Constitutive Expression of a Dominant-Negative TGF-ß Type II Receptor in the Posterior Left Atrium Leads to Beneficial Remodeling of Atrial Fibrillation Substrate.

RATIONALE: Fibrosis is an important structural contributor to formation of atrial fibrillation (AF) substrate in heart failure. Transforming growth factor-ß (TGF-ß) signaling is thought to be intricately involved in creation of atrial fibrosis. OBJECTIVE: We hypothesized that gene-based expression of dominant-negative type II TGF-ß receptor (TGF-ß-RII-DN) in the posterior left atrium in a canine heart failure model will sufficiently attenuate fibrosis-induced changes in atrial conduction and restitution to decrease AF. Because AF electrograms are thought to reflect AF substrate, we further hypothesized that TGF-ß-RII-DN would lead to increased fractionation and decreased organization of AF electrograms. METHODS AND RESULTS: Twenty-one dogs underwent injection+electroporation in the posterior left atrium of plasmid expressing a dominant-negative TGF-ß type II receptor (pUBc-TGFß-DN-RII; n=9) or control vector (pUBc-LacZ; n=12), followed by 3 to 4 weeks of right ventricular tachypacing (240 bpm). Compared with controls, dogs treated with pUBC-TGFß-DN-RII demonstrated an attenuated increase in conduction inhomogeneity, flattening of restitution slope and decreased duration of induced AF, with AF electrograms being more fractionated and less organized in pUBc-TGFß-DN-RII versus pUBc-LacZ dogs. Tissue analysis revealed a significant decrease in replacement/interstitial fibrosis, p-SMAD2/3 and p-ERK1/2. CONCLUSIONS: Targeted gene-based reduction of TGF-ß signaling in the posterior left atrium-with resulting decrease in replacement fibrosis-led to beneficial remodeling of both conduction and restitution characteristics of the posterior left atrium, translating into a decrease in AF and increased complexity of AF electrograms. In addition to providing mechanistic insights, this data may have important diagnostic and therapeutic implications for AF.

Necroptosis in spontaneously-mutated hematopoietic cells induces autoimmune bone marrow failure in mice.

Acquired aplastic anemia is an autoimmune-mediated bone marrow failure syndrome. The mechanism by which such an autoimmune reaction is initiated is unknown. Whether and how the genetic lesions detected in patients cause autoimmune bone marrow failure have not yet been determined. We found that mice with spontaneous deletion of the TGFß-activated kinase-1 gene in a small subset of hematopoietic cells developed bone marrow failure which resembled the clinical manifestations of acquired aplastic anemia patients. Bone marrow failure in such mice could be reversed by depletion of CD4+ T lymphocytes or blocked by knockout of interferon-γ, suggesting a Th1-cell-mediated autoimmune mechanism. The onset and progression of bone marrow failure in such mice were significantly accelerated by the inactivation of tumor necrosis factor-α signaling. Tumor necrosis factor-α restricts autoimmune bone marrow failure by inhibiting type-1 T-cell responses and maintaining the function of myeloid-derived suppressor cells. Furthermore, we determined that necroptosis among a small subset of mutant hematopoietic cells is the cause of autoimmune bone marrow failure because such bone marrow failure can be prevented by deletion of receptor interacting protein kinase-3 Our study suggests a novel mechanism to explain the pathogenesis of autoimmune bone marrow failure.

Phase II neoadjuvant clinical trial of carboplatin and eribulin in women with triple negative early-stage breast cancer (NCT01372579).

The purpose of this study is to evaluate the efficacy and safety of neoadjuvant treatment with carboplatin and eribulin in patients with early-stage triple negative breast cancer (TNBC), and to explore biomarkers based on DNA and protein expression profiles as predictors of response. Patients with histologically confirmed early-stage TNBC received carboplatin AUC 6 iv every 21 days, and eribulin 1.4 mg/m(2) day 1 and day 8 every 21 days for four cycles. The primary endpoint of the study was pathologic complete response (pCR), with secondary endpoints including clinical response and safety of the combination. Exploratory studies assessed DNA-based biomarkers [homologous recombination deficiency (HRD) score, and HR deficiency status (HRD score + BRCA1/BRCA2 mutation status)], protein-based biomarkers (Ki67, TP53, androgen receptor, Cyclin E, CDK2, Cyclin D, CDK4, Pin1 and Smad3), and clinical pretreatment factors as predictors of pCR. 13/30 (43.3 %) patients enrolled in the study achieved pCR. 24 (80.0 %) had a clinical complete or partial response. The combination was safe with mostly grade 1 and 2 toxicities. HRD score (P = 0.0024) and HR deficiency status (P = 0.0012) significantly predicted pCR. Pretreatment cytoplasmic CDK2 was also associated with pCR (P = 0.021). Significant differences in pre- versus post-treatment expression levels of nuclear Cyclin D (P = 0.020), nuclear CDK4 (P = 0.0030), and nuclear Smad3 (P = 0.015) were detected. The combination of carboplatin and eribulin is safe and efficacious in the treatment of early-stage TNBC. HRD score, HR deficiency status, and cytoplasmic CDK2 predicted pCR in this patient population.

