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Representative images of each genotype of PyMT mice with mammary tumors obtained from 12-16-week old female (S1); Histological sections of the lungs from PyMT mice of different OGG1 genotypes (S2); OGG1 activity in mammary tumors isolated from B-nuclear, and D- mitochondrial fractions from PyMT/KO and PyMT/Tg animals (S3); LDH activity in mammary tumors isolated from PyMT/KO, PyMT/WT, PyMT/Tg and PyMT/ KOTg mice (S4).

Background: We previously found that QNBC tumors are more frequent in African Americans compared to TNBC tumors. To characterize this subtype further, we sought to determine the miRNA-mRNA profile in QNBC patients based on race. Methods: Both miRNA and mRNA expression data were analyzed from TCGA and validated using datasets from the METABRIC, TCGA proteomic, and survival analysis by KMPLOT. Results: miRNA-mRNAs which include FOXA1 and MYC (mir-17/20a targets); GATA3 and CCNG2 (mir-135b targets); CDKN2A, CDK6, and B7-H3 (mir-29c targets); and RUNX3, KLF5, IL1-β, and CTNNB1 (mir-375 targets) were correlated with basal-like and immune subtypes in QNBC patients and associated with a worse survival. Conclusion: Thus, QNBC tumors have an altered gene signature implicated in racial disparity and poor survival.

Production of mitochondrial reactive oxygen species and integrity of mitochondrial DNA (mtDNA) are crucial in breast cancer progression and metastasis. Therefore, we evaluated the role of mtDNA damage in breast cancer by genetically modulating the DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1) in the PyMT transgenic mouse model of mammary tumorigenesis. We generated mice lacking OGG1 (KO), mice overexpressing human OGG1 subunit 1α in mitochondria (Tg), and mice simultaneously lacking OGG1 and overexpressing human OGG1 subunit 1α in mitochondria (KO/Tg). We found that Tg and KO/Tg mice developed significantly smaller tumors than KO and wildtype mice after 16 weeks. Histological analysis revealed a roughly two-fold decrease in the incidence of lung metastases in Tg mice (33.3%) compared to wildtype mice (62.5%). Furthermore, lungs from Tg mice exhibited nearly a 15-fold decrease in the average number of metastatic foci compared to WT mice (p≤0.05). Primary tumors isolated from Tg mice also demonstrated reduced total and mitochondrial oxidative stress, diminished mtDNA damage, and increased mitochondrial function. Targeting hOGG1 to the mitochondria protected cells from mtDNA damage resulting in downregulation of HIF-1α and attenuated phosphorylation of Akt. Collectively, we demonstrate proof of concept that mtDNA damage results in breast cancer progression and metastasis in vivo. Moreover, our findings offer new therapeutic strategies for modulating the levels of mtDNA repair enzymes to delay or stall metastatic progression.

Background Human epidermal growth factor 2 (HER2) amplification and/or overexpression occurs in 12% to 25% of breast cancers. Accurate detection of HER2 is critical in predicting response to HER2-targeted therapy. Both immunohistochemistry (IHC) and in situ hybridization (ISH) are FDA-approved methods for detecting HER2 status because its protein overexpression is largely attributable to gene amplification. However, variable discordant results between IHC and ISH have been reported. Methods We determined the frequency of HER2 IHC/ISH discordance in these patients and also performed a pooled literature review analysis. Results Of the 1125 consecutive primary or metastatic breast cancers with HER2 IHC and ISH performed simultaneously between 2015 and 2020, 84.6% had an unequivocal HER2 status. Discordance was found in 30 cases from 26 patients, including 13 IHC−/ISH+ and 17 IHC+/ISH−, representing 1.6% and 11.9% of IHC− and IHC+ cases, respectively. Review of the literature between 2001 and 2020 identified 46 relevant studies, with a total of 43,468 cases with IHC and ISH performed. The IHC−/ISH+ and IHC+/ISH− discordances were seen in all antibody clones and ISH methods used. The IHC+/ISH− discordance was significantly higher than IHC−/ISH+ (13.8% vs. 3%, P

