Therapeutic potential of the poly(ADP-ribose) polymerase inhibitor rucaparib for the treatment of sporadic human ovarian cancer.

Here, we investigate the potential role of the PARP inhibitor rucaparib (CO-338, formerly known as AG014699 and PF-01367338) for the treatment of sporadic ovarian cancer. We studied the growth inhibitory effects of rucaparib in a panel of 39 ovarian cancer cell lines that were each characterized for mutation and methylation status of BRCA1/2, baseline gene expression signatures, copy number variations of selected genes, PTEN status, and sensitivity to platinum-based chemotherapy. To study interactions with chemotherapy, we used multiple drug effect analyses and assessed apoptosis, DNA fragmentation, and γH2AX formation. Concentration-dependent antiproliferative effects of rucaparib were seen in 26 of 39 (67%) cell lines and were not restricted to cell lines with BRCA1/2 mutations. Low expression of other genes involved in homologous repair (e.g., BCCIP, BRCC3, ATM, RAD51L1), amplification of AURKA or EMSY, and response to platinum-based chemotherapy was associated with sensitivity to rucaparib. Drug interactions with rucaparib were synergistic for topotecan, synergistic, or additive for carboplatin, doxorubicin or paclitaxel, and additive for gemcitabine. Synergy was most pronounced when rucaparib was combined with topotecan, which resulted in enhanced apoptosis, DNA fragmentation, and γH2AX formation. Importantly, rucaparib potentiated chemotherapy independent of its activity as a single agent. PARP inhibition may be a useful therapeutic strategy for a wider range of ovarian cancers bearing deficiencies in the homologous recombination pathway other than just BRCA1/2 mutations. These results support further clinical evaluation of rucaparib either as a single agent or as an adjunct to chemotherapy for the treatment of sporadic ovarian cancer.

Frequent EVI1 translocations in myeloid blast crisis CML that evolves through tyrosine kinase inhibitors.

Clinical variables associated with ecotropic viral integration site 1 (EVI1) translocations were evaluated in 42 consecutive chronic myeloid leukemia (CML) patients in myeloid blast crisis (MBC). Translocations were confirmed with fluorescence in situ hybridization, and Western blot analysis demonstrated EVI1 expression. Translocations of EVI1 were present in 3 of 24 (12%) patients whose disease evolved MBC before tyrosine kinase inhibitor (TKI) exposure, and 7 of 18 (39%) patients who had received one or more TKIs. Univariate analysis showed that prior TKI therapy was the only clinical variable that was significantly associated with EVI1 translocation (P = 0.047). TKI-resistant BCR-ABL1 mutations were present in 71% of MBC patients with EVI1 translocations at the time of disease progression. These observations suggest that EVI1 overexpression collaborates with BCR-ABL1 in the evolution of TKI-resistant MBC. Inhibition of c-ABL kinase-mediated DNA double-strand repair by TKIs may predispose to EVI1 translocation in this setting.

Developmental or degenerative--NR2E3 gene mutations in two patients with enhanced S cone syndrome.

PURPOSE: Enhanced S Cone Syndrome is a rare autosomal recessive disorder characterized clinically by an absence of rod function, a replacement of most L and M cone function by S cone activity (Goldmann-Favre Syndrome) and by variable degrees of retinal degeneration in different families. The causative gene, nuclear receptor subfamily 2, group E, member 3 (NR2E3), controls the developmental sequence for rods and cones. The purpose of this study was to compare the nature and implications of mutations in two subjects with Enhanced S Cone Syndrome who have significantly different degrees of degenerative damage. METHODS: A direct sequencing approach was used to identify the mutations. Genomic DNA was amplified from all the exons of NR2E3 and used as a template for sequencing. Of the two families studied, Case 1 is of Persian ethnicity while Case 2 is Brazilian. A total of six individuals within the two families were studied. RESULTS: Case 1 (original propositus of the syndrome) has the characteristic developmental rod/cone abnormality with large amplitude electroretinogram responses and no retinal degeneration. She was homozygous for a novel mutation, c.[del196-201del6] (p.G66-C67del), which lies entirely within the P-box for this gene. By comparison, Case 2 had Goldmann-Favre Syndrome with retinal degeneration and low electroretinogram signals. She was a compound heterozygote for c.[119-2A>C]+[del194-202del9] (p.N65-C67del), mutations that have been reported previously. Her second mutation overlaps that of Case 1 within the P-box. CONCLUSIONS: The novel in-frame homozygous deletion of Case 1, within the P-box motif of the DNA binding domain, caused a developmental abnormality without retinal degeneration. Case 2, with more traditional Goldmann-Favre Syndrome with retinal degeneration, was a compound heterozygote where one allele had a similar P-box deletion but the other was a splicing defect. Case 1 is the first reported homozygous deletion within the P-box. This is the first report of NR2E3 mutations in a Persian and a Brazilian family.

