Proteomic analysis of archival breast cancer clinical specimens identifies biological subtypes with distinct survival outcomes.

Despite advances in genomic classification of breast cancer, current clinical tests and treatment decisions are commonly based on protein level information. Formalin-fixed paraffin-embedded (FFPE) tissue specimens with extended clinical outcomes are widely available. Here, we perform comprehensive proteomic profiling of 300 FFPE breast cancer surgical specimens, 75 of each PAM50 subtype, from patients diagnosed in 2008-2013 (n = 178) and 1986-1992 (n = 122) with linked clinical outcomes. These two cohorts are analyzed separately, and we quantify 4214 proteins across all 300 samples. Within the aggressive PAM50-classified basal-like cases, proteomic profiling reveals two groups with one having characteristic immune hot expression features and highly favorable survival. Her2-Enriched cases separate into heterogeneous groups differing by extracellular matrix, lipid metabolism, and immune-response features. Within 88 triple-negative breast cancers, four proteomic clusters display features of basal-immune hot, basal-immune cold, mesenchymal, and luminal with disparate survival outcomes. Our proteomic analysis characterizes the heterogeneity of breast cancer in a clinically-applicable manner, identifies potential biomarkers and therapeutic targets, and provides a resource for clinical breast cancer classification.

A Rapid and Cost-Effective Gene Expression Assay for the Diagnosis of Well-Differentiated and Dedifferentiated Liposarcomas.

Histologic examination neither reliably distinguishes benign lipomas from atypical lipomatous tumor/well-differentiated liposarcoma, nor dedifferentiated liposarcoma from other pleomorphic sarcomas, entities with different prognoses and management. Molecular confirmation of pathognomonic 12q13-15 amplifications leading to MDM2 overexpression is a diagnostic gold standard. Currently the most commonly used assay for this purpose is fluorescence in situ hybridization (FISH), but this is labor intensive. This study assessed whether newer NanoString-based technology could allow for more rapid and cost-efficient diagnosis of liposarcomas on standard formalin-fixed tissues through gene expression. Leveraging large-scale transcriptome data from The Cancer Genome Atlas, 20 genes were identified, most from the 12q13-15 amplicon, that distinguish dedifferentiated liposarcoma from other sarcomas and can be measured within a single NanoString assay. Using 21 cases of histologically ambiguous low-grade adipocytic tumors with available MDM2 amplification status, a machine learning-based analytical pipeline was built that assigns a given sample as negative or positive for liposarcoma based on quantitative gene expression. The effectiveness of the assay was validated on an independent set of 100 sarcoma samples (including 40 incident prospective cases), where histologic examination was considered insufficient for clinical diagnosis. The NanoString assay had a 93% technical success rate, and an accuracy of 97.8% versus an MDM2 amplification FISH gold standard. NanoString had a considerably faster turnaround time and was cheaper than FISH.

Basal biomarkers nestin and INPP4b identify intrinsic subtypes accurately in breast cancers that are weakly positive for oestrogen receptor.

AIMS: Recent evidence indicates that weakly positive immunohistochemical staining of oestrogen receptor (ER) is not associated reliably with a luminal subtype, with the majority reclassified as basal-like by gene expression profile. In this study we assessed the capacity of recently identified immunohistochemical markers of basal-like subtype not dependent upon ER status - positive expression of nestin or loss of inositol polyphosphate-4-phosphatase (INPP4b) - to discriminate intrinsic subtypes, focusing on clinically problematic cases with weak ER positivity. METHODS AND RESULTS: Formalin-fixed paraffin-embedded blocks, enriched for large proportions of ER-negative and ER weakly positive breast cancers, were selected from two previous studies conducted in the period 2008-13 and used for (i) RNA extraction for 50-gene subtype predictor (PAM50) intrinsic subtyping and (ii) tissue microarray construction for immunohistochemical assessment of nestin and INPP4b. Fifty-eight cases were weakly positive for ER (Allred 3-5), among which 28 (48%) were assigned as basal-like by PAM50 gene expression. In these 58 cases, the nestin/INPP4b panel identified 23 basal-like cases with a positive predictive value of 87% [95% confidence interval (CI) 78-95%] and excluded luminal subtype with a negative predictive value of 95% (95% CI 88-100%). Weakly positive ER patients assigned as basal-like by nestin/INPP4b definition demonstrated a median survival time of 45.8 months, significantly lower than 65 months among other ER weakly positive cases (P = 0.012). CONCLUSIONS: Immunohistochemical assessment of nestin and INPP4b provides an accurate, accessible and inexpensive tool to identify basal-like breast cancer subtype in the clinically problematic setting of weak ER positivity. This panel identifies poor prognosis patients who might need strong considerations for non-endocrine-based therapies.

Upregulation of endothelial and inducible nitric oxide synthase expression by reactive oxygen species.