Context.—Advancements in genomic, computing, and imaging technology have spurred new opportunities to use quantitative image analysis (QIA) for diagnostic testing.Objective.—To develop evidence-based recommendations to improve accuracy, precision, and reproducibility in the interpretation of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) for breast cancer where QIA is used.Design.—The College of American Pathologists (CAP) convened a panel of pathologists, histotechnologists, and computer scientists with expertise in image analysis, immunohistochemistry, quality management, and breast pathology to develop recommendations for QIA of HER2 IHC in breast cancer. A systematic review of the literature was conducted to address 5 key questions. Final recommendations were derived from strength of evidence, open comment feedback, expert panel consensus, and advisory panel review.Results.—Eleven recommendations were drafted: 7 based on CAP laboratory accreditation requirements and 4 based on expert consensus opinions. A 3-week open comment period received 180 comments from more than 150 participants.Conclusions.—To improve accurate, precise, and reproducible interpretation of HER2 IHC results for breast cancer, QIA and procedures must be validated before implementation, followed by regular maintenance and ongoing evaluation of quality control and quality assurance. HER2 QIA performance, interpretation, and reporting should be supervised by pathologists with expertise in QIA.

Production of mitochondrial reactive oxygen species and integrity of mitochondrial DNA (mtDNA) are crucial in breast cancer progression and metastasis. Therefore, we evaluated the role of mtDNA damage in breast cancer by genetically modulating the DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1) in the PyMT transgenic mouse model of mammary tumorigenesis. We generated mice lacking OGG1 (KO), mice overexpressing human OGG1 subunit 1α in mitochondria (Tg), and mice simultaneously lacking OGG1 and overexpressing human OGG1 subunit 1α in mitochondria (KO/Tg). We found that Tg and KO/Tg mice developed significantly smaller tumors than KO and wild-type (WT) mice after 16 weeks. Histologic analysis revealed a roughly 2-fold decrease in the incidence of lung metastases in Tg mice (33.3%) compared to WT mice (62.5%). Furthermore, lungs from Tg mice exhibited nearly a 15-fold decrease in the average number of metastatic foci compared with WT mice (P ≤ 0.05). Primary tumors isolated from Tg mice also demonstrated reduced total and mitochondrial oxidative stress, diminished mtDNA damage, and increased mitochondrial function. Targeting hOGG1 to the mitochondria protected cells from mtDNA damage, resulting in downregulation of HIF1α and attenuated phosphorylation of Akt. Collectively, we demonstrate proof of concept that mtDNA damage results in breast cancer progression and metastasis in vivo. Moreover, our findings offer new therapeutic strategies for modulating the levels of mtDNA repair enzymes to delay or stall metastatic progression. Cancer Res; 76(1); 30–34. ©2015 AACR.

Pulmonary arterial hypertension (PAH) is a debilitating and deadly disease with no known cure. Heart failure is a major comorbidity and a common cause of the premature death of patients with PAH. Increased asymmetrical right ventricular hypertrophy and septal wall thickening compress the left ventricular cavity and elicit diastolic heart failure. In this study, we used the Sugen5416/hypoxia/normoxia-induced PAH rat to determine whether altered pyridine nucleotide signaling in the failing heart contributes to 1) increased oxidative stress, 2) changes in metabolic phenotype, 3) autophagy, and 4) the PAH-induced failure. We found that increased reactive oxygen species, metabolic maladaptation, and autophagy contributed to the pathogenesis of right ventricular remodeling and hypertrophy that lead to left ventricular diastolic dysfunction. In addition, arterial elastance increased in PAH rats. Glucose-6-phosphate dehydrogenase is a major source of pyridine molecule (nicotinamide adenine dinucleotide phosphate), which is a substrate for nicotinamide adenine dinucleotide phosphate oxidases in the heart. Dehydroepiandrosterone, a 17-ketosteroid that reduces pulmonary hypertension and right ventricular hypertrophy, inhibited glucose-6-phosphate dehydrogenase, decreased oxidative stress, increased glucose oxidation and acetyl-coA, and reduced autophagy in the hearts of PAH rats. It also decreased arterial stiffness and improved left ventricular diastolic function. These findings demonstrate that pyridine nucleotide signaling, at least partly, mediates PAH-induced diastolic heart failure, and that reduction of glucose-6-phosphate dehydrogenase-derived nicotinamide adenine dinucleotide phosphate is beneficial to improve left ventricle diastolic function.

