Long-term outcomes after high dose therapy and autologous haematopoietic cell rescue for refractory/relapsed Hodgkin lymphoma.

The standard treatment for patients with refractory or relapsed Hodgkin lymphoma (HL) is high-dose chemotherapy and/or radiation with autologous haematopoietic cell rescue (AHCR). In this study, we assessed quality of life and evaluated the risk of late morbidity and mortality for HL patients who underwent AHCR. One hundred and fifty-four patients who underwent AHCR at Stanford University from 1988 to 2002 and survived ≥2 years were evaluated. Median follow-up was 10·2 years. There were 54 deaths, 34 from HL, 20 from other causes. The 10-year cumulative incidence of death from HL or other causes was 21·7% and 12·7%, respectively. Thirteen deaths were from second malignancies. The risk ratio of second malignancies was 8·0 [95% confidence interval (CI), 4·7-12·6] compared with the general population, and 3·0 (95% CI, 1·8-4·8) compared with HL patients not undergoing AHCR. The risk ratio of second malignancies was 1·5 (95% CI, 0·9-2·4) compared with HL patients receiving non-AHCR therapy. Overall quality of life did not differ from the general population, but AHCR survivors did note reduced functioning and some worse symptoms. AHCR survivors may be at increased risk of death from HL and other causes compared with the general population, but not compared with the HL population as a whole.

No evidence of familial correlation in breast cancer metastasis.

Animal experiments support the hypothesis that the metastatic potential of breast cancer is a heritable trait of the host. Our objective was to evaluate correlations in metastasis occurrence in large families with multiple cases of breast cancer. We evaluated correlation among pairs of relatives in the occurrence and timing of distant metastasis using retrospective cohort data from 743 female breast cancer patients in 242 families. We adjusted for correlation in their age at diagnosis, year of diagnosis, educational level, lymph node involvement, and estrogen receptor status. Distant metastasis occurred in 255 patients (34.3%) during mean followup of 11.7 years. None of the correlation coefficients for metastasis in blood relatives differed significantly from zero. The estimated correlation coefficient in first-degree relatives was -0.03 (95% confidence interval -0.11 to 0.06). These findings suggest that a family history of metastatic breast cancer does not contribute substantially to risk of metastasis for breast cancer patients.

Osteonecrosis of the maxilla and mandible in patients with advanced cancer treated with bisphosphonate therapy.

Cases of osteonecrosis of the jaw (ONJ) have been reported with an increasing frequency over the past 5 years. ONJ is most often identified in patients with cancer who are receiving intravenous bisphosphonate (IVBP) therapy, but it has also been diagnosed in patients receiving oral bisphosphonates for nonmalignant conditions. To further categorize risk factors associated with ONJ and potential clinical outcomes of this condition, we performed a retrospective study of patients with metastatic bone disease treated with intravenous bisphosphonates who have been evaluated by the Memorial Sloan-Kettering Cancer Center Dental Service between January 1, 1996 and January 31, 2006. We identified 310 patients who met these criteria. Twenty-eight patients were identified as having ONJ at presentation to the Dental Service and an additional 7 patients were subsequently diagnosed with ONJ. Statistically significant factors associated with increased likelihood of ONJ included type of cancer, duration of bisphosphonate therapy, sequential IVBP treatment with pamidronate followed by zoledronic acid, comorbid osteoarthritis or rheumatoid arthritis, and benign hematologic conditions. Our data do not support corticosteroid use or oral health as a predictor of risk for ONJ. Clinical outcomes of patients with ONJ were variable with 11 patients demonstrating improvement or healing with conservative management. Our ONJ experience is presented here.

Orthopaedic surgery core curriculum: the spine.

