Co-active receptor tyrosine kinases mitigate the effect of FGFR inhibitors in FGFR1-amplified lung cancers with low FGFR1 protein expression.

Targeted therapies are effective in subsets of lung cancers with EGFR mutations and anaplastic lymphoma kinase (ALK) translocations. Large-scale genomics have recently expanded the lung cancer landscape with FGFR1 amplification found in 10-20% of squamous cell carcinomas (SCCs). However, the response rates have been low for biomarker-directed fibroblast growth factor receptor (FGFR) inhibitor therapy in SCC, which contrasts to the relatively high rates of response seen in EGFR mutant and ALK-translocated lung cancers treated with epidermal growth factor receptor (EGFR) inhibitors and ALK inhibitors, respectively. In order to better understand the low response rates of FGFR1-amplified lung cancers to FGFR inhibitors, relationships between gene copy number, mRNA expression and protein expression of FGFR1 were assessed in cell lines, tumor specimens and data from The Cancer Genome Atlas. The importance of these factors for the sensitivity to FGFR inhibitors was determined by analyzing drug screen data and conducting in vitro and in vivo experiments. We report that there was a discrepancy between FGFR1 amplification level and FGFR1 protein expression in a number of these cell lines, and the cancers with unexpectedly low FGFR1 expression were uniformly resistant to the different FGFR inhibitors. Further interrogation of the receptor tyrosine kinase activity in these discordant cell lines revealed co-activation of HER2 and platelet-derived growth factor receptor-α (PDGFRα) caused by gene amplification or ligand overexpression maintained phosphoinositide 3-kinase (PI3K) and MEK/ERK signaling even in the presence of FGFR inhibitor. Accordingly, co-inhibition of FGFR1 and HER2 or PDGFRα led to enhanced drug responses. In contrast, FGFR1-amplified high FGFR1 protein-expressing lung cancers are sensitive to FGFR inhibitor monotherapy by downregulating ERK signaling. Addition of a PI3K inhibitor to these high FGFR1 protein-expressing cancers further sensitized them to FGFR inhibitor. These data reveal that biomarker-directed trials for FGFR1-amplified SCC require assessment of FGFR1 protein expression and uncover novel therapeutic strategies for FGFR1-amplified SCC with low FGFR1 protein expression.

Hysteroscopic selective salpingography.

OBJECTIVE: To evaluate the effectiveness of hysteroscopic selective salpingography (HSS) as a method for diagnosing the tubal proximal occlusion shown by hysterosalpingography (HSG). DESIGN: Prospective study. SETTING: Outpatient Department of Obstetrics and Gynecology, Social Insurance Saitama Chuo Hospital, Urawa, Japan. PATIENT(S): A total of 572 infertile women underwent HSG. Forty-seven of 50 women with unilateral or bilateral proximal tubal occlusion demonstrated by HSG underwent HSS. INTERVENTION(S): Hysteroscopic selective salpingography was performed for the diagnosis of tubal occlusion in cases in which the proximal tubal occlusion was shown by HSG. MAIN OUTCOME MEASURE(S): Number of patients who underwent HSS and pregnancy rate after HSS. RESULT(S): Twenty-seven (79.4%) of 34 patients with unilateral occlusion diagnosed by HSG were shown to have normal patency by HSS. Of 12 women with bilaterally normal patent tubes confirmed by HSS, 8 (66.7%) achieved normal pregnancies within 1 year. Seven (53.8%) of 13 patients with bilateral occlusion found by HSG were shown to have normally patent tubes by HSS. CONCLUSION: The simple method of HSS was clinically effective for evaluating the presence of proximal tubal occlusion.

Gestational choriocarcinoma whose responsible pregnancy was a complete hydatidiform mole identified by PCR analysis with new sequence tagged site primers.

We report a case where the pregnancy responsible for a gestational choriocarcinoma was not the antecedent pregnancy or the second normal term delivery, but a complete hydatidiform mole that had advanced to clinically invasive mole. This responsible pregnancy was identified by polymerase chain reaction analysis (PCR). PCR analysis was performed by using five new sets of sequence-tagged site (STS) primers on four chromosomes (chr. 1, D1S225; chr. 3, D3S1744; chr. 12, D12S1090; chr. 18, D18S849 and D18S877). The constitution of alleles of choriocarcinoma was shown to be almost identical with that of the husband on every marker. The allele patterns of choriocarcinoma on D3S1744 and D12S1090 were not observed with DNA from the patient. The band pattern originating from molar DNA was also identical with those of the husband and choriocarcinomas on D18S849 and D1S225.

