Fifth Ovarian Cancer Consensus Conference of the Gynecologic Cancer InterGroup: recurrent disease.

This manuscript reports the consensus statements regarding recurrent ovarian cancer (ROC), reached at the fifth Ovarian Cancer Consensus Conference (OCCC), which was held in Tokyo, Japan, in November 2015. Three important questions were identified: (i) What are the subgroups for clinical trials in ROC? The historical definition of using platinum-free interval (PFI) to categorise patients as having platinum-sensitive/resistant disease was replaced by therapy-free interval (TFI). TFI can be broken down into TFIp (PFI), TFInp (non-PFI) and TFIb (biological agent-free interval). Additional criteria to consider include histology, BRCA mutation status, number/type of previous therapies, outcome of prior surgery and patient reported symptoms. (ii) What are the control arms for clinical trials in ROC? When platinum is considered the best option, the control arm should be a platinum-based therapy with or without an anti-angiogenic agent or a poly (ADP-ribose) polymerase (PARP) inhibitor. If platinum is not considered the best option, the control arm could include a non-platinum drug, either as single agent or in combination. (iii) What are the endpoints for clinical trials in ROC? Overall survival (OS) is the preferred endpoint for patient cohorts with an expected median OS < or = 12 months. Progression-free survival (PFS) is an alternative, and it is the preferred endpoint when the expected median OS is > 12 months. However, PFS alone should not be the only endpoint and must be supported by additional endpoints including pre-defined patient reported outcomes (PROs), time to second subsequent therapy (TSST), or time until definitive deterioration of quality of life (TUDD).

Two novel mutations causing familial benign hypocalciuric hypercalcaemia in three Scottish families.

BACKGROUND AND AIMS: Familial benign hypocalciuric hypercalcaemia (FBHH) is a benign autosomal dominantly inherited condition which results in elevated serum calcium and low urinary calcium. This condition is of clinical interest because it can be mistakenly diagnosed as primary hyperparathyroidism (PHP). In most cases FBHH can be shown to be due to a mutation in the calcium sensing receptor (CASR) gene and we aimed to find the causative mutation in three Scottish kindreds with FBHH. METHODS: We used a combination of denaturing gradient gel electrophoresis and direct DNA sequencing to detect mutations in the CASR gene. RESULTS: We detected a mutation in the CASR gene in each of the three kindreds. Two different mutations were detected (the same one was present in two kindreds). Neither mutation has been reported previously. All hypercalcaemic individuals from each kindred had the appropriate mutation while all normocalcaemic individuals did not. CONCLUSION: In the vast majority of kindreds with FBHH which have been reported previously, the CASR mutation responsible is private to that kindred. In three Scottish kindreds we have identified two new mutations.

Changes in gene expression induced by chemoradiation in advanced cervical carcinoma: a microarray study of RTOG C-0128.