Although it is known that blood vessels undergo remodeling in type 2 diabetes (T2D), the signaling pathways that underlie the structural and functional changes seen in diabetic arteries remain unclear. Our objective was to determine whether the remodeling in type 2 diabetic Goto-Kakizaki (GK) rats is evoked by elevated reactive oxygen species (ROS). Our results show that aortas from GK rats produced greater force ( P < 0.05) in response to stimulation with KCl and U46619 than aortas from Wistar rats. Associated with these changes, aortic expression of contractile proteins (measured as an index of remodeling) and the microRNA (miR-145), which act to upregulate transcription of contractile protein genes, was twofold higher ( P < 0.05) in GK than Wistar (age-matched control) rats, and there was a corresponding increase in ROS and decrease in nitric oxide signaling. Oral administration of the antioxidant Tempol (1 mmol/l) to Wistar and GK rats reduced ( P < 0.05) myocardin and calponin expression. Tempol (1 mmol/l) decreased expression of miR-145 in Wistar and GK rat aorta. To elucidate the mechanism through which ROS increases miR-145, we measured their levels in freshly isolated aorta and cultured aortic smooth muscle cells incubated for 12 h in the presence of H2O2 (300 μmol/l). H2O2 increased expression of miR-145, and there were corresponding nuclear increases in myocardin, a miR-145 target protein. Intriguingly, H2O2-induced expression of miR-145 was decreased by U0126 (10 μmol/l), a MEK1/2 inhibitor, and myocardin was decreased by anti-miR-145 (50 nmol/l) and U0126 (10 μmol/l). Our novel findings demonstrate that ROS evokes vascular wall remodeling and dysfunction by enhancing expression of contractile proteins in T2D.

Introduction Primary smooth muscle tumors (SMT) of the breast are rare with leiomyosarcomas representing less than 0.1% of all malignant breast tumors. Case Presentation A 58-year-old female with no significant past medical history noted on screening mammography to have a circumscribed 6-mm nodule in the right breast, upper outer quadrant. Core needle biopsy showed a spindle cell neoplasm with smooth muscle differentiation. The excisional biopsy showed a 6-mm lesion composed of atypical hyperchromatic spindle cells in fascicles, marked nuclear pleomorphism, and 5 mitoses per 10 high-power fields. By immunohistochemistry, the spindle cells were positive for smooth muscle actin, desmin, and negative for S-100 and cytokeratin AE1/AE3. Positron emission tomography/computed tomography of head/neck, chest, abdomen, and pelvis did not identify other neoplasms. Despite the lesion size, findings were supportive of a leiomyosarcoma. Discussion Breast SMTs have nonspecific clinical or imaging features. Histologically, these present as spindle cell tumors with smooth muscle differentiation. Initial workup starts with distinction between benign and malignant neoplasms. The malignant SMTs are usually large tumors with cytologic atypia and mitotic activity used as diagnostic criteria. In addition, the distinction between primary and metastasis is important and frequently relies on clinical history and exclusion of other primary origins by radiographic survey. In the current case, although the tumor size is unusually small, the histological features and absence of other primary malignancies support the diagnosis of a leiomyosarcoma. Conclusion Primary leiomyosarcoma of breast is extremely uncommon with less than 70 cases reported in the literature. Although they are usually large tumors, this diagnosis should be included in the differential diagnosis when smooth muscle differentiation, significant atypia, and mitoses are encountered in a spindle cell tumor of the breast.