Double PIK3CA Alterations and Parallel Evolution in Colorectal Cancers.

OBJECTIVES: To demonstrate clinicopathologic features and evaluate the clonality of double PIK3CA alterations in colorectal cancers (CRCs). METHODS: Clonality was examined in 13 CRCs with double PIK3CA alterations (1.7% of CRCs or 9.6% of PIK3CA-mutated CRCs). Multiregional analyses were performed to confirm subclonal PIK3CA alterations. RESULTS: PIK3CA alterations were detected within exon 9 (51%), exon 20 (23%), exon 1 (15%), and exon 7 (6.0%). CRCs with exon 7 alterations showed a significantly higher incidence of double PIK3CA alterations. Most double PIK3CA alterations consisted of a hotpsot alteration and an uncommon alteration; they were often clonal and present within a single tumor population. Multiregional analyses of CRCs with predicted subclonal double-alterations revealed multiclonal CRCs with divergent PIK3CA variant status originating from a common APC- and KRAS-mutated founder lineage of adenoma. CONCLUSIONS: The findings supported multiclonal CRCs resulting from parallel evolution during the progression from adenoma to adenocarcinoma within the mitogen-activated protein kinase pathway, as previously demonstrated, or the mammalian target of rapamycin pathway. Further studies are warranted to elucidate clinical significance and potential targeted therapy for CRC patients with double PIK3CA alterations and impacts on clinical decision-making in patients with multiclonal CRCs harboring divergent PIK3CA mutational status.

IDH1 and IDH2 Mutations in Colorectal Cancers.

OBJECTIVES: To elucidate clinicopathologic and molecular characteristics of IDH1 and IDH2 (IDH1/2) mutations in colorectal cancers (CRCs). METHODS: We evaluated IDH1/2 mutations in 1,623 CRCs using a next-generation sequencing assay. RESULTS: IDH1/2 mutations, predominantly IDH1 p.R132C, were detected in 15 (0.9%) CRCs and in 5 (3.0%) of 167 BRAF p.V600E-mutated CRCs. Three IDH1/2-mutated CRCs were associated with inflammatory bowel disease. They were significantly associated with old age, mucinous or signet ring cell adenocarcinoma, and high-grade histomorphology. Concordance of variant allele frequency between IDH1/2 mutants and other trunk drivers in CRCs and presence of IDH1/2 mutation in the adenoma and early adenocarcinoma indicated IDH1/2 mutations could be trunk drivers suitable for targeted therapy. CONCLUSIONS: IDH1/2 mutations in CRCs were uncommon but enriched in BRAF p.V600E-mutated CRCs and perhaps colitis-associated CRCs. Further studies on IDH1/2-mutated CRCs are needed to clarify their clinicopathologic features and implications for targeted therapy.

Nuclear size measurement for distinguishing urothelial carcinomas from reactive urothelium on tissue sections.

BACKGROUND: Pathological diagnosis of urothelial carcinoma (UC) is primarily based on cytological atypia. It has previously been shown that high-grade (HG) UC, particularly UC in situ cells (CIS), can be over five times the size of a lymphocyte. However, this has not been demonstrated in comparison to reactive urothelium. The objective of this study was to empirically compare the difference in nuclear size of UC cells with reactive urothelial cells. METHODS: Using CellSens imaging software, we measured urothelial nuclear length (l) and width (w) on digital images of H&E sections. The area (a) of a nucleus was calculated based on the oval shape of most urothelial cells. Lymphocytes were measured to calculate normalized urothelial linear and area ratios. RESULTS: A total of 1085 urothelial cell nuclei from 60 cases were measured from reactive urothelium, low grade (LG) UC, HG UC and CIS. CIS nuclei were found to have an a 2.75 times larger than reactive nuclei (p < 0.001). A nuclear size cut-off of 11 um for l and 7 um for w was found to be sensitive [98.09 % (95 % CI: 95.60-99.38 %) and 89.31 % (95 % CI: 83.6-91.82 %) for l and w, respectively] and specific [92.60 % (95 % CI: 87.13-95.82 %) and 85.71 % (95 % CI: 79.49-90.63 %) for l and w, respectively] for distinguishing CIS from reactive atypia. CONCLUSIONS: Nuclear morphometry can be used to differentiate CIS from reactive atypia. A l over 11 um and a w over 7 um and is highly sensitive and specific for CIS compared to reactive urothelium. This difference in nuclear size may be used as a tool for differentiating the flat urothelial lesions from reactive urothelium in daily practice.