PURPOSE: To evaluate gene expression patterns in patients with advanced cervix cancer before and during chemoradiation in a multi-institutional cooperative group setting. METHODS: RTOG C0128 was designed as a Phase II trial of radiation therapy with concomitant chemotherapy and Celecoxib at 400 mg twice daily for one year. Tumor samples were obtained for microarray gene expression analysis before treatment and at the time of the first implant (paired sample). RNA was extracted, linearly amplified, and purity was assessed by gel electrophoresis. Each sample was hybridized against a universal RNA mixture on a customized spotted array consisting of >10,000 genes. Gene expression pre-treatment was compared with clinical characteristics. Changes in gene expression following radiation were assessed within the paired samples (same patient) and then compared across all paired samples. Data were normalized using the AROMA software, and clustering analysis was performed using Ward's method in Spotfire. Differences in paired samples were calculated with Significance Analysis of Microarrays (SAM). RESULTS: From August 2001 to March 2004, 84 patients were accrued to the trial. Tissue was obtained prior to initiation of therapy from 34 patients (40%). FIGO stages of the patients providing tissue were IB (23%), II (57%), and IIIA-IVA (20%). RNA quality was sufficient in 22 pre-treatment and 14 post-treatment samples. Among pre-treatment samples, no significant differences in gene expression were observed by FIGO stage, age, or race. However, between comparison of histologic subtypes (adenocarcinoma, n=5; squamous cell carcinoma, n=17) demonstrated 45 genes differentially expressed with a false discovery rate of 0.018. Cluster analysis segregated unpaired samples into 2 groups: 18/22 comprising pre-treatment samples and 10/14 in group 2 representing post-treatment samples. In all 13 paired samples, gene expression after chemoradiation was significantly upregulated in 91 genes and downregulated in 251 genes (false discovery rate of 0.0018). Genes significantly upregulated included bax, cdk inhibitor 1, MMP2, and adhesion molecules PECAM1, VCAM1, and ICAM2. Genes significantly downregulated included topoisomerase II alpha, myc, H2AX, MSH2, RAD51, RAD53, PCNA, and cell cycle-regulating molecules chk1, CDK2, cyclinB1, cyclin D3, cdc2, and cdc25. CONCLUSIONS: Microarray analysis was successfully performed in a multi-institutional cooperative group trial. Gene expression significantly correlated with histology, but not stage, age or race. Cluster analysis identified two groups of gene expression profiles correlating with pre or post-treatment acquisition of tissue. Notably, paired samples showed significant changes in gene expression following chemoradiation, including several downregulated radiation response genes. Further analysis comparing gene expression to clinical outcomes, acute and late toxicities awaits maturation of clinical data. Hopefully, this data will lead to the development of molecularly based therapies.

Survival impact of lymph node dissection in endometrial adenocarcinoma: a surveillance, epidemiology, and end results analysis.

The therapeutic benefit of lymph node dissection (LND) in women with endometrial cancer remains controversial. The purpose of this study is to analyze the impact of LND on survival. Data were obtained from the Surveillance, Epidemiology, and End Results program of the US National Cancer Institute for the years 1988-2003. Women with adenocarcinoma of the endometrium who underwent surgery as primary management of their disease were eligible. Multivariate analyses of pertinent variables were performed for the end points of overall survival and cause-specific survival. Women included in the analysis were 42,184. The average frequency of LND was 31%, 40%, 47%, and 53%, for the years 1988-1991, 1992-1995, 1996-1999, and 2000-2003, respectively (P < 0.0001). On multivariate analysis, presence of LND was associated with overall and uterine-specific survival benefits with hazard ratios (HR) of 0.81 (P < 0.0001) and 0.78 (P < 0.0001) and removal of greater than 11 lymph nodes (LN) associated with a HR of 0.74 (P < 0.0001) and 0.69 (P < 0.0001), respectively. Further multivariate analyses demonstrated greater than 11 LN to associate with all other cause-specific and cardiac-specific survival benefits, with HR of 0.77 (P < 0.0001) and 0.82 (P = 0.0062), respectively. We conclude that the presence of LND and increased number of nodes dissected predicted for improved overall and uterine-specific survival in women with adenocarcinoma of the endometrium. Improved cause-specific survival was most pronounced for greater than 11 nodes removed and stage II or higher disease. The improvement in noncancer-related mortality with LND predicted by this data suggests the presence of inherit biases, and the need for caution in analyzing retrospective data.

Imaging and cancer of the cervix in low- and middle-income countries.

Cervix cancer is the fourth most common cancer globally but the second most cancer in women in resource-limited countries. It has remained a clinically-staged neoplasm as per the International Federation of Gynecology and Obstetrics staging classification. As the imaging machines are becoming more available worldwide, the resource-stratified guidelines recommended the inclusion of imaging whenever possible to guide treatment planning. In this report, the utility of imaging in low- and middle-income countries for diagnosis and treatment of cancer of the cervix will be reviewed.

Assessment of thiopurine methyltransferase enzyme activity is superior to genotype in predicting myelosuppression following azathioprine therapy in patients with inflammatory bowel disease.