Objective Primary sarcomas of the vulva are very rare malignancies, accounting for 1% to 3% of all gynecologic sarcomas. Leiomyosarcoma is the most common histologic type. They present with nonspecific symptoms, aggressive behavior, and high metastatic potential. Methods A 63-year-old female patient presented with a 6-month history of a slow-growing, painless vulvar mass at the right posterior fourchette. CT scan revealed an asymmetric, heterogeneous vulvar mass extending to the distal vagina, without lymphadenopathy or evidence of distant metastasis. Focal prominence of the uterus suggestive of leiomyoma was also noted, but primary uterine neoplasm with vulvar metastasis could not be excluded. The patient underwent wide local excision of the vulvar mass with hysterectomy and bilateral salpingo-oophorectomy. Results Gross examination of the vulvar specimen showed an ulcerated 2.1-cm solid white mass with irregular borders. Microscopically, the vulvar tumor consisted of a focally infiltrative spindle and pleomorphic neoplastic proliferation with interlacing fascicular pattern and increased mitotic activity up to 25/10 HPF, without necrosis or lymphovascular invasion. The tumor focally extended to resection margins. Immunohistochemically, the neoplastic cells were positive for SMA, ER, and desmin and negative for S-100 and cytokeratin AE1/AE3. These findings, in conjunction with the clinical presentation, were consistent with high-grade primary vulvar leiomyosarcoma. Radiation therapy was subsequently started. Conclusion Primary vulvar leiomyosarcomas are rare and secondary involvement by uterine primary should always be ruled out. In addition, these tumors can be mistaken clinically as benign processes such as Bartholin gland cysts, infectious granuloma, fibroma, or myoma. Hence, it is vital to consider vulvar sarcomas in the differential diagnosis of nonspecific vulvar lesions in order to avoid delay in diagnosis and treatment. The rarity of this diagnosis makes this case worth reporting.

We present the case of a 55-year-old African American male who was admitted for an acute right posterior middle cerebral artery ischemic infarction. Appropriate management of thrombotic stroke was ...

Splenic lymphangioma is a rare malformation of the splenic lymphatic channels, mostly seen in children. It is characterized by the presence of cysts, resulting from increases in the size and number of thin-walled lymphatic vessels that are abnormally interconnected and dilated. The condition may be restricted to the spleen, but in most cases it involves multiple organs (systemic lymphangiomatosis). The clinical picture is variable; small lesions are often incidentally detected through imaging studies, while larger lesions can result in compression of organs, causing pain or rupture even after minor trauma. Therefore, splenic lymphangiomas should be considered in the differential diagnosis of splenomegaly or left upper quadrant pain even among adults and should be immediately treated with splenectomy; delay in the therapeutic intervention can lead to life-threatening complications.

CD30 is a marker of lymphoid activation that is characteristically expressed in lymphoproliferative disorders such as anaplastic large cell lymphoma (ALCL), lymphomatoid papulosis (LyP) and Hodgkin lymphoma, but also in other T- and B-cell lymphomas, and in cutaneous lymphoid hyperplasia. However, CD30 expression can also be found in a wide variety of inflammatory and infectious skin conditions including immune-mediated disorders (eg, atopic dermatitis, pityriasis lichenoides, scleroderma, graft versus host disease), drug reactions (eg, antibiotics, chemotherapeutic agents), viral infections (eg, molluscum contagiosum, parapoxvirus, herpes simplex virus, human immunodeficiency virus), bacterial and fungal infections (eg, abscesses, cellulitis, chromomycosis), environmental conditions (eg, scabies, insect and spider bites), peritumoral host responses (eg, basal cell carcinoma, cutaneous lymphadenoma) and in other miscellaneous disorders (eg, hidradenitis suppurativa, ruptured cysts, pressure ulcers, granulation tissue). Moreover, CD30 expression can also be found in nonhematolymphoid cells such as germ cells and mesothelial cells. In inflammatory skin conditions, atypical CD30+ lymphoid cells may cause differential diagnostic problems with CD30+ lymphoproliferative disorders. It is obvious that the sole presence of CD30+ atypical cells in the skin can not in itself support a malignant diagnosis. The differentiation between benign and malignant cutaneous lymphoid infiltrates is only possible through the combined use of multiple clinical, histologic, immunophenotypic and molecular genetic analyses, as there is no single reliable criterion that would allow this distinction. Large atypical CD30+ lymphoid cells are a part of cutaneous reactive infiltrates and do not qualify alone for the diagnosis of malignancy.

The case of a 53-year-old female with abdominal pain is presented. Abdominal ultrasound was unremarkable, a HIDA scan demonstrated biliary dyskinesia, and a computed tomography scan of the abdomen and pelvis revealed an intraluminal gallbladder hyperdensity suspicious for a gallstone. A laparoscopic cholecystectomy was performed and, on pathologic examination, no gallstones were present. Microscopically, a focus of bone metaplasia of the gallbladder mucosa was noted. No other lesions were identified. Bone metaplasia of the gallbladder is a rare event of unknown clinical significance that may be clinically confused with cholelithiasis affecting treatment options.