Expression of p16 and retinoblastoma determines response to CDK4/6 inhibition in ovarian cancer.

PURPOSE: PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer. EXPERIMENTAL DESIGN: We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and array CGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort of ovarian cancer patients. RESULTS: Concentration-dependent antiproliferative effects of PD-0332991 were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb-proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, RB, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration-and time-dependent manner, and enhanced the effects of chemotherapy. Rb-proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and was independently associated with poor progression-free survival (adjusted relative risk 1.49, 95% CI 1.00-2.24, P = 0.052). CONCLUSIONS: PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer.

Clonal hematopoiesis in Philadelphia chromosome-negative bone marrow cells of chronic myeloid leukemia patients receiving dasatinib.

A clonal cytogenetic abnormality was observed in Philadelphia chromosome-negative bone marrow cells of 6/27 chronic myeloid leukemia patients (+8 in 4, -7 in 1, and 20q- in 1) with dasatinib-induced remissions. The X-linked human androgen receptor gene assay demonstrated clonality in one additional patient. Single nucleotide polymorphism array analysis revealed somatic uniparental disomy involving chromosome 17(p12-pter) in another patient. The TP53 gene had a 5' splice site deletion of exon 6 that caused alternative splicing, frame shifting and introduction of a premature stop codon. After three years, no patient developed myelodysplastic syndrome or acute myeloid leukemia.

Candidate gene screening for posterior polymorphous dystrophy.

PURPOSE: To perform candidate gene screening for posterior polymorphous corneal dystrophy (PPCD). The initial 3 genes chosen, ID1, BCL2L1, and VSX1, lie within the region on chromosome 20 to which the PPCD gene has been linked, and mutations in VSX1 have previously been identified in patients with PPCD. METHODS: DNA extraction, PCR amplification, and direct sequencing of the VSX1, BCL2L1, and ID1 genes were performed in 14 affected patients (12 families) as well as in unaffected family members and healthy control subjects. RESULTS: No coding region mutations in the BCL2L1 or ID1 genes were identified in affected patients. In the VSX1 gene, the previously identified Gly160Asp missense change was not present in any of our 12 probands, and the Asp144Glu mutation was identified in 1 affected patient as well as 1 unaffected control individual. Additionally, 2 synonymous substitutions were identified, Ala182Ala (8 affected patients from 8 families) and Gly239Gly (1 affected patient and 1 unaffected patient from the same family). In the ID1 gene, the synonymous substitution Gly216Gly was observed in 2 affected patients (2 families) who also demonstrated a single nucleotide change in both the 5'UTR (2129T>C) and 3'UTR (3267A>G). Another 5'UTR change, 2177T>C, was identified in 1 affected patient and his unaffected parent, both of whom also demonstrated the 2129T>C and 3267A>G changes. CONCLUSIONS: None of the 12 probands with PPCD demonstrated the previously described Gly160Asp mutation within the VSX1 gene. The Asp144Glu missense change, present in an affected patient as well as an unaffected control individual, appears to be a rare polymorphism, not a disease-causing mutation. No coding region changes were identified in the ID1 or BCL2L1 genes. Therefore, although we report a number of novel polymorphisms in the VSX1 and ID1 genes, the failure to identify any sequence variants that sort with the disease phenotype suggests that other genetic factors are involved in PPCD.

Keratoconus--no association with the transforming growth factor beta-induced gene in a cohort of American patients.