BACKGROUND: Myelosuppression occurs in 2-7% of inflammatory bowel disease (IBD) patients treated with azathioprine, and can be associated with reduced activity of thiopurine methyltransferase (TPMT) in some patients. It has been proposed that pretreatment assessment of TPMT status reduces the incidence of toxicity and is cost-effective. AIMS: To determine if screening for TPMT status predicts side-effects to azathioprine in patients with IBD and to ascertain whether screening by TPMT enzyme activity or genotype is superior. METHODS: Sequential IBD patients were identified and azathioprine tolerance recorded. Blood was collected for measurement of TPMT activity and TPMT*3C, TPMT*3A and TPMT*2 genotypes. RESULTS: Of 130 patients, 25% stopped azathioprine because of toxicity. Four patients experienced severe myelosuppression (WCC < 2). Eleven of 17 patients with reduced TPMT activity were heterozygotes, including one patient with marked TPMT deficiency who experienced severe myelosuppression. There was no association between intermediate TPMT deficiency and any side-effect. CONCLUSIONS: Moderate reduction of TPMT activity in heterozygotes was not associated with toxicity, but very low TPMT activity caused severe myelosuppression in one patient. This would have been predicted by measuring TPMT activity but not by genotyping. Measurement of TPMT activity may therefore be superior to genotype in predicting severe myelosuppression.

Intraoperative high-dose-rate brachytherapy: An American Brachytherapy Society consensus report.

PURPOSE: This report presents recommendations from the American Brachytherapy Society for the use of intraoperative high-dose-rate (IOHDR) brachytherapy. METHODS AND MATERIALS: Members of the American Brachytherapy Society with expertise in IOHDR formulated this document based on their clinical experience and a review of the literature. This report covers the use of IOHDR in colorectal cancer, soft tissue sarcoma, gynecologic cancers, head and neck cancers, and pediatric cancers. This report does not cover intraoperative brachytherapy for breast cancer. Details about treatment planning and delivery are emphasized so this document can serve as a guide to practices implementing this technique. RESULTS: IOHDR brachytherapy is generally most beneficial for patients with either close or positive margins and/or recurrent disease in a previous resection bed or previously irradiated area. IOHDR brachytherapy requires a well-coordinated multidisciplinary team. IOHDR brachytherapy is recommended in the treatment of both recurrent and primary locally advanced disease for colorectal and gynecologic malignancies, soft tissue sarcoma, and selected head and neck and pediatric malignancies. Other techniques such as perioperative fractionated brachytherapy are also acceptable in many cases with some advantages and disadvantages compared to IOHDR. CONCLUSIONS: IOHDR brachytherapy is a specialized technique in radiation therapy with unique properties and advantages in cancer control. Special considerations for treatment planning and delivery are outlined herein.

Merocyanine 540-sensitized photoinactivation of soluble and membrane-bound enzymes in L1210 leukemia cells.

Merocyanine 540 (MC 540) is a photosensitizing dye that is used clinically for the purging of autologous bone marrow grafts and preclinically for the inactivation of enveloped viruses in blood products. Its mechanism of action is not yet well understood. This paper investigates the sites of MC 540-mediated photodamages in L1210 leukemia cells by examining the effects of MC 540-sensitized photoirradiation on several soluble and membrane-bound marker enzymes. When exposed to MC 540 and white light under a standard set of conditions, the activities of Na+/K(+)-ATPase, Mg2(+)-ATPase, and 5'-nucleotidase (three plasma membrane-bound enzymes) were reduced by 54, 49, and 55%, respectively. None of the intracellular enzymes included in this survey was affected by MC 540-sensitized photoirradiation as long as the plasma membrane remained intact. The two soluble enzymes, lactate dehydrogenase and malate dehydrogenase, remained refractory to MC 540-sensitized photoirradiation even after the plasma membrane had been disrupted. By contrast, the activities of the membrane-bound enzymes, NADPH-cytochrome c reductase and succinate dehydrogenase, were reduced in cell lysates by 55 and 81%, respectively. Purified NADPH-cytochrome c reductase was about 3 times less sensitive than the microsomal enzyme, suggesting that the membrane environment facilitated photoinactivation. The MC 540-sensitized photoinactivation of enzymes was accelerated in the presence of deuterium oxide and inhibited if oxygen in the medium was displaced by nitrogen or azide was added to the medium. Taken together, these data support the view that the plasma membrane is a major target of MC 540-mediated photodamages, that the inactivation of membrane-bound enzymes is an oxidative process, and that at least some photodynamic damages are mediated by type II chemistry.

