Arleen D. Auerbach

A note on competing risks in survival data analysis

Survival analysis encompasses investigation of time to event data. In most clinical studies, estimating the cumulative incidence function (or the probability of experiencing an event by a given time) is of primary interest. When the data consist of patients who experience an event and censored individuals, a nonparametric estimate of the cumulative incidence can be obtained using the Kaplan–Meier method. Under this approach, the censoring mechanism is assumed to be noninformative. In other words, the survival time of an individual (or the time at which a subject experiences an event) is assumed to be independent of a mechanism that would cause the patient to be censored. Often times, a patient may experience an event other than the one of interest which alters the probability of experiencing the event of interest. Such events are known as competing risk events. In this setting, it would often be of interest to calculate the cumulative incidence of a specific event of interest. Any subject who does not experience the event of interest can be treated as censored. However, a patient experiencing a competing risk event is censored in an informative manner. Hence, the Kaplan–Meier estimation procedure may not be directly applicable. The cumulative incidence function for an event of interest must be calculated by appropriately accounting for the presence of competing risk events. In this paper, we illustrate nonparametric estimation of the cumulative incidence function for an event of interest in the presence of competing risk events using two published data sets. We compare the resulting estimates with those obtained using the Kaplan–Meier approach to demonstrate the importance of appropriately estimating the cumulative incidence of an event of interest in the presence of competing risk events.

Biallelic mutations in PALB2 cause Fanconi anemia subtype FA-N and predispose to childhood cancer

PALB2 was recently identified as a nuclear binding partner of BRCA2. Biallelic BRCA2 mutations cause Fanconi anemia subtype FA-D1 and predispose to childhood malignancies. We identified pathogenic mutations in PALB2 (also known as FANCN) in seven families affected with Fanconi anemia and cancer in early childhood, demonstrating that biallelic PALB2 mutations cause a new subtype of Fanconi anemia, FA-N, and, similar to biallelic BRCA2 mutations, confer a high risk of childhood cancer.

The BRCA1-interacting helicase BRIP1 is deficient in Fanconi anemia

Seven Fanconi anemia–associated proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG and FANCL) form a nuclear Fanconi anemia core complex that activates the monoubiquitination of FANCD2, targeting FANCD2 to BRCA1-containing nuclear foci. Cells from individuals with Fanconi anemia of complementation groups D1 and J (FA-D1 and FA-J) have normal FANCD2 ubiquitination. Using genetic mapping, mutation identification and western-blot data, we identify the defective protein in FA-J cells as BRIP1 (also called BACH1), a DNA helicase that is a binding partner of the breast cancer tumor suppressor BRCA1.

Fanconi Anemia and its Diagnosis

Fanconi anemia (FA) is a genetically and phenotypically heterogeneous recessive disorder characterized by diverse congenital malformations, progressive pancytopenia, and predisposition to both hematologic malignancies and solid tumors. Congenital anomalies vary from patient to patient and may affect skeletal morphogenesis as well as any of the major organ systems. Although this highly variable phenotype makes accurate diagnosis on the basis of clinical manifestations difficult in some patients, laboratory study of chromosomal breakage induced by diepoxybutane (DEB) or other crosslinking agents provides a unique cellular marker for the diagnosis of the disorder either prenatally or postnatally. Diagnosis based on abnormal response to DNA crosslinking agents can be used to identify the pre-anemia patient as well as patients with aplastic anemia or leukemia who may or may not have the physical stigmata associated with the syndrome. This overview will present our current knowledge regarding the varied phenotypic manifestations of FA and procedures for diagnosis based upon abnormal DNA damage responses.

Leukemia and preleukemia in fanconi anemia patients : a review of the literature and report of the international fanconi anemia registry

Fanconi anemia (FA) is an autosomal recessive disorder characterized clinically by a progressive pancytopenia, diverse congenital abnormalities, and increased predisposition to malignancy. Although a variable phenotype makes accurate diagnosis on the basis of clinical manifestations difficult in some patients, the unique sensitivity of FA cells to the clastogenic effect of DNA cross-linking agents such as diepoxybutane (DEB) can be used to facilitate the diagnosis. We review all cases of FA reported to have leukemia, preleukemia, or a bone marrow (BM) clonal chromosomal abnormality and include for the first time an analysis of these conditions observed in patients in the International Fanconi Anemia Registry (IFAR). The incidence of acute myelogenous leukemia (AML) in FA patients is more than 15,000 times that observed in children in the general population. Cytogenetic studies of FA-associated leukemias disclose a high frequency of monosomy 7 and duplications involving 1q. There were no occurrences of t(8;21), t(15;17), or abnormalities of 11q, which are associated with M2, M3, and M5 leukemias, respectively, but not with preleukemia. Development of leukemia in FA patients was associated with an exceedingly poor prognosis, with a mean age of death of 15 years. We suggest that all FA patients may be considered preleukemic and that this disorder presents a model for study of the etiology of AML.

FANCI is a second monoubiquitinated member of the Fanconi anemia pathway

Activation of the Fanconi anemia (FA) DNA damage–response pathway results in the monoubiquitination of FANCD2, which is regulated by the nuclear FA core ubiquitin ligase complex. A FANCD2 protein sequence–based homology search facilitated the discovery of FANCI, a second monoubiquitinated component of the FA pathway. Biallelic mutations in the gene coding for this protein were found in cells from four FA patients, including an FA-I reference cell line.