A study on the correlation between maturation and quality of human oocytes.

The success ratio of live birth after human in vitro fertilization and embryo transfer program is still around 12% Per oocyte retrieval cycle. A large number of mature oocytes became degenerate and unable to develop after insemination or implantation. In order to determine this discrepancy, assessment of maturation process as well as cytoplasmic maturity of the oocyte was performed. Initiation and completion of maturation were dependent on steroid metabolism and cyclic AMP in the oocyte cytoplasm. Extracellular calcium is another determinant factor of oocyte maturation. Phosphorylation of 23.5kD and 29.5kD protein was observed in the cytoplasm during maturation. In addition to cAMP, nuclear maturation is regulated by protein tyrosine phosphorylation in the cytoplasm. An inhibitor of Na+/H+ antiport appears to be effective in the procedure of cryopreservation but has no relationship with oocyte maturation. Quality of the oocyte consists of nuclear and cytoplasmic maturation.

Effect of KN-62, a selective inhibitor of calmodulin-dependent kinase II, on mouse oocyte activation.

PURPOSE: Our purpose was to determine the association of calmodulin-dependent protein kinase II (CaMKII) with oocyte activation and to explore the network of protein kinases during mammalian fertilization. METHODS: Mouse M-II oocytes were collected after superovulation induced by PMSG-hCG injection. The oocytes were inseminated or artificially activated by Ca ionophore (A23187) or 12-O-tetradecanoyl phorbol 13-acetate (TPA). The effects of KN-62, a specific and selective inhibitor of calmodulin-dependent protein kinase II, on second polar body emission (2PBE), pronuclear formation (PF), and cortical granule exocytosis (CGE) during fertilization or after artificial oocyte activation were investigated. RESULTS: KN-62 inhibited 2PBE and PF after sperm or Ca ionophore inducing activation. Additionally, PF was inhibited by KN-62 after TPA activation, whereas KN-62 did not inhibit CGE in any case. KN-04, an inactive form of KN-62, did not inhibit significantly 2PBE, CGE, or PF. When oocytes were exposed to KN-62 after Ca ionophore or TPA activation, no inhibitory effects on 2PBE or PF were observed. CONCLUSIONS: The CaMKII activation that occurs after fertilization or artificial activation of mouse oocytes is presumably secondary to increases in the intracellular free calcium concentration. As determined by the use of inhibitor, CaMKII activity is associated with 2PBE and PF but not with CGE.

Egg activation induced by osmotic pressure change and the effects of amiloride on the cryopreservation of mouse oocytes.

Activation of oocytes is caused by osmotic pressure change in some species. However, cryopreservation of oocytes occurs in the presence of osmotic pressure change induced by cryoprotectants. We investigated the effect of 5-(N,N,-dimethyl)-amiloride (NNDMA), a selective inhibitor of Na+/H+ exchange, on the cryopreservation and osmotic activation of mouse oocytes. The percentage (23.2%) of degenerate oocytes after cryopreservation in the presence of NNDMA was found to be lower than that (39.5%) of untreated oocytes. After thawing, the percentage (23.6%) of oocytes which could be fertilized following cryopreservation in the presence of NNDMA was significantly higher than that of untreated (18.0%) oocytes. These results suggest that amiloride increased the survival rate after thawing following cryopreservation. To investigate the effect of NNDMA on oocyte activation caused by the cryoprotectant, dimethyl sulphoxide (DMSO) was used to induce osmotic pressure change. NNDMA was found to inhibit cortical granule exocytosis, the second polar body emission and pronuclear formation which occurs upon activation due to osmotic pressure change. It also inhibited the increase in phosphorylation of many proteins including 33 and 45 kDa proteins, which occurs, during fertilization and chemical oocyte activation. In contrast, protein phosphorylation was not inhibited by W7, a calmodulin inhibitor. The actions of these inhibitors suggest that oocyte activation induced by osmotic pressure change involves a pathway mediated by Na+/H+ exchange which may be distinct from the Ca-calmodulin pathway. Amiloride may be a useful drug for increasing the rate of survival of cryopreserved oocytes.

The acceptability of prenatal diagnosis in Japan.