OBJECTIVE: To develop a core curriculum for orthopaedic surgery and to conduct a national survey to assess the importance of 281 items in the curriculum. Attention was focused specifically on 24 items pertaining to the curriculum that are pertinent to the spine. STUDY DESIGN: A cross-sectional survey of a random sample of orthopaedic surgeons whose primary affiliation was non-academic, representing the provinces and territories of Canada METHODS: A questionnaire containing 281 items was developed. A random group of 131 (out of 156) orthopaedic surgeons whose primary affiliation is non-academic completed the questionnaire. The data were analysed quantitatively using average mean scores, histograms, the modified Hotelling's T2 test and the Benjimini-Hochberg procedure. RESULTS: 131 of 156 (84%) orthopaedic surgeons participated, in this study. 14 of 24 items were ranked at no less than 3 out of 4 thus suggesting that 58% of the items are important or probably important to know by the end of residency (SD< or =0.07). Residents need to learn the diagnosis and principles of managing patients with common conditions of the spine. CONCLUSIONS: The study shows, with reliable statistical evidence, that orthopaedic residents are no longer expected to be able to perform spinal fusions with proficiency on completion of residency. Is the exposure to surgical spine problems and the ability to be comfortable with operating expectations specific to the fellowship level? If so, the focus during residency or increasing accredited spine fellowships needs to be addressed to ensure that enough spine surgeons are educated to meet the future healthcare demands projected for Canada.

Orthopaedic surgery core curriculum: foot and ankle reconstruction.

BACKGROUND: The purpose of this study was to develop a core curriculum for orthopaedic surgery and to conduct a national survey to assess the importance of 281 curriculum items. Attention was focused on 45 items pertaining to the foot and ankle. METHODS: A 281-item curriculum was developed. A content review and cross-sectional survey of a random selection of orthopaedic surgeons with primary nonacademic affiliations was completed. Data were analyzed descriptively and quantitatively using histograms, modified Hotelling's T(2)-statistic, and the Benjamini-Hochberg procedure. Our analyses assumed that each respondent answered questions independently of the answers of any other respondent but that the answers to different questions by the same respondent might be dependent. RESULTS: Of the 156 orthopaedic surgeons contacted, 131 (86%) participated in this study. Eighty-two percent (37 of 45) of the items were ranked by respondents with an average mean score higher than 3.5/4.0 and 42 higher than 3.0/40, thus suggesting that 93% of the items are important or probably important to know by the end of residency (p

Two-stage sampling designs for external validation of personal risk models.

We propose a cost-effective sampling design and estimating procedure for validating personal risk models using right-censored cohort data. Validation involves using each subject's covariates, as ascertained at cohort entry, in a risk model (specified independently of the data) to assign him/her a probability of an adverse outcome within a future time period. Subjects are then grouped according to the magnitudes of their assigned risks, and within each group, the mean assigned risk is compared with the probability of outcome occurrence as estimated using the follow-up data. Such validation presents two complications. First, in the presence of right-censoring, estimating the probability of developing the outcomes before death requires competing risk analysis. Second, for rare outcomes, validation using the full cohort requires assembling covariates and assigning risks to thousands of subjects. This can be costly when some covariates involve analyzing biological specimens. A two-stage sampling design addresses this problem by assembling covariates and assigning risks only to those subjects most informative for estimating key parameters. We use this design to estimate the outcome probabilities needed to evaluate model performance and we provide theoretical and bootstrap estimates of their variances. We also describe how to choose two-stage designs with minimal efficiency loss for a parameter of interest when the quantities determining optimality are unknown at the time of design. We illustrate these methods by using subjects in the California Teachers Study to validate ovarian cancer risk models. We find that a design with optimal efficiency for one performance parameter need not be so for others, and trade-offs will be required. A two-stage design that samples all outcome-positive subjects and more outcome-negative than censored subjects will perform well in most circumstances. The methods are implemented in Risk Model Assessment Program, an R program freely available at http://med.stanford.edu/epidemiology/two-stage.html.

Implant prosthodontic rehabilitation of fibula free-flap reconstructed mandibles: a Memorial Sloan-Kettering Cancer Center review of prognostic factors and implant outcomes.

