Optimal sequencing of ibrutinib, idelalisib, and venetoclax in chronic lymphocytic leukemia: results from a multicenter study of 683 patients.

Background: Ibrutinib, idelalisib, and venetoclax are approved for treating CLL patients in the United States. However, there is no guidance as to their optimal sequence. Patients and methods: We conducted a multicenter, retrospective analysis of CLL patients treated with kinase inhibitors (KIs) or venetoclax. We examined demographics, discontinuation reasons, overall response rates (ORR), survival, and post-KI salvage strategies. Primary endpoint was progression-free survival (PFS). Results: A total of 683 patients were identified. Baseline characteristics were similar in the ibrutinib and idelalisib groups. ORR to ibrutinib and idelalisib as first KI was 69% and 81%, respectively. With a median follow-up of 17 months (range 1-60), median PFS and OS for the entire cohort were 35 months and not reached. Patients treated with ibrutinib (versus idelalisib) as first KI had a significantly better PFS in all settings; front-line [hazard ratios (HR) 2.8, CI 1.3-6.3, P = 0.01], relapsed-refractory (HR 2.8, CI 1.9-4.1, P < 0.001), del17p (HR 2.0, CI 1.2-3.4, P = 0.008), and complex karyotype (HR 2.5, CI 1.2-5.2, P = 0.02). At the time of initial KI failure, use of an alternate KI or venetoclax had a superior PFS when compared with chemoimmunotherapy. Furthermore, patients who discontinued ibrutinib due to progression or toxicity had marginally improved outcomes if they received venetoclax (ORR 79%) versus idelalisib (ORR 46%) (PFS HR .6, CI.3-1.0, P = 0.06). Conclusions: In the largest real-world experience of novel agents in CLL, ibrutinib appears superior to idelalisib as first KI. Furthermore, in the setting of KI failure, alternate KI or venetoclax therapy appear superior to chemoimmunotherapy combinations. The use of venetoclax upon ibrutinib failure might be superior to idelalisib. These data support the need for trials testing sequencing strategies to optimize treatment algorithms.

Relaxin increases secretion of matrix metalloproteinase-2 and matrix metalloproteinase-9 during uterine and cervical growth and remodeling in the pig.

Matrix metalloproteinases are proteolytic enzymes that degrade the extracellular matrix and are essential for tissue remodeling. Uterine and cervical growth require remodeling of structural barriers to cell invasion and matrix metalloproteinase-2 and -9 degrade type IV collagen, the major component of basement membranes. Relaxin stimulates uterine and cervical growth and remodeling, which includes remodeling of support elements such as basement membranes. The objective of this study was to determine whether relaxin alters the production and/or activity of matrix metalloproteinase-2 and -9 in the uterus or cervix of the pig. The growth-promoting effects of relaxin were elicited by administering relaxin to prepubertal gilts every 6 h for 54 h. The expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 was characterized by gel zymography, and proteins were quantified by immunoblotting. Total enzyme activity was measured using matrix metalloproteinase-specific fluorescent substrate assays. In both uterine and cervical tissues, immunoreactive matrix metalloproteinase-2 and matrix metalloproteinase-9 protein expression was similar in relaxin-treated and control animals. However, tissue-associated gelatinase activity was attenuated by relaxin (P < 0.05). In contrast, relaxin significantly increased the secretion of active matrix metalloproteinase-2 and -9 protein into uterine fluid (P < 0.05). Given the importance of matrix metalloproteinases in extracellular matrix degradation, the observation that relaxin promotes uterine secretion of matrix metalloproteinase-2 and -9 supports the concept that relaxin facilitates the growth and remodeling of reproductive tissues by increasing extracellular proteolysis in the pig reproductive tract.

Expression of uterine and cervical epithelial cadherin during relaxin-induced growth in pigs.

Epithelial cadherin (E-cadherin), a member of the cadherin family of calcium-dependent adhesion molecules, is present in reproductive tissues. Relaxin, a hormone important for uterine and cervical growth in pigs, increases the expression of E-cadherin in the MCF-7 mammary epithelial cell line. The objective of this study was to characterize the expression of E-cadherin during relaxin-induced growth of the uterus and cervix in an immature pig model, independent of high circulating steroids. After administration of relaxin to prepubertal gilts, the uterus and cervix were collected. E-cadherin mRNA and protein were measured by northern and western blot analysis, respectively. A 120 kDa protein band, corresponding to E-cadherin, was detected in all tissues examined. Relaxin significantly (P < 0.05) increased the amount of E-cadherin protein in the uterus (P < 0.05), whereas no significant changes were observed in E-cadherin protein in the cervix. A 4.2 kb E-cadherin transcript was detected in all tissues and E-cadherin mRNA was significantly higher (P < 0.05) in uteri from relaxin-treated gilts compared with control gilts. E-cadherin was localized by immunocytochemistry to the epithelial cells of the uterine and cervical lumen, and the uterine glandular epithelium. Quantitative analysis revealed that administration of relaxin significantly increased (P < 0.05) the height of the uterine luminal epithelium compared with that of the controls. This is the first report of the expression of E-cadherin in the uterus and cervix of pigs. The findings from this study indicate that relaxin increases the expression of uterine E-cadherin in the reproductive tract of pigs. Administration of relaxin to prepubertal gilts in vivo increased uterine epithelial cell growth independent of circulating steroids, with a concomitant increase in E-cadherin expression.