PURPOSE: Keratoconus is a noninflammatory, corneal thinning disorder leading to mixed myopic and irregular astigmatism and implicated as a major reason for cornea transplantations in the Western world. Genetic factors have been suggested as a cause of keratoconus. The levels of transforming growth factor beta-induced (TGFBI) protein have been reported to be altered in keratoconus tissues. Mutations in this gene are responsible for causing various corneal dystrophies. Given this strong evidence of the involvement of this gene in corneal dystrophies, we investigated possible mutations within this gene in 15 probands of families with keratoconus. METHODS: All patients and control individuals had complete ophthalmological examination by a corneal specialist to determine their affectation status. The entire transcript of the TGFBI gene was analyzed by direct sequencing from patient DNA. RESULTS: We found 8 sequence variations within the gene, none of which was protein-altering changes. These changes were also observed in control individuals, and 4 are previously known polymorphisms. CONCLUSIONS: We concluded that the TGFBI gene is not responsible for causing keratoconus in these patients.

Comparative analysis of the FOXL2 gene and characterization of mutations in BPES patients.

Bleparophimosis ptosis epicanthus inversus syndrome (BPES) is a rare disorder characterized by eyelid malformation and in some cases associated with premature ovarian failure. Although the familial form is autosomal dominant, many cases are also sporadic. The mutations causing this disorder were found in a winged/forkhead transcription factor gene named FOXL2. We have sequenced the mouse homolog for the FOXL2 gene and identified the Fugu rubripes (pufferfish) ortholog from the database. By alignment of the three sequences, we found an almost complete conservation of the forkhead domain in the three species. There is 95% and 61% conservation at the protein level between human-mouse and human-pufferfish, respectively. The polyalanine and polyproline tracts within the gene are absent in Fugu rubripes. An overview identifies four breaks in the conservation of the gene within these species. Using a direct sequencing approach, we performed mutation analysis from DNA of nine affected individuals from familial and sporadic cases. The mutations are distributed throughout the coding region of the FOXL2 gene. We identified five novel mutations: g.292delG (E19fsX149); g.530G>A (W98X); g.548A>G (H104R); g.652G>T (E139X); and g.1178_1185del8 (A314fsX530). In addition we also identified two known mutations g.823C>T (Q196X) and g.1092_1108dup17, the latter in individuals from three unrelated pedigrees.

Identification of GUCY2D gene mutations in CORD5 families and evidence of incomplete penetrance.

Cone rod dystrophy 5 (CORD5) is an autosomal dominant retinal disease that primarily affects cone function. The locus has previously been mapped to human chromosome 17p12-p13 between the markers D17S926/D17S849 and D17S945/D17S804. One of our "unaffected" recombinant individual from family 1175 was subsequently found to cross through this interval. Reexamination revealed that he was in fact mildly affected. This expanded the minimum candidate region. Direct sequencing of the GUCY2D and other candidate genes within this interval was carried out on 2 American families affected with CORD5. There was an R838C missense mutation within the GUCY2D gene in one and a R838H missense mutation in another families. The previously reported mutations for CORD6 are clustered at the same position within the gene. These results indicate that both CORD5 (MIM# 600977) and CORD6 (MIM# 601777) are actually the same disease. We conclude that significant variability in expression and incomplete penetrance exists even within one family.

A pooled case-control study of the apolipoprotein E (APOE) gene in age-related maculopathy.

Age-related maculopathy (ARM) is a multifactorial disorder known to have a substantial genetic component. The epsilon4 allele of the apolipoprotein E gene (APOE-4) has previously been reported to have a protective effect on ARM risk, while the APOE-2 allele may increase disease risk. This study combined four independent data sets (three US and one European) of Caucasian ARM patients and controls in order to obtain better statistical power to examine the role of APOE in ARM. APOE genotype and allele frequencies were compared for 617 ARM cases and 1260 controls, adjusting for age and sex differences between the two groups via multiple logistic regression. The protective effect of the APOE-4 allele on ARM risk was confirmed (age- and sex-adjusted odds ratio (OR) for APOE-4 carriers 0.54, 95% confidence interval (CI) 0.41-0.70, p < 0.0001). The effect of APOE-4 did not differ significantly between males and females and was observed consistently for both atrophic and neovascular ARM. Evidence for an increased risk of ARM due to the APOE-2 allele was found for men, but not for women (OR for men 1.54, 95% CI 0.97-2.45; OR for women 0.74, 95% CI 0.52-1.06, p = 0.01 for interaction of sex and APOE-2 carrier status). These data confirm that the APOE-4 allele, or an allele in linkage disequilibrium with it, reduces the risk of ARM. They also suggest that the effect of the APOE-2 allele may vary by gender, and that APOE-2 may confer an increased risk only to males.