PURPOSE: This study aimed to estimate the cumulative survival rates (CSRs) of implants placed in reconstructed mandibles and to identify prognostic factors that may influence implant survival. MATERIALS AND METHODS: The charts of 24 patients (10 male, 14 female) who had undergone mandibular resection and reconstruction with fibula free-flaps treated with implant-supported prostheses from April 1986 through December 2001 were reviewed. Information on demographics, surgical characteristics, treatment modalities, dentition, implant parameters, prostheses, and hyperbaric oxygen therapy (HBO) was gathered. Kaplan-Meier survival estimates were generated for the 100 implants that satisfied the inclusion criteria. Multivariate Cox proportional hazards regression models accounting for correlated implants within subjects were developed to identify prognostic factors for implant survival. RESULTS: Ninteen implants had been placed in native mandible (3 in irradiated bone) and 81 in fibula bone flap. Six implants failed during the follow-up period (mean 51.7 months). The overall 5- and 10-year CSRs were 97.0% and 79.9%, respectively. In the univariate analysis, variables associated with implant survival were age, gender, chemotherapy, radiation therapy, HBO, irradiated bone, implant diameter, xerostomia, trismus, opposing dentition, and type of prosthesis. At 5 years, the CSR of implants in patients with HBO was 86.7%; HBO was statistically associated with an increased risk for implant failure (P = .005, hazard ratio = 19.79, 95% CI: 2.42 to 161.71). DISCUSSION: The CSR was lower when implants were placed in a previously irradiated mandible. There is still a lack of reliable clinical evidence to support the effectiveness of HBO in these patients. CONCLUSIONS: A high survival rate was demonstrated for implants placed in fibula free-flap reconstructed mandibles. The finding that HBO was a risk factor can probably be attributed to the small sample size; further study is needed in this patient population.

Risk of early-onset prostate cancer in relation to germ line polymorphisms of the vitamin D receptor.

Vitamin D inhibits prostate cancer cell growth, angiogenesis, and metastasis. These actions are mediated by the vitamin D receptor. We examined associations between prostate cancer risk and five polymorphisms in the VDR gene: four single nucleotide polymorphisms (FokI, BsmI, ApaI, and TaqI restriction sites) and the polyadenylic acid microsatellite. Specifically, we genotyped population-based samples of young African Americans (113 cases and 121 controls) and Whites (232 cases and 171 controls) and members of 98 predominantly White families with multiple cases of prostate cancer. Among Whites, there was no evidence for association between prostate cancer risk and alleles at any of the five polymorphic sites regardless of how the men were ascertained. Moreover, estimated five-locus haplotype frequencies were similar in White cases and controls. Among African Americans, prostate cancer risk was associated with homozygosity for the F allele at the FokI site (odds ratio 1.9, 95% confidence interval 1.0-3.3). In addition, estimated haplotype frequencies differed significantly (P < 0.01) between African American cases and controls. These findings need replication in other studies of African Americans. Homozygosity for the F allele at the FokI site is more prevalent in the African American population than in U.S. Whites. If the FokI association noted here were causal, this difference could account for some of the disease burden among African Americans and some of the excess risk in African Americans compared with Whites.

Phase II double-blind randomized study comparing oral aloe vera versus placebo to prevent radiation-related mucositis in patients with head-and-neck neoplasms.

PURPOSE: In a single-institution, double-blind, prospective, randomized trial, we determined whether oral aloe vera gel can reduce radiation-induced mucositis in head-and-neck cancer patients. METHODS AND MATERIALS: We randomized 58 head-and-neck cancer patients between oral aloe vera and placebo. To be included in this Phase II protocol, patients had to be treated with radiotherapy with curative intent at Stanford University between February 1999 and March 2002. We examined patients biweekly for mucositis at 15 head-and-neck subsites and administered quality-of-life questionnaires. RESULTS: Patients in the aloe and placebo groups were statistically identical in baseline characteristics. By the end of treatment, the two groups were also statistically identical in maximal grade of toxicity, duration of Grade 2 or worse mucositis, quality-of-life scores, percentage of weight loss, use of pain medications, hydration requirement, oral infections, and prolonged radiation breaks. CONCLUSION: In our randomized study, oral aloe vera was not a beneficial adjunct to head-and-neck radiotherapy. The mean quality-of-life scores were greater in the aloe vera group, but the differences were not statistically significant. Oral aloe vera did not improve tolerance to head-and-neck radiotherapy, decrease mucositis, reduce soreness, or otherwise improve patient well-being.

A genome-wide association study of early-onset breast cancer identifies PFKM as a novel breast cancer gene and supports a common genetic spectrum for breast cancer at any age.