Twenty-four novel mutations in Wilson disease patients of predominantly European ancestry.

Wilson disease (WND), an autosomal recessive disorder of copper transport, shows wide genotypic and phenotypic variability, with hepatic and/or neurological symptoms. The WND gene, ATP7B, encodes a copper transporting ATPase that is involved in the transport of copper into the plasma protein ceruloplasmin, and in the excretion of copper from the liver. ATP7B mutations result in copper storage in liver and brain. From 247 WND patients worldwide whose DNA has been sequenced in our laboratory, we have identified 24 new mutations. The origins of the patients were European white (one deletion, one nonsense, one splice site, and 18 missense), Chinese (one deletion, one missense) and Bangladeshi (one missense). Most of these had strong support as disease causing mutations, based on conservation between species, structural changes, and absence in controls. One missense mutation in a Chinese patient was considered uncertain because of its conservative nature and position in the protein. We also identified 15 nucleotide substitutions (11 of them new) causing silent or intronic changes, none of which produce an additional splice site that could lead to disease. Characterization of mutations, both disease-causing and normal variants, is essential for accurate molecular diagnosis of this condition.

The role of serum and serum components in the merocyanine 540-sensitized photoinactivation of K562 leukemia cells.

Serum is known to inhibit the merocyanine 540 (MC540)-sensitized photoinactivation of cells and enveloped viruses in a concentration-dependent manner. In diagnostic applications of MC540, a moderate amount of serum or serum albumin is frequently added to the staining solution because it enhances the contrast between intensely staining cells (e.g., electrically excitable cells or leukemia cells) and cells with a lower affinity for the dye (e.g., nonexcitable cells, red cells, normal leukocytes). In this communication we report on a quantitative analysis of the interactions of MC540 with serum and serum components. Human serum inhibited the MC540-sensitized photoinactivation of K562 leukemia cells most effectively, followed in order of decreasing potency by calf, newborn calf, horse, and fetal bovine serum. The photoprotective capacity of these five sera was directly proportional to their albumin content. Gel filtration experiments and differential spectroscopy showed that MC540 bound to serum albumin and lipoproteins. Both delipidated and lipidated albumin were capable of binding MC540. However, lipidated albumin had a considerably higher binding capacity and affinity for dye molecules.

The Xba1 polymorphism of the apolipoprotein B gene influences the degradation of low density lipoprotein in vitro.

This study examines the influence of variation in the apolipoprotein B (apoB) gene, the major protein of low-density lipoprotein (LDL), on the LDL degradation rate in vitro. Previously we have shown (Demant et al. (1988) J. Clin. Invest. 82, 797-802) that there is an association between the fractional catabolic rate of LDL in vivo and the apoB polymorphism detected using the Xba1 restriction enzyme. Subjects with genotype X1X1 (X1 = absence of cutting site) cleared LDL more rapidly from the plasma compartment than those with the X2X2 genotype. In this study, the LDL degradation rate on dermal fibroblasts was measured for 33 individuals of genotype X1X1 or X2X2. These were subdivided into three groups: (1) young normolipidaemic, (2) older normolipidaemic and (3) older hypercholesterolaemic subjects, because age is known to markedly affect the plasma LDL concentration and may independently influence the population of LDL particles under study. In all experiments, the degradation rate of one type of LDL was compared directly in the cell culture dish with that from an individual of the alternate genotype by labelling them separately with the two iodine isotopes 125I and 131I. In the group of young normals (mean cholesterol 5.03 mmol/l, mean age 31 years), no significant difference was observed between the degradation rates of LDL derived from X1X1 individuals versus X2X2. However, in the older group of normals (mean cholesterol 5.4 mmol/l, mean age 48 years), LDL from subjects with X1X1 genotype was catabolised 17% faster than that from X2X2 subjects (P less than 0.001). A similar result was seen in hypercholesterolaemics (mean cholesterol 8.3 mmol/l, mean age 57 years) with LDL isolated from X1X1 subjects being degraded 22% more rapidly than that from X2X2 subjects. This in vitro evidence adds further weight to the hypothesis that genetic variation in the apoB gene leads to structural changes in LDL than alter its potential for degradation via the LDL receptor.