A randomized phase II presurgical trial of transdermal 4-hydroxytamoxifen gel versus oral tamoxifen in women with ductal carcinoma in situ of the breast.

PURPOSE: Local transdermal therapy to the breast may achieve effective target-organ drug delivery, while diminishing systemic effects. We conducted a randomized, double-blind, placebo-controlled phase II trial comparing transdermal 4-hydroxytamoxifen gel (4-OHT) to oral tamoxifen (oral-T) in women with ductal carcinoma in situ (DCIS). METHODS: Twenty-seven pre- and postmenopausal women were randomized to 4-OHT (4 mg/day) or oral-T (20 mg/day) for 6 to 10 weeks before surgery. Plasma, nipple aspirate fluid, and breast adipose tissue concentrations of tamoxifen and its major metabolites were determined by liquid chromatography/tandem mass spectrometry. The primary endpoint was Ki67 labeling in DCIS lesions, measured by immunohistochemistry. In plasma, insulin-like growth factor-1 (IGFI), sex hormone-binding globulin (SHBG), and coagulation protein concentrations were determined. RESULTS: Posttherapy Ki67 decreased by 3.4% in the 4-OHT and 5.1% in the oral-T group (P ≤ 0.03 in both, between-group P = 0. 99). Mean plasma 4-OHT was 0.2 and 1.1 ng/mL in 4-OHT and oral groups, respectively (P = 0.0003), whereas mean breast adipose tissue concentrations of 4-OHT were 5.8 ng/g in the 4-OHT group and 5.4 ng/g in the oral group (P = 0.88). There were significant increases in plasma SHBG, factor VIII, and von Willebrand factor and a significant decrease in plasma IGFI with oral-T, but not with 4-OHT. The incidence of hot flashes was similar in both groups. CONCLUSIONS: The antiproliferative effect of 4-OHT gel applied to breast skin was similar to that of oral-T, but effects on endocrine and coagulation parameters were reduced. These findings support the further evaluation of local transdermal therapy for DCIS and breast cancer prevention.

Lung transplantation in patients 60 years and older: results, complications, and outcomes.

BACKGROUND: Advanced recipient age is reported to negatively affect survival after lung transplantation (LTX). We hypothesized that LTX in patients aged > or = 60 years could be performed with acceptable outcomes. METHODS: We identified 182 consecutive LTX recipients from 1995 to 2005. Outcomes were analyzed and survival compared with results in recipients aged < 60, as well as with United Network for Organ Sharing (UNOS) registry outcomes for the same age and study period. Actuarial survivals were calculated by the Kaplan-Meier method. RESULTS: During the study period, 29% (52/182) of LTX recipients were > or = 60 years old (range, 60 to 69 years). Median follow-up was 2.9 years (range, 0 to 10 years). All patients but one received a single lung. Indications included chronic obstructive pulmonary disease in 63% (33/52), idiopathic pulmonary fibrosis in 27% (14/52), and other in 10% (5/52). In-hospital mortality was 12% (6/52) for those aged > or = 60 compared with 7% (9/130) for those aged < 60 (p = NS). Complications included reoperation in 10% (5/52), requirement for extracorporeal membrane oxygenation in 6% (3/52), renal failure in 12% (6/52), and stroke in 4% (2/52). Actuarial survivals at 30 days, and 1, 3, and 5 years were 90% (82, 98), 86% (76, 96), 71% (56, 85), and 55% (37, 73), respectively. No significant difference in survival was observed between age cohorts for our institutional data by Kaplan-Meier analysis (p = 0.34) or by Cox proportional hazard model (p = 0.15). A significant survival advantage was noted for our institution compared with UNOS for this cohort (p = 0.018). CONCLUSIONS: In carefully selected recipients > or = 60 years of age, LTX offers acceptable outcomes and survival.