Prenatal diagnosis has been performed more frequently in Japan recently, but is still less popular than in the United States or Europe. Legal arrangements, insufficient economic support, and insufficient medical information provided to future parents may explain this difference. The acceptability of prenatal diagnosis, based on the concept of the cost of side effects and elective abortion, was found to be similar when examined through decision analysis and direct survey. Amniocentesis was considered useful for more than half of couples in Japan when the incidence of chromosomal abnormality is more than 0.5% and proper information about the decision to undergo this examination is provided to the pregnant woman and her family.

Analysis of cytoplasmic factors in developmental cleavage of mouse embryo.

One-cell embryos from certain mouse strains were found incapable of developing beyond the 2-cell stage in vitro (2-cell block), but a microinjection of EDTA effectively overcame this block. When 2-cell arrested embryos were fused with embryos that had developed to the late 2-cell stage in vivo, the fusants developed beyond the 2-cell stage. Microinjection of cytoplasm of in vivo 2-cell embryos into 1-cell embryos also obviated the 2-cell block. Analyses of 35S-labeled embryos by 2-dimensional polyacrylamide gel electrophoresis indicated changes in synthetic protein patterns possibly related to this block.

Cooperative inhibitory effect of follicular fluid and cAMP on hamster oocyte maturation.

Porcine or human follicular fluid inhibited the spontaneous maturation of isolated hamster oocytes in vitro during the first 1.5 h of culture. Moreover, the presence of 50% follicular fluid combined with 100 microM dbcAMP cooperatively reduced the incidence of germinal vesicle breakdown. The addition of FSH also inhibited the resumption of meiosis, and the presence of LH did not overcome the inhibitory effects of follicular fluid and tended to impede isolated hamster oocyte maturation in vitro.

Microvasculature of preovulatory follicles: comparison of in situ and in vitro perfused rabbit ovaries following stimulation of ovulation.

Perifollicular vasculature undergoes significant morphologic changes as ovulation approaches. These vascular changes were observed in in vitro perfused and in situ rabbit ovaries by means of scanning electron microscopy of microcorrosion casts. Casts were made in in situ unstimulated ovaries, in situ ovaries stimulated with human chorionic gonadotropin, in vitro perfused unstimulated ovaries, and in vitro perfused ovaries after an ovulation-inducing dose of human chorionic gonadotropin, prostaglandin F2 alpha, histamine, or norepinephrine. Dilated vessels, extravasation of resin from weakened vessels, and filling defects at the apex of the follicle were observed in in situ ovaries 9 to 12 hours after stimulation and in in vitro perfused ovaries 4 to 6 hours after human chorionic gonadotropin. In vitro perfused ovaries stimulated with prostaglandin F2 alpha or histamine demonstrated dilated capillaries with extravasation of the resin and filling defects at the apex of large follicles. Norepinephrine-stimulated ovaries showed incomplete filling of vessels, although some large follicles showed extravasation of resin. The occurrence of dilated vessels, extravasation of resin, and filling defects is indicative of preovulatory vascular changes in both in situ and in vitro perfused ovaries, regardless of the ovulatory stimulus.

Examination of the role of calcium in ovulation in the in vitro perfused rabbit ovary with use of ethyleneglycol-bis(beta-aminoethyl ether)-n,n'-tetraacetic acid and verapamil.

The in vitro perfused rabbit ovary preparation was used to examine the role of calcium in the ovulatory process. Two groups of rabbits were studied. In the first group, verapamil hydrochloride (10(-4) mol/L), a calcium channel blocker, was used together with human chorionic gonadotropin (50 IU) in the perfusate. Verapamil had no apparent effect on human chorionic gonadotropin-induced ovulation. Verapamil treatment, however, significantly reduced the percentage of ovulated ova that were mature (68.8%) in comparison to ovulated ova from human chorionic gonadotropin-treated control ovaries (95.0%). In a second experimental group, ethyleneglycol-bis(beta-aminoethyl ether)-n,n'-tetraacetic acid (2.0 mmol/L), a calcium ion chelator, was included in the perfusate with gonadotropin. The ethyleneglycol-bis(beta-aminoethyl ether)-n,n'-tetraacetic acid significantly reduced ovulatory efficiency (16.7% +/- 9.43%) in comparison to that of controls exposed to human chorionic gonadotropin alone (79.5% +/- 11.1%). In addition, ovulation occurred at an earlier time in ovaries perfused with ethyleneglycol-bis(beta-aminoethyl ether)-n,n'-tetraacetic acid; however, only four ovulations occurred in these ovaries. These four ovulated ova were immature, probably reflecting the early time of ovulation. Furthermore, both verapamil and ethyleneglycol-bis(beta-aminoethyl ether)-n,n'-tetraacetic acid blocked ovarian smooth muscle contractions during ovarian perfusion. These data provide additional support for the concept that calcium dynamics influence the processes of ovulation and ovum maturation. Furthermore ovarian smooth muscle contractions do not appear to be essential for ovulation in this model.