Somatic mosaicism in Fanconi anemia: Evidence of genotypic reversion in lymphohematopoietic stem cells

Somatic mosaicism has been observed previously in the lymphocyte population of patients with Fanconi anemia (FA). To identify the cellular origin of the genotypic reversion, we examined each lymphohematopoietic and stromal cell lineage in an FA patient with a 2815–2816ins19 mutation in FANCA and known lymphocyte somatic mosaicism. DNA extracted from individually plucked peripheral blood T cell colonies and marrow colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells revealed absence of the maternal FANCA exon 29 mutation in 74.0%, 80.3%, and 86.2% of colonies, respectively. These data, together with the absence of the FANCA exon 29 mutation in Epstein–Barr virus-transformed B cells and its presence in fibroblasts, indicate that genotypic reversion, most likely because of back mutation, originated in a lymphohematopoietic stem cell and not solely in a lymphocyte population. Contrary to a predicted increase in marrow cellularity resulting from reversion in a hematopoietic stem cell, pancytopenia was progressive. Additional evaluations revealed a partial deletion of 11q in 3 of 20 bone marrow metaphase cells. By using interphase fluorescence in situ hybridization with an MLL gene probe mapped to band 11q23 to identify colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells with the 11q deletion, the abnormal clone was exclusive to colonies with the FANCA exon 29 mutation. Thus, we demonstrate the spontaneous genotypic reversion in a lymphohematopoietic stem cell. The subsequent development of a clonal cytogenetic abnormality in nonrevertant cells suggests that ex vivo correction of hematopoietic stem cells by gene transfer may not be sufficient for providing life-long stable hematopoiesis in patients with FA.

Stem Cell Collection and Gene Transfer in Fanconi Anemia

Fanconi anemia (FA) is a rare genetic syndrome characterized by progressive bone marrow failure (BMF), congenital anomalies, and a predisposition to malignancy. Successful gene transfer into hematopoietic stem cells (HSCs) could reverse BMF in this disease. We developed clinical trials to determine whether a sufficient number of CD34+ stem cells could be collected for gene modification and to evaluate the safety and efficacy of HSC-corrective gene transfer in FA genotype A (FANCA) patients. Here, we report that FA patients have significant depletion of their BM CD34+ cell compartment even before severe pancytopenia is present. However, oncoretroviral-mediated ex vivo gene transfer was efficient in clinical scale in FA-A cells, leading to reversal of the cellular phenotype in a significant percentage of CD34+ cells. Re-infusion of gene-corrected products in two patients was safe and well tolerated and accompanied by transient improvements in hemoglobin and platelet counts. Gene correction was transient, likely owing to the low dose of gene-corrected cells infused. Our early experience shows that stem cell collection is well tolerated in FA patients and suggests that collection be considered as early as possible in patients who are potential candidates for future gene transfer trials.

FAAP100 is essential for activation of the Fanconi anemia-associated DNA damage response pathway

The Fanconi anemia (FA) core complex plays a central role in the DNA damage response network involving breast cancer susceptibility gene products, BRCA1 and BRCA2. The complex consists of eight FA proteins, including a ubiquitin ligase (FANCL) and a DNA translocase (FANCM), and is essential for monoubiquitination of FANCD2 in response to DNA damage. Here, we report a novel component of this complex, termed FAAP100, which is essential for the stability of the core complex and directly interacts with FANCB and FANCL to form a stable subcomplex. Formation of this subcomplex protects each component from proteolytic degradation and also allows their coregulation by FANCA and FANCM during nuclear localization. Using siRNA depletion and gene knockout techniques, we show that FAAP100‐deficient cells display hallmark features of FA cells, including defective FANCD2 monoubiquitination, hypersensitivity to DNA crosslinking agents, and genomic instability. Our study identifies FAAP100 as a new critical component of the FA‐BRCA DNA damage response network.

Re: Human Papillomavirus DNA and p53 Polymorphisms in Squamous Cell Carcinomas From Fanconi Anemia Patients

Fanconi anemia is an autosomal recessive disorder characterized by congenital malformations, bone marrow failure, and the development of squamous cell carcinomas (SCCs) and other cancers. Recent clinicopathologic evidence has raised the possibility that an environmental factor such as human papillomavirus (HPV) may be involved in the pathogenesis of SCCs in Fanconi anemia patients. Given the high prevalence of p53 mutations in SCCs among the general population and the lack of p53 mutations in HPV-related carcinogenesis, we evaluated the role of HPV and p53 mutations and polymorphisms in SCC from Fanconi anemia patients. We used polymerase chain reaction (PCR) screening and real-time PCR to detect and quantify HPV DNA in DNA extracted from microdissected SCCs obtained from 24 Fanconi anemia patients (n = 25 SCCs; case subjects) and 50 age-, sex-, and tumor site-matched SCC patients without Fanconi anemia (n = 50 SCCs; control subjects). We PCR-amplified and sequenced exons 4-9 of the p53 gene from SCC DNA. We detected HPV DNA in 84% of the SCC specimens from the case subjects and in 36% of the SCC specimens from the control subjects (P<.001). The prevalence of p53 mutations in SCCs from the case subjects (0%, 0/25) was statistically significantly lower than that of SCCs from the control subjects (36%, 12/33; P<.001). A greater proportion of patients with Fanconi anemia and SCC were homozygous for Arg72, a p53 polymorphism that may be associated with increased risk for HPV-associated human malignancies, than an ethnically-matched cohort of Fanconi anemia patients without SCC (75% versus 51%; P =.05). These data suggest that Fanconi anemia is associated with increased susceptibility to HPV-induced carcinogenesis.