Early-onset breast cancer (EOBC) causes substantial loss of life and productivity, creating a major burden among women worldwide. We analyzed 1,265,548 Hapmap3 single-nucleotide polymorphisms (SNP) among a discovery set of 3,523 EOBC incident cases and 2,702 population control women ages ≤ 51 years. The SNPs with smallest P values were examined in a replication set of 3,470 EOBC cases and 5,475 control women. We also tested EOBC association with 19,684 genes by annotating each gene with putative functional SNPs, and then combining their P values to obtain a gene-based P value. We examined the gene with smallest P value for replication in 1,145 breast cancer cases and 1,142 control women. The combined discovery and replication sets identified 72 new SNPs associated with EOBC (P < 4 × 10(-8)) located in six genomic regions previously reported to contain SNPs associated largely with later-onset breast cancer (LOBC). SNP rs2229882 and 10 other SNPs on chromosome 5q11.2 remained associated (P < 6 × 10(-4)) after adjustment for the strongest published SNPs in the region. Thirty-two of the 82 currently known LOBC SNPs were associated with EOBC (P < 0.05). Low power is likely responsible for the remaining 50 unassociated known LOBC SNPs. The gene-based analysis identified an association between breast cancer and the phosphofructokinase-muscle (PFKM) gene on chromosome 12q13.11 that met the genome-wide gene-based threshold of 2.5 × 10(-6). In conclusion, EOBC and LOBC seem to have similar genetic etiologies; the 5q11.2 region may contain multiple distinct breast cancer loci; and the PFKM gene region is worthy of further investigation. These findings should enhance our understanding of the etiology of breast cancer.

Intravenous bisphosphonate-related osteonecrosis of the jaw: long-term follow-up of 109 patients.

OBJECTIVE: We report long-term follow-up of patients with intravenous bisphosphonate-related osteonecrosis of the jaw (BRONJ). STUDY DESIGN: Medical and dental histories, including type and duration of bisphosphonate treatment and comorbidities, were analyzed and compared with clinical course of 109 patients with BRONJ at Memorial Sloan-Kettering Cancer Center Dental Service. RESULTS: Median onset of BRONJ in months was 21 (zoledronic acid), 30 (pamidronate), and 36 (pamidronate plus zoledronic acid), with a significant difference between the pamidronate plus zoledronic acid and zoledronic acid groups (P = .01; Kruskal-Wallis). The median number of doses for BRONJ onset was significantly less with zoledronic acid (n = 18) than pamidronte plus zoledronic acid (n = 36; P = .001), but not pamidronate alone (n = 29). An association between diabetes (P = .05), decayed-missing-filled teeth (P = .02), and smoking (P = .03) and progression of BRONJ was identified through χ(2) test. CONCLUSIONS: This long-term follow-up of BRONJ cases enhances the literature and contributes to the knowledge of BRONJ clinical course.

Persistence versus reversion of 3TC resistance in HIV-1 determine the rate of emergence of NVP resistance.

When HIV-1 is exposed to lamivudine (3TC) at inhibitory concentrations, resistant variants carrying the reverse transcriptase (RT) substitution M184V emerge rapidly. This substitution confers high-level 3TC resistance and increased RT fidelity. We established a novel in vitro system to study the effect of starting nevirapine (NVP) in 3TC-resistant/NNRTI-naïve clinical isolates, and the impact of maintaining versus dropping 3TC pressure in this setting. Because M184V mutant HIV-1 seems hypersusceptible to adefovir (ADV), we also tested the effect of ADV pressure on the same isolates. We draw four conclusions from our experiments simulating combination therapy in vitro. (1) The presence of low-dose (1 µM) 3TC prevented reversal to wild-type from an M184V mutant background. (2) Adding low-dose 3TC in the presence of NVP delayed the selection of NVP-associated mutations. (3) The presence of ADV, in addition to NVP, led to more rapid reversal to wild-type at position 184 than NVP alone. (4) ADV plus NVP selected for greater numbers of mutations than NVP alone. Inference about the "selection of mutation" is based on two statistical models, one at the viral level, more telling, and the other at the level of predominance of mutation within a population. Multidrug pressure experiments lend understanding to mechanisms of HIV resistance as they bear upon new treatment strategies.

Nonparametric linkage analysis using person-specific covariates.