Effect of radiotherapy after breast-conserving treatment in women with breast cancer and germline BRCA1/2 mutations.

PURPOSE: Recent laboratory data suggest a role for BRCA1/2 in the cellular response to DNA damage. There is a paucity of clinical data, however, examining the effect of radiotherapy (RT), which causes double-strand breaks, on breast tissue from BRCA1/2 mutation carriers. Thus the goals of this study were to compare rates of radiation-associated complications, in-breast tumor recurrence, and distant relapse in women with BRCA1/2 mutations treated with breast-conserving therapy (BCT) using RT with rates observed in sporadic disease. PATIENTS AND METHODS: Seventy-one women with a BRCA1/2 mutation and stage I or II breast cancer treated with BCT were matched 1:3 with 213 women with sporadic breast cancer. Conditional logistic regression models were used to compare matched cohorts for rates of complications and recurrence. RESULTS: Tumors from women in the genetic cohort were associated with high histologic (P =.0004) and nuclear (P =.009) grade and negative estrogen (P=.0001) and progesterone (P=.002) receptors compared with tumors from the sporadic cohort. Using Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer toxicity scoring, there were no significant differences in acute or chronic morbidity in skin, subcutaneous tissue, lung, or bone. The 5-year actuarial overall survival, relapse-free survival, and rates of tumor control in the treated breast for the patients in the genetic cohort were 86%, 78%, and 98%, respectively, compared with 91%, 80%, and 96%, respectively, for the sporadic cohort (P = not significant). CONCLUSION: There was no evidence of increased radiation sensitivity or sequelae in breast tissue heterozygous for a BRCA1/2 germline mutation compared with controls, and rates of tumor control in the breast and survival were comparable between BRCA1/2 carriers and controls at 5 years. Although additional follow-up is needed, these data may help in discussing treatment options in the management of early-stage hereditary breast cancer and should provide reassurance regarding the safety of administering RT to carriers of a germline BRCA1/2 mutation.

Regulation of the F conjugation genes studied by hybridization and tra-lacZ fusion.

Hybridization experiments and tra-lacZ fusions were used to obtain further insight into the complex series of control systems that affect F conjugation. We confirmed that the regular IncF FinOP control system represses transcription of traJ, and found that the traJ product is required for transcription of traM as well as of the traY-Z operon. The chromosomal sfrA gene product may be required to prevent premature termination of traJ transcription, while the sfrB gene product prevents premature termination at two sites within the traY-Z operon. The FinQ inhibition system determined by several IncI plasmids caused termination at three different sites in the operon, and that of JR66a at one further site. JR66a and R485 strongly inhibit F transfer, but have weak, or no (respectively) effects on transcription: they may inhibit function of one or more transfer gene products.

Predominance of a 6 bp deletion in exon 2 of the LDL receptor gene in Africans with familial hypercholesterolaemia.