Expression of connexin-26, -32, and -43 gap junction proteins in the porcine cervix and uterus during pregnancy and relaxin-induced growth.

Connexin (CX) proteins participate in growth, differentiation, and tissue remodeling. Relaxin-stimulated reproductive tissue growth and remodeling may be facilitated by enhanced intracellular communication. This study was an examination of the effects of relaxin in vivo on expression of CX-26, CX-32, and CX-43 in the cervix and uterus of prepubertal pigs. In addition, expression of these proteins was monitored in the sow uterus during pregnancy. Relaxin was administered to prepubertal gilts every 6 h for 54 h. CX expression was characterized by immunoblotting and localized by immunofluorescence. Significant increases in all three CXs were observed in the cervix following relaxin treatment (P < 0.05). Uterine CX proteins were also significantly higher (P < 0.05) in relaxin-treated animals compared to controls. The CX protein level in relaxin-treated animals was similar to that observed during the second half of pregnancy, but below levels found in mature, nonpregnant sows. This is the first evidence for specific CX expression in the porcine cervix, and the first study to show that relaxin increases the expression of CX proteins in the porcine uterus and cervix. The data show that CX proteins are differentially regulated in the uterus of the pig during pregnancy. These data support a role for CX-mediated communication during relaxin-induced reproductive tissue growth and remodeling.

Estrogen regulation of angiotensin-converting enzyme mRNA.

Estrogen replacement therapy is cardioprotective in postmenopausal women; however, the precise molecular mechanisms for this modulation are not fully elucidated. We previously showed that chronic estrogen replacement therapy reduced angiotensin-converting enzyme (ACE) activity in tissue extracts and serum with an associated reduction in plasma angiotensin II. A reverse transcriptase-polymerase chain reaction assay was developed to determine whether estrogen treatment regulates tissue ACE mRNA concentration. Total RNA was isolated from kidney cortex, kidney medulla, lung, and aorta of ovariectomized Sprague-Dawley rats after 21 days of chronic 17beta-estradiol replacement therapy (5 mg pellet per rat SC) or placebo. A marked decrease in densitometric intensity ratios of amplified ACE cDNA to elongation factor-1alpha control cDNA was observed in all tissues from placebo-treated rats compared with the estradiol-treated rats (renal cortex: 0.29+/-0.04 versus 0.14+/-0.02; renal medulla: 0. 37+/-0.04 versus 0.24+/-0.03; lung: 4.49+/-0.37 versus 2.49+/-0.59; and aorta: 0.41+/-0.04 versus 0.29+/-0.02; all P<0.05). A comparable reduction in ACE activity was detected in tissue extracts from kidney cortex, kidney medulla, and lung of hormone-treated animals. Incubation of purified rat lung ACE with 1 or 10 micromol/L 17beta-estradiol had no effect on enzyme activity. These results suggest that estrogen treatment regulates tissue ACE activity by reducing ACE mRNA concentrations. Thus, the beneficial cardiovascular effects of estrogen may be mediated in part by downregulation of ACE with a consequent reduction in the circulating levels of the vasoconstrictor angiotensin II, a decrease in the metabolism of the vasodilator bradykinin, and an increase in the production of the vasorelaxant angiotensin-(1-7).

Relaxin increases insulin-like growth factors (IGFs) and IGF-binding proteins of the pig uterus in vivo.

Relaxin promotes growth of reproductive tissues, including the uterus. Although we have evidence of a role for insulin-like growth factor I (IGF-I) in mediating relaxin-induced growth of porcine granulosa cells in vitro, the mechanism of action by which relaxin enhances uterine growth has not been identified. To investigate a role for the uterine insulin-like growth factor (IGF) system in relaxin-induced uterine growth, we monitored the effects of relaxin on porcine IGFs and IGF-binding proteins (IGFBPs) in vivo. The trophic effects of relaxin on the uterus were elicited by administering relaxin or saline to prepubertal gilts every 6 h for 54 h. Three hours after the last injection, uterine flushes, uteri, follicular fluid, and ovaries were collected. Estradiol was measured in plasma and follicular fluid to confirm the prepubertal status of each animal. Significantly higher concentrations of uterine lumen IGF-I (P < 0.05) and IGF-II (P < 0.01) were observed in animals treated with relaxin. However, relaxin administration did not affect uterine IGF-I and -II gene expression, as determined by a ribonuclease protection assay and Northern analysis, respectively. In uterine flushes, relaxin treatment increased an IGFBP doublet (33 and 34.5 kDa) and IGFBP-3. The uterine IGFBP doublet was identified as IGFBP-2 by immunoprecipitation. Plasma or follicular fluid IGFs and IGFBPs were unaffected by relaxin administration. In addition, relaxin did not influence IGF-I binding to its uterine receptor. This is the first study to demonstrate regulation of the pig uterine IGF system by relaxin. In conclusion, the data point to IGF-I, IGF-II, IGFBP-2, and IGFBP-3 as putative mediators of relaxin-induced uterine growth in the pig.