We present the biochemical characterization of two amyloid proteins, including their partial amino terminal sequence, isolated from localized forms of ureteral amyfoidosis. The major component of amyloid NAv extracted from milligram quantities of biopsy tissue, had a molecular mass of 16 kDa and the 20 first amino acids showed homology to immunoglobulin (Ig) light chain of the subgroup λ II. In addition, amyloid P component co-purified with amyloid NA V as determined by Western blot analysis. Amyloid MAI was extracted from formalin fixed, paraffin embedded tissue. It had a molecular mass of 14 kDa and its amino terminal sequence (17 steps) revealed homology to Ig light chain of the subgroup λ III. These results provide additional evidence for the association between amyloidogenic Ig light chains and localized, tumor-like forms of amyloidosis. Moreover, the two simple methods presented here may facilitate the characterization of amyloid proteins from small samples of frozen tissue and rare specimens stored...

Secretogranin II (SgII) is a member of the granin family of proteins found in neuroendocrine and endocrine cells. The expression and storage of SgII in the pituitary gland of Old World primates and rodents have been linked with those of luteinizing hormone (LH). However, New World primates including squirrel monkeys do not express LH in the pituitary gland, but rather CG is expressed. If CG takes on the luteotropic role of LH in New World primates, SgII may be associated with the expression and storage of CG in the pituitary gland. The goal of this study was to evaluate the regulation and distribution of CG and SgII in the squirrel monkey. A DNA fragment containing approximately 750 bp of squirrel monkey SgII promoter was isolated from genomic DNA and found to contain a cyclic AMP response element that is also present in the human SgII promoter and important for GnRH responsiveness. The squirrel monkey and human SgII promoters were similarly activated by GnRH in luciferase reporter gene assays in LβT2 cells. Double immunofluorescence microscopy demonstrated close association of SgII and CG in gonadotrophs of squirrel monkey pituitary gland. These results suggest that CG and SgII have a similar intercellular distribution and are coregulated in squirrel monkey pituitary gland.

Isolated renal infections with fungal organisms of the class Zygomycetes are rare, but these infections are most frequently seen in patients who are immunocompromised. We report the case of a 45-year-old African American man who presented with symptoms of right-sided pyelonephritis, including fever, dysuria, and flank pain. The patient's history was significant only for sickle cell trait, and no evidence of immunosuppression was identified. Renal ultrasound imaging revealed a hypoechoic lesion in the superior pole of the right kidney, and the radiologic differential diagnoses included neoplasm, abscess, and infarct. Urine cultures were negative, but urinalysis showed white blood cells, which were too numerous to count. A computed tomography scan of the abdomen and pelvis performed 2 weeks after the initial presentation showed a slight increase in the renal mass, despite antimicrobial therapy, and a right nephrectomy was subsequently performed. On gross sectioning, an 8.5-cm well-circumscribed lesion was identified in the upper pole of the kidney. Microscopic sections showed extensive necrosis of the renal parenchyma, and, in areas of both infarcted and viable renal tissue, large, broad, aseptate fungal hyphae with irregular branching. Angioinvasion with associated thrombosis was seen in the renal tissue. The morphologic features of the organism were most compatible with that of a zygomycete. No evidence of disseminated fungal disease was identified on imaging studies. This case represents a successful outcome of a rarely reported isolated renal zygomycosis in a patient with no known underlying risk factors for the infection and illustrates the wide range of clinical presentations with which zygomycotic infections may present.

Lymph node mapping and sentinel lymph node biopsy are currently used to stage patients with cutaneous malignant melanoma. Immunohistochemical stains contribute to the detection of micrometastases; however, molecular biology techniques are associated with better diagnostic sensitivity. Sixty sentinel lymph nodes were included in this study. The primary lesions were malignant melanoma stage I or II, with a follow-up of longer than 2 years. Sentinel lymph nodes were studied with hematoxylin-eosin, immunohistochemistry for S-100 and HMB-45, and molecular biology techniques (reverse transcription (RT)-PCR) for the detection of tyrosinase messenger RNA. In 15 of 60 cases (25%), tyrosinase was detected by RT-PCR; three of these cases were also positive by immunohistochemistry. The population was divided into three groups: (i) hematoxylin-eosin-/immunohistochemistry+/molecular biology techniques+ (3 cases); (ii) hematoxylin-eosin-/immunohistochemistry-/molecular biology techniques+ (12 cases); (iii) hematoxylin-eosin-/immunohistochemistry-/molecular biology techniques- (45 cases). Correlation of the groups with overall survival showed the following: (i) 2 of 3 patients died (67%); (ii) 5 of 12 died (42%), and (iii) all 45 patients are alive, with no lymphadenectomy and a median follow-up of 84 months. The inclusion of molecular biology techniques appears to be of great value for the detection of sentinel lymph node micrometastases in patients with cutaneous malignant melanoma. In our series, those patients who showed negativity with all the three methods had a null recurrence rate. Therefore, this triple negativity could be a positive prognostic factor for overall survival. Our findings suggest the possibility of molecular oncological staging, which would allow the selection of patients with submicroscopic metastases for a complete treatment.

