular features. It occurs predominantly in the extremities of young adults but has been reported in many other anatomic sites. Histologically, it is classified as biphasic, monophasic, and poorly differentiated. The latter category, which includes tumors with a rhabdoid morphology, has been associated with a more aggressive behavior. Generally, the biphasic variant does not pose any diagnostic problem because of its typical histologic appearance; in contrast, the monophasic and poorly differentiated variants may represent a diagnostic challenge because their microscopic features can be confused with those of other spindle cell tumors with rhabdoid features. The application of molecular techniques, such as reverse transcriptase polymerase chain reaction to detect the fusion transcript associated with the characteristic t(X;18) translocation of synovial sarcoma, has enabled the confirmation of this diagnosis, even in cases of unusual localization, such as the one we present here.

Carcinogen 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis is accelerated in Smad3 heterozygous mice compared to Smad3 wild type mice.

Previous studies based on cell culture and xenograft animal models suggest that Smad3 has tumor suppressor function for breast cancer during early stages of tumorigenesis. In this report, we show that DMBA (7,12-dimethylbenz[a]anthracene), a chemical carcinogen, induces mammary tumor formation at a significantly higher frequency in the Smad3 heterozygous mice than in the Smad3 wild type mice. This is the first genetic evidence showing that Smad3 inhibits mammary tumor formation in a mouse model. Our findings support the notion that Smad3 has important tumor suppressor function for breast cancer.

Toxicity characterisation of sediment porewaters collected from UK estuaries using a Tisbe battagliai bioassay.

The lethal toxicity of sediment porewater isolated from samples collected from six United Kingdom estuaries was tested using a Tisbe battagliai bioassay. A selection of Phase 1 toxicity identification evaluation (TIE) procedures was then used to characterise the substances responsible for the measured toxicity. In samples collected in 2000, cationic metals, ammonia, organic compounds were identified as the cause of toxicity in the most toxic sample collected. Some of the toxicity remained uncharacterised. It was shown that the toxicity in samples collected from the same locations in 2001 was due to ammonia. The successful application of the T. battagliai bioassay demonstrates that it is a suitable assay for marine sediment porewater TIE and that combined with simple characterisation procedures allow an initial assessment to be made on the type of substances responsible for the measured toxicity.

Characterisation of potentially genotoxic compounds in sediments collected from United Kingdom estuaries.

The mutagenic activity bioassay Mutatox was used to assess the mutagenic activity associated with sediments collected from five UK estuaries. Assays were performed on extracts of sediment pore water and residual particulate material isolated from sediment samples collected from the rivers Tyne, Tees, Mersey, and Thames as well as Southampton Water. No mutagenic activity was associated with the pore water extracts, however, 7 of the 28 organic solvent extracts of sediment particulate material contained potential genotoxins. By using Mutatox in association with bioassay-directed fractionation, attempts were made to identify the mutagenic compounds present in the extracts. The fractionation procedure used normal phase solid phase extraction, C18 reverse phase HPLC and cyano/amino bonded silica normal phase HPLC. GC-MS (EI and NICI) analysis was used to identify polycyclic aromatic hydrocarbons (PAH), alkyl substituted PAH, nitro-polycyclic aromatic compounds (nitro-PACs), polycyclic aromatic ketones, oxygenated-PACs, and other known mutagens contributing to the genotoxicity measured in the samples. Some potentially genotoxic compounds remain unidentified.

Metabotropic glutamate receptor-1 contributes to progression in triple negative breast cancer.

TNBC is an aggressive breast cancer subtype that does not express hormone receptors (estrogen and progesterone receptors, ER and PR) or amplified human epidermal growth factor receptor type 2 (HER2), and there currently exist no targeted therapies effective against it. Consequently, finding new molecular targets in triple negative breast cancer (TNBC) is critical to improving patient outcomes. Previously, we have detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in TNBC and observed that targeting glutamatergic signaling inhibits TNBC growth both in vitro and in vivo. In this study, we explored how mGluR1 contributes to TNBC progression, using the isogenic MCF10 progression series, which models breast carcinogenesis from nontransformed epithelium to malignant basal-like breast cancer. We observed that mGluR1 is expressed in human breast cancer and that in MCF10A cells, which model nontransformed mammary epithelium, but not in MCF10AT1 cells, which model atypical ductal hyperplasia, mGluR1 overexpression results in increased proliferation, anchorage-independent growth, and invasiveness. In contrast, mGluR1 knockdown results in a decrease in these activities in malignant MCF10CA1d cells. Similarly, pharmacologic inhibition of glutamatergic signaling in MCF10CA1d cells results in a decrease in proliferation and anchorage-independent growth. Finally, transduction of MCF10AT1 cells, which express c-Ha-ras, using a lentiviral construct expressing GRM1 results in transformation to carcinoma in 90% of resultant xenografts. We conclude that mGluR1 cooperates with other factors in hyperplastic mammary epithelium to contribute to TNBC progression and therefore propose that glutamatergic signaling represents a promising new molecular target for TNBC therapy.

ATG proteins mediate efferocytosis and suppress inflammation in mammary involution.

Involution is the process of post-lactational mammary gland regression to quiescence and it involves secretory epithelial cell death, stroma remodeling and gland repopulation by adipocytes. Though reportedly accompanying apoptosis, the role of autophagy in involution has not yet been determined. We now report that autophagy-related (ATG) proteins mediate dead cell clearance and suppress inflammation during mammary involution. In vivo, Becn1(+/-) and Atg7-deficient mammary epithelial cells (MECs) produced 'competent' apoptotic bodies, but were defective phagocytes in association with reduced expression of the MERTK and ITGB5 receptors, thus pointing to defective apoptotic body engulfment. Atg-deficient tissues exhibited higher levels of involution-associated inflammation, which could be indicative of a tumor-modulating microenvironment, and developed ductal ectasia, a manifestation of deregulated post-involution gland remodeling. In vitro, ATG (BECN1 or ATG7) knockdown compromised MEC-mediated apoptotic body clearance in association with decreased RAC1 activation, thus confirming that, in addition to the defective phagocytic processing reported by other studies, ATG protein defects also impair dead cell engulfment. Using two different mouse models with mammary gland-associated Atg deficiencies, our studies shed light on the essential role of ATG proteins in MEC-mediated efferocytosis during mammary involution and provide novel insights into this important developmental process. This work also raises the possibility that a regulatory feedback loop exists, by which the efficacy of phagocytic cargo processing in turn regulates the rate of engulfment and ultimately determines the kinetics of phagocytosis and dead cell clearance.

Autophagy opposes p53-mediated tumor barrier to facilitate tumorigenesis in a model of PALB2-associated hereditary breast cancer.

Hereditary breast cancers stem from germline mutations in susceptibility genes such as BRCA1, BRCA2, and PALB2, whose products function in the DNA damage response and redox regulation. Autophagy is an intracellular waste disposal and stress mitigation mechanism important for alleviating oxidative stress and DNA damage response activation; it can either suppress or promote cancer, but its role in breast cancer is unknown. Here, we show that similar to Brca1 and Brca2, ablation of Palb2 in the mouse mammary gland resulted in tumor development with long latency, and the tumors harbored mutations in Trp53. Interestingly, impaired autophagy, due to monoallelic loss of the essential autophagy gene Becn1, reduced Palb2-associated mammary tumorigenesis in a Trp53-wild-type but not conditionally null background. These results indicate that, in the face of DNA damage and oxidative stress elicited by PALB2 loss, p53 is a barrier to cancer development, whereas autophagy facilitates cell survival and tumorigenesis.