Linkage analysis provides an important tool for mapping genes for complex disease. However its usefulness has been limited by inadequate marker density, inadequate sample sizes and the possibility that different genes account for different subtypes of the disease (phenotypic heterogeneity). The first two limitations can be addressed by high-density single nucleotide polymorphism (SNP) genotyping and the pooling of large sets of multiple-case families. Phenotypic heterogeneity can be addressed by analyses that weigh the contributions of affected family members according to characteristics of their disease phenotypes. Here we introduce a method for including such person-specific weights in nonparametric linkage analysis. We show with simulations that such weighting can provide stronger linkage signals when a causal polymorphism affects some manifestations of the disease more than others. We applied the method to prostate cancer linkage data in a region on chromosome 19p, and obtained higher lod scores by assigning weights of one to men with early-onset aggressive cancers, weights of zero to those with late-onset nonaggressive cancers, and intermediate weights to all other affected men. We have developed a modified version of GENEHUNTER that allows inclusion of person-specific weights in the nonparametric analyses. This program is freely available at http://med.stanford.edu/epidemiology/statisticalSoftware/weightedKAC.

Pooled genome linkage scan of aggressive prostate cancer: results from the International Consortium for Prostate Cancer Genetics.

While it is widely appreciated that prostate cancers vary substantially in their propensity to progress to a life-threatening stage, the molecular events responsible for this progression have not been identified. Understanding these molecular mechanisms could provide important prognostic information relevant to more effective clinical management of this heterogeneous cancer. Hence, through genetic linkage analyses, we examined the hypothesis that the tendency to develop aggressive prostate cancer may have an important genetic component. Starting with 1,233 familial prostate cancer families with genome scan data available from the International Consortium for Prostate Cancer Genetics, we selected those that had at least three members with the phenotype of clinically aggressive prostate cancer, as defined by either high tumor grade and/or stage, resulting in 166 pedigrees (13%). Genome-wide linkage data were then pooled to perform a combined linkage analysis for these families. Linkage signals reaching a suggestive level of significance were found on chromosomes 6p22.3 (LOD = 3.0), 11q14.1-14.3 (LOD = 2.4), and 20p11.21-q11.21 (LOD = 2.5). For chromosome 11, stronger evidence of linkage (LOD = 3.3) was observed among pedigrees with an average at diagnosis of 65 years or younger. Other chromosomes that showed evidence for heterogeneity in linkage across strata were chromosome 7, with the strongest linkage signal among pedigrees without male-to-male disease transmission (7q21.11, LOD = 4.1), and chromosome 21, with the strongest linkage signal among pedigrees that had African American ancestry (21q22.13-22.3; LOD = 3.2). Our findings suggest several regions that may contain genes which, when mutated, predispose men to develop a more aggressive prostate cancer phenotype. This provides a basis for attempts to identify these genes, with potential clinical utility for men with aggressive prostate cancer and their relatives.

Covariate adjustment in family-based association studies.

Family-based tests of association between a candidate locus and a disease evaluate how often a variant allele at the locus is transmitted from parents to offspring. These tests assume that in the absence of association, an affected offspring is equally likely to have inherited either one of the two homologous alleles carried by a parent. However, transmission distortion was documented in families in which the offspring are unselected for phenotype. Moreover, if offspring genotypes are associated with a risk factor for the disease, transmission distortion to affected offspring can occur in the absence of a causal relation between gene and disease risk. We discuss the appropriateness of adjusting for established risk factors when evaluating association in family-based studies. We present methods for adjusting the transmission/disequilibrium test for risk factors when warranted, and we apply them to data on CYP19 (aromatase) genotypes in nuclear families with multiple cases of breast cancer. Simulations show that when genotypes are correlated with risk factors, the unadjusted test statistics have inflated size, while the adjusted ones do not. The covariate-adjusted tests are less powerful than the unadjusted ones, suggesting the need to check the relationship between genotypes and known risk factors to verify that adjustment is needed. The adjusted tests are most useful for data containing a large proportion of families that lack disease-discordant sibships, i.e., data for which multiple logistic regression of matched sibships would have little power. Software for performing the covariate-adjusted tests is available at http://www.stanford.edu/dept/HRP/epidemiology/COVTDT.

A combined genomewide linkage scan of 1,233 families for prostate cancer-susceptibility genes conducted by the international consortium for prostate cancer genetics.