BACKGROUND: The effect of reactive oxygen species (ROS) on nitric oxide synthase (NOS) expression remains uncertain. This study explored the effect of increased ROS activity on NOS expression in vitro in human coronary artery endothelial cells (HCAECs) grown in culture and in intact animals. METHODS: Endothelial NOS (eNOS) expression and nuclear factor kappaB (NFkappaB) activation were determined in HCAECs grown in culture and exposed to oxidative stress with xanthine-xanthine oxidase (X-XO) generated superoxide, H(2)O(2), or glutathione depletion with buthionine sulfoximine (BSO) for 24 h. In parallel experiments, cells were treated with a nitric oxide (NO) scavenger (hemoglobin), and with an NO donor S-nitroso-N-acetyl penicillamine (SNAP)]. In addition, eNOS and inducible NOS (iNOS) expressions were determined in rats treated with either BSO or vehicle for 48 h. RESULTS: Increases in ROS activity, achieved by exogenous superoxide and H(2)O(2) or by glutathione depletion, upregulated the expression of eNOS at both transcriptional and translational levels in HCAECs. Similar effects were seen with the non-radical NO scavenger, hemoglobin. The upregulatory action of hemoglobin on eNOS messenger RNA (mRNA) and protein expressions was overcome by the NO donor, SNAP, thereby suggesting that there is a negative feedback regulation of eNOS by NO. Nuclear translocation of NFkappaB (p65) was noted within 5 min of exposure to H(2)O(2) and at least 15 min after exposure to superoxide or BSO. Induction of oxidative stress by glutathione depletion led to upregulation of renal and aorta eNOS and iNOS in live animals. CONCLUSIONS: An increase in ROS activity upregulates NOS expression in vitro in HCAECs grown in culture, and also in vivo in animals. This effect appears to be, in part, mediated by limiting the availability of NO, thereby exerting a negative feedback influence on NOS expression through activation of NFkappaB.

Association of renal injury with nitric oxide deficiency in aged SHR: prevention by hypertension control with AT1 blockade.

BACKGROUND: Aged spontaneously hypertensive rats (SHR) develop end-stage renal disease resembling that of uncontrolled essential hypertension in humans. Nitric oxide (NO) and angiotensin II (Ang II) play an important role in the regulation of blood pressure and the growth of vascular smooth muscle and renal mesangial cells. The relationship between renal NO system, Ang II activity and renal injury in aged SHR is not fully understood. METHODS: The 8-week-old SHR were randomized into losartan-treated (30 mg/kg/day for 55 weeks) and vehicle treated groups. The age-matched Wistar-Kyoto rats (WKY) served as controls. Renal histology and tissue expressions of endothelial and inducible NO synthases (eNOS and iNOS) and nitrotyrosine were examined at 63-weeks of age. RESULTS: Compared to the WKY group, untreated SHR showed severe hypertension, proteinuria, renal insufficiency, a twofold decrease in renal tissue eNOS and iNOS expressions and massive nitrotyrosine accumulation. This was associated with severe glomerulosclerosis, tubular atrophy and interstitial fibrosis. Losartan therapy normalized blood pressure, prevented proteinuria and renal insufficiency, abrogated the fall in renal eNOS and iNOS protein contents, mitigated renal nitrotyrosine accumulation, and prevented the histological abnormalities found in the untreated SHR. CONCLUSIONS: Aged SHR exhibit severe renal lesions with acquired NO deficiency that are prevented by hypertension control with AT1 blockade. These findings point to the possible role of NO deficiency in the pathogenesis of renal lesions in aged SHR.

Nitric oxide synthase expression in hypertension induced by inhibition of glutathione synthase.

Induction of chronic oxidative stress by glutathione (GSH) depletion has been shown to cause hypertension in normal rats. This was accompanied by and perhaps in part due to inactivation and sequestration of NO by reactive oxygen species (ROS), leading to diminished NO bioavailability. This study was designed to examine renal histology, nitric oxide synthase (NOS) isotype expression, and nitrotyrosine distribution in this model. Sprague-Dawley rats were subjected to oxidative stress by administration of the GSH synthase inhibitor buthionine sulfoximine (BSO; 30 mM/l in drinking water) for 2 weeks. The controls were given tap water. Blood pressure, renal histology, tissue expression of endothelial and inducible NOS (eNOS and iNOS) and nitrotyrosine, tissue GSH content, and urinary excretion of NO metabolites (NOx) were examined. The BSO-treated group showed a 3-fold decrease in tissue GSH content, a marked elevation in blood pressure, and a significant reduction in the urinary excretion of NOx. Histological examination of kidneys revealed no significant abnormalities in either group. In addition, no significant differences were observed in either intensities or localizations of eNOS and iNOS in the kidney. However, the BSO-treated group exhibited intense accumulation in the renal tissue of nitrotyrosine, which is the footprint of NO oxidation by ROS. These observations suggest that oxidative stress-induced hypertension is not caused by either structural abnormality of or depressed NOS expression by the kidney in this model. Instead, it is associated with and perhaps partially related to enhanced renal NO inactivation by ROS and diminished NO bioavailability.