Although the role of the ErbB2/HER2 oncogene in cancers has been extensively studied, how ErbB2 is regulated remains poorly understood. A novel microRNA, mir-4728, was recently found within an intron of the ErbB2 gene. However, the function and clinical relevance of this intronic miRNA are completely unknown. Here, we demonstrate that mir-4728 is a negative regulator of MAPK signaling through directly targeting the ERK upstream kinase MST4 and exerts numerous tumor-suppressive properties in vitro and in animal models. Importantly, our patient sample study shows that mir-4728 was under-expressed in breast tumors compared with normal tissue, and loss of mir-4728 correlated with worse overall patient survival. These results strongly suggest that mir-4728 is a tumor-suppressive miRNA that controls MAPK signaling through targeting MST4, revealing mir-4728’s significance as a potential prognostic factor and target for therapeutic intervention in cancer. Moreover, this study represents a conceptual advance by providing strong evidence that a tumor-suppressive miRNA can antagonize the canonical signaling of its host oncogene.

Introduction: Lymph node status in patients with cutaneous malignant melanoma is the most important prognostic factor. Patients with clinically positive nodes (stage III) should undergo therapeutic lymphadenectomy; however, the surgical approach to the regional disease in patients with negative clinical examination (stage I and II) is still controversial. Selective lymphadenectomy consists of the intraoperative identification of the first node in the nodal basin, the sentinel lymph node (SLN). Routine examination, serial sectioning, and immunohistochemistry may underestimate the presence of tumor cells. PCR is a molecular biology technique that may be useful for the detection of malignant melanoma nodal metastases in the SLN. Aim: The aim of this study was to use tyrosinase messenger RNA (mRNA) amplification for the detection of micrometastases in fresh frozen SLNs. Methods: 46 hematoxylin-eosin (HE)-negative sentinel node samples from 42 patients with malignant melanoma were included in this study. Formalin-fixed paraffin-embedded sections were immunostained with S-100 protein and HMB-45. A central portion of the node was submitted for PCR. This method was accomplished with a combination of reverse transcription and amplification of the tyrosinase complementary DNA and double-round PCR (nested reverse transcriptase [RT]-PCR). Results: In 1 of the 42 SLN-negative patients, immunohistochemistry stains allowed the detection of micrometastases. With molecular biology, 14 of the 42 SLN patients were positive (33%); in another 12 (29%), only the nested RT-PCR was positive. Of the 42 patients, 24 were put into 3 groups and followed for a 5-year period with 1, 7, and 16 patients, respectively, in the groups. The first group involved 1 patient who had provided 2 SLN samples that were found to be SLN-positive using both techniques, immunohistochemistry stains and nested RT-PCR (he had hepatic metastasis and died 24 months after diagnosis). The second group, with only nested RT-PCR positive SLN samples, included 7 of 12 patients who were followed and had a median survival of 37 months; 4 died of widespread metastatic disease, the other 3 patients had event-free survival, but 1 consented to undergo a therapeutic lymphadenectomy as a result of a positive test. The last group consisting of 16 of 32 patients, with complete 5-year survival, who were SLN-negative with both techniques, immunohistochemistry stains and nested RT-PCR. Fourteen of the 16 (88%) were event-free survival during the follow-up, and 2 had local relapse. Conclusion: Tyrosinase mRNA amplification may be a negative prognostic factor for the detection of micrometastases in fresh frozen SLNs using molecular biology techniques.