The relationship between prostaglandins and histamine in the ovulatory process as determined with the in vitro perfused rabbit ovary.

The process of follicle rupture has been described as an inflammatory reaction in which prostaglandins (PGs) and/or histamine may be involved. With an in vitro perfused rabbit ovary preparation, experiments were carried out for determination of whether a relationship exists among PGs, histamine, and ovulation. PGF2 alpha alone was capable of inducing ovulation when added to the perfusion fluid at 1, 10, and 100 ng/ ml. Effectiveness in achieving ovulation varied directly with the dosage; however, the ovulatory efficiency of PGF2 alpha-treated ovaries was lower than that of ovaries exposed to human chorionic gonadotropin (hCG, 100 IU). PGF2 alpha-induced ovulation could not be blocked by the H2 receptor antagonist, cimetidine. The PG synthesis inhibitor, indomethacin, did not prevent histamine-induced ovulation. Ovulation induced by hCG was partially blocked by the administration of indomethacin; however, the concomitant administration of cimetidine was not associated with further reduction in ovulation. In all but one experimental group, the majority of ovulated ova did not progress beyond the intact germinal vesicle stage unless the ovaries had been exposed to hCG. On the basis of these experiments, PGs and histamine do not appear to be interdependent in their effects on the ovulatory process in vitro.

Direct ovarian effect of clomiphene citrate in the rabbit.

The effects of clomiphene citrate (CC) on ovulation and ovum maturation were studied using the isolated perfused rabbit ovary. CC (10(-5) M) added to the perfusate with human chorionic gonadotropin (50 IU) did not affect ovulatory efficiency, ovulation time, oocyte maturation, or degeneration of ovulated ova and follicular oocytes. During perfusion without human chorionic gonadotropin, the percentage of follicular oocytes with germinal vesicle breakdown was significantly increased in response to CC (10(-5) M or 10(-7) M); a greater percentage of follicular oocytes was degenerated. Estradiol (100 ng/ml) added to the perfusate reversed the effect of CC on degeneration of follicular oocytes. Of follicular oocytes from ovaries perfused with CC, 79.3% were degenerated; in contrast, 25% were degenerated in ovaries treated with CC plus estradiol. These data suggest that CC has a direct ovarian effect and that ovum degeneration associated with CC may be related to an antiestrogenic action.

Influence of calcium and magnesium deprivation on ovulation and ovum maturation in the perfused rabbit ovary.

The role of calcium (Ca++) and magnesium (Mg++) in the ovulation process was studied using in vitro perfused rabbit ovaries. Ovaries were perfused with or without human chorionic gonadotropin (hCG) in Ca++/Mg++-free medium (M199) alone or combined with standard M199 to yield varying concentrations of Ca++ and/or Mg++. In all ovaries perfused with hCG, ovulatory efficiency was similar regardless of the concentration of Ca++ and/or Mg++. In ovaries perfused in Ca++/Mg++-free medium without hCG, ovulatory efficiency was similar to that in ovaries perfused with hCG. As Ca++/Mg++ levels were increased without hCG, ovulatory efficiency declined. Ovulation time was significantly accelerated in ovaries perfused in Ca++/Mg++-free medium with or without hCG. Most ovulated ova from ovaries perfused without hCG were immature. With hCG, degree of ovum maturity was directly related to ovulation time. Ovarian smooth muscle contractions were undetectable in 3 ovaries perfused in Ca++/Mg++-free M199 despite occurrence of ovulation. Smooth muscle contractions were recorded in 2 of 3 ovaries perfused in standard M199 with hCG. These results indicate: 1) Ca++/Mg++ exclusion results in rapid follicle rupture and immature ova; 2) oocyte maturation appears to be gonadotropin-dependent; 3) ovulation occurs in the absence of ovarian smooth muscle contractions during perfusion with Ca++/Mg++-free medium.