Evidence of the existence of major prostate cancer (PC)-susceptibility genes has been provided by multiple segregation analyses. Although genomewide screens have been performed in over a dozen independent studies, few chromosomal regions have been consistently identified as regions of interest. One of the major difficulties is genetic heterogeneity, possibly due to multiple, incompletely penetrant PC-susceptibility genes. In this study, we explored two approaches to overcome this difficulty, in an analysis of a large number of families with PC in the International Consortium for Prostate Cancer Genetics (ICPCG). One approach was to combine linkage data from a total of 1,233 families to increase the statistical power for detecting linkage. Using parametric (dominant and recessive) and nonparametric analyses, we identified five regions with "suggestive" linkage (LOD score >1.86): 5q12, 8p21, 15q11, 17q21, and 22q12. The second approach was to focus on subsets of families that are more likely to segregate highly penetrant mutations, including families with large numbers of affected individuals or early age at diagnosis. Stronger evidence of linkage in several regions was identified, including a "significant" linkage at 22q12, with a LOD score of 3.57, and five suggestive linkages (1q25, 8q13, 13q14, 16p13, and 17q21) in 269 families with at least five affected members. In addition, four additional suggestive linkages (3p24, 5q35, 11q22, and Xq12) were found in 606 families with mean age at diagnosis of < or = 65 years. Although it is difficult to determine the true statistical significance of these findings, a conservative interpretation of these results would be that if major PC-susceptibility genes do exist, they are most likely located in the regions generating suggestive or significant linkage signals in this large study.

Genetic association tests for family data with missing parental genotypes: a comparison.

We consider three tests for genetic association in data from nuclear families (the Family-Based Association Test (FBAT) test proposed by Rabinowitz and Laird ([2000] Hum. Hered. 50:211-223), a second test proposed by Rabinowitz ([2002] J. Am. Stat. Assoc. 97:742-758), and the Family Genotype Analysis Program (FGAP) nonfounder or partial score test proposed by Clayton ([1999] Am. J. Hum. Genet. 65:1170-1177) and Whittemore and Tu ([2000] Am. J. Hum. Genet. 66:1329-1340)). We show that each test statistic arises from the efficient score of the family data as the solution to a set of constraints on its null expectation. Moreover, the FBAT and Rabinowitz tests (but not the FGAP test) are locally the most powerful among all tests satisfying their constraints. We used simulations to examine how the three tests perform in situations when their assumptions are violated and the number of families is not huge. We found that the FBAT test tended to have less power than the other two tests, particularly when applied to families in whom all offspring were affected. The Rabinowitz and FGAP tests performed similarly, although the latter tended to extract more information from families containing one typed parent. While none of the tests showed good power to detect rare, recessively acting genes, the Rabinowitz test with a sample variance estimate performed particularly poorly in this case. However, the Rabinowitz test with a model-based variance had power comparable to that of the FGAP test, and more accurate type I error rates. We conclude that for the situations we considered, the Rabinowitz test with model-based variance has good power without forfeiting robustness against misspecification of parental genotype probabilities. However, its utility is limited by the lack of a simple algorithm to apply it to families with varying structures and phenotypes.

Logistic regression of family data from retrospective study designs.

We wish to study the effects of genetic and environmental factors on disease risk, using data from families ascertained because they contain multiple cases of the disease. To do so, we must account for the way participants were ascertained, and for within-family correlations in both disease occurrences and covariates. We model the joint probability distribution of the covariates of ascertained family members, given family disease occurrence and pedigree structure. We describe two such covariate models: the random effects model and the marginal model. Both models assume a logistic form for the distribution of one person's covariates that involves a vector beta of regression parameters. The components of beta in the two models have different interpretations, and they differ in magnitude when the covariates are correlated within families. We describe ascertainment assumptions needed to estimate consistently the parameters beta(RE) in the random effects model and the parameters beta(M) in the marginal model. Under the ascertainment assumptions for the random effects model, we show that conditional logistic regression (CLR) of matched family data gives a consistent estimate beta(RE) for beta(RE) and a consistent estimate for the covariance matrix of beta(RE). Under the ascertainment assumptions for the marginal model, we show that unconditional logistic regression (ULR) gives a consistent estimate for beta(M), and we give a consistent estimator for its covariance matrix. The random effects/CLR approach is simple to use and to interpret, but it can use data only from families containing both affected and unaffected members. The marginal/ULR approach uses data from all individuals, but its variance estimates require special computations. A C program to compute these variance estimates is available at http://www.stanford.edu/dept/HRP/epidemiology. We illustrate these pros and cons by application to data on the effects of parity on ovarian cancer risk in mother/daughter pairs, and use simulations to study the performance of the estimates.