In South Africa, the high prevalence of familial hypercholesterolaemia (FH) among Afrikaners, Jews, and Indians as a result of founder genes is in striking contrast to its reported virtual absence in the black population in general. In this study, the molecular basis of primary hypercholesterolaemia was studied in 16 Africans diagnosed with FH. DNA analysis using three screening methods resulted in the identification of seven different mutations in the coding region of the low density lipoprotein (LDLR) gene in 10 of the patients analysed. These included a 6 bp deletion (GCGATG) accounting for 28% of defective alleles, and six point mutations (D151H, R232W, R385Q, E387K, P678L, and R793Q) detected in single families. The Sotho patient with missense mutation R232W was also heterozygous for a de novo splicing defect 313+1G-->A. Several silent mutations/polymorphisms were detected in the LDLR and apolipoprotein B genes, including a base change (g-->t) at nucleotide position -175 in the FP2 LDLR regulatory element. This promoter variant was detected at a significantly higher (p<0.05) frequency in FH patients compared to controls and occurred in cis with mutation E387K in one family. Analysis of four intragenic LDLR gene polymorphisms showed that the same chromosomal background was identified at this locus in the four FH patients with the 6 bp deletion. Detection of the 6 bp deletion in Xhosa, Pedi, and Tswana FH patients suggests that it is an ancient mutation predating tribal separation approximately 3000 years ago.

Merocyanine 540-sensitized photoinactivation of leukemia cells: role of oxygen and effects on plasma membrane integrity and mitochondrial respiration.

Merocyanine 540 (MC 540) is a photosensitizing dye that is used clinically for the purging of autologous bone marrow grafts and preclinically for the inactivation of enveloped viruses in blood products. In this paper we present evidence that the MC 540-sensitized photoinactivation of leukemia cells is an oxygen-dependent process and that unsaturated plasma membrane lipids are substrates for singlet oxygen and/or other activated oxygen species generated by photoirradiated MC 540. A comparison of the inhibition of clonal growth, the inhibition of mitochondrial respiration, and the exclusion of trypan blue by the plasma membrane after exposure to MC 540 and graded doses of light showed that mitochondrial respiration is compromised relatively early in the course of the dye-mediated photoinactivation of cells, well before the plasma membrane loses its capacity to exclude trypan blue. It also showed that trypan blue exclusion assays can greatly underestimate the cytotoxic effects of MC 540-sensitized photoirradiation.

Plasma membrane properties regulating the sensitivity of leukemia, lymphoma, and solid tumor cells to merocyanine 540-sensitized photoirradiation.

Merocyanine 540 (MC 540) is a photosensitizing dye that has been used in a phase I clinical trial for the purging of leukemia and lymphoma cells from autologous bone marrow grafts. In this paper we examine the role of plasma membrane negative charge, plasma membrane fluidity, and plasma membrane hydrophobicity in the regulation of a cell's susceptibility to MC 540-sensitized photoirradiation. Among solid tumor cells, we found an inverse correlation between surface electronegativity, affinity for dye molecules, and susceptibility to MC 540-sensitized photoinactivation. That is, the least electronegative cells bound the highest amount of dye and were the most susceptible to dye-sensitized photoirradiation. By contrast, no such correlations were found among leukemia/lymphoma cells. This suggested that dye binding and susceptibility to MC 540-mediated photodynamic damages are regulated differently in hematopoietic/lymphopoietic and solid tumor cells.

Genetic variation in the cholesteryl ester transfer protein and apolipoprotein A-I genes and its relation to coronary heart disease in a Sri Lankan population.

The influence of variation in the genes for cholesteryl ester transfer protein and apolipoprotein A-I was investigated in 95 patients with coronary heart disease and 95 matched control subjects of South East Asian extraction. Restriction fragment length polymorphisms (RFLPs) linked to the cholesteryl ester transfer protein gene TaqIA and TaqIB, and to the apolipoprotein A-I gene SstI, were examined to investigate the extent of genetic variation at these loci. None of the alleles defined by these RFLPs were associated with increased coronary risk. Analysis of the data by division of high density lipoprotein-cholesterol levels into tertiles showed a trend of a higher frequency of B1 allele (presence of the TaqIB site) with reduced high density lipoprotein levels. The B1 allele was more frequent in control subjects, with low high density lipoprotein levels (P less than 0.02), but not in coronary heart disease patients. The differences became significant for both groups (P less than 0.05) when the data of non-smokers were analysed separately.