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Lower expression of ATM and gene deletion is more frequent in adrenocortical carcinomas than adrenocortical adenomas
Abstract
Adrenocortical carcinoma (ACC) is a rare endocrine malignancy accounting for approximately 0.02-0.2% of all cancer deaths. The molecular pathogenesis of ACC has been the hot topic of recent reviews but it is still poorly understood. It is imperative to have a better understanding on the pathophysiology of ACC so as to establish precise diagnosis and effective treatment. This study aims to identify the molecular markers between ACCs and adrenocortical adenomas (ACAs). With MLPA, we checked on 10 ACA and 9 ACC tissue samples. The MLPA results showed deletion on chromosomes 18q, 11q, 11p, and 13q and duplication on chromosomes 3q, 4q, 6p, and 19p. There was a significant difference in the number of aberration copies of the ataxia telangiectasia-mutated (ATM) gene located on chromosome 11q22-q23 between ACCs and ACAs. Five out of 9 (56%) ACC specimens had deletion of ATM (P = 0.011). RT-PCR result then demonstrated that ATM mRNA level is lower in ACCs than in ACAs (P < 0.001). In addition, immunohistochemistry (IHC) study of the 19 ACA and 18 ACC samples confirmed lower expression of ATM protein in ACCs than in ACAs (P < 0.001). The study demonstrated that ATM expression was diminished in ACC than in ACA, suggesting an important role of ATM in the tumorigenesis of ACC.
... Increased access to NGS has facilitated genomic sequencing of rare cancers including ACC. Previous studies have reported decreased expression, deletions, and gene alterations in the ATM gene with potential to contribute to malignancy [21][22][23] . De Martino et al. reported higher frequency of ATM variants than the general population, while additional studies have reported genomic alterations, and decreased copies of the ATM gene in ACC shedding light into potential alterations for targeted therapy [21][22][23] . ...
... Previous studies have reported decreased expression, deletions, and gene alterations in the ATM gene with potential to contribute to malignancy [21][22][23] . De Martino et al. reported higher frequency of ATM variants than the general population, while additional studies have reported genomic alterations, and decreased copies of the ATM gene in ACC shedding light into potential alterations for targeted therapy [21][22][23] . Here, we report the first case of a patient with ACC and a germline heterozygous pathogenic variant in the ATM gene. ...
Background
: Adrenocortical carcinoma (ACC) is a rare malignancy arising from the adrenal cortex. ACC carries a dismal prognosis and surgery offers the only chance for a cure. Germline pathogenic variants among certain oncogenes have been implicated in ACC. Here, we report the first case of ACC in a patient with a pathogenic variant in the Ataxia Telangiectasia Mutated (ATM) gene.
Patients and Methods
: A 56-year-old Caucasian woman with biopsy proven ACC deemed unresectable and treated with etoposide, doxorubicin and cisplatin (EDP), and mitotane presented to our institution for evaluation. The tumor specimen was examined pathologically, and genetic analyses were performed on the tumor and germline using next-generation sequencing.
Results
: Pathologic evaluation revealed an 18.0 × 14.0 × 9.0 cm low-grade ACC with tumor free resection margins. Immunohistochemistry stained for inhibin, melan-A, and chromogranin. ClinOmics analysis revealed a germline pathogenic deletion mutation of one nucleotide in ATM is denoted as c.1215delT at the cDNA level and p.Asn405LysfsX15 (N405KfsX15) at the protein level. Genomic analysis of the tumor showed loss of heterozygosity (LOH) of chromosome 11 on which the ATM resides.
Conclusion
: ACC is an aggressive malignancy for which surgical resection currently offers the only curative option. Here we report a heterozygous loss-of-function mutation in germline DNA and LOH of ATM in tumor in an ACC patient, a classic two-hit scenario in a well-known cancer suppresser gene, suggesting a pathogenic role of the ATM gene in certain ACC cases.
... 6 As observed for the other cited genes, reduced expression of the ATM gene has been reported in different carcinomas, such as breast carcinoma, 36 gastric carcinoma, 37 adrenocortical carcinoma, 38 and pancreatic carcinoma. 39 Decreased expression has been attributed to deletions or rearrangement within or close to the ATM gene. 36 In OSCC, the mean expression of ATM was found to be 2.33 times lower in carcinomatous tissue compared with normal oral tissue; a reduction in ATM expression can lead to lack of DNA damage detection and contribute to tumor cell proliferation. ...
Objective:
The aim of this study was to evaluate the expression of DNA repair genes in cases of oral squamous cell carcinoma (OSCC).
Study design:
Expression of the MLH1, MSH2, MLH3, ATM, MRE11A, XRCC1, and PMS2 genes was evaluated by reverse transcription-quantitative polymerase chain reaction in the OSCC group (32 patients) and the control group (15 patients). The groups were compared by using the Mann-Whitney test, with Bonferroni correction. Associations between gene expression levels and clinical data were explored by using Pearson's and Spearman's correlation coefficients, with P value less than .05 indicating a significant difference.
Results:
The MLH1, MSH2, MLH3, ATM, MRE11A, XRCC1, and PMS2 genes were downregulated in the OSCC group compared with the control group, with significant values for MLH1 (P < .0001); MSH2 (P = .038); MLH3 (P < .0001); ATM (P < .0001); MRE11A (P < .0001); XRCC1 (P = .0004); and PMS2 (P = .008). Analysis of the correlation between gene expression and clinical data only revealed a significant negative correlation between age and expression of the PMS2 gene.
Conclusions:
Expression of the DNA repair genes MLH1, MSH2, MLH3, ATM, MRE11 AMRE11A, XRCC1, and PMS2 was reduced in OSCC.
... Adrenocortical carcinoma (ACC) is a rare, heterogeneous malignancy with a very poor prognosis. The overall incidence of ACC is approximately 0.5-2 cases per million per year [1], and it accounts for approximately 0.02-0.2% of all cancer-related deaths [2]. ACCs are classified as functional or nonfunctional based on the hormonal syndromes they produce. ...
Background:
Adrenocortical carcinoma (ACC) is a rare, heterogeneous malignancy with a poor prognosis. ACCs are classified as functioning and non-functioning. The pathogenesis of ACC remains elusive, and diagnosis of ACC is currently based on pathology. In the absence of other effective approaches, surgical resection is the preferred treatment option.
Case presentation:
Here, we report a case of ACC in the retroperitoneum. The patient underwent radical adrenalectomy and remained disease-free throughout a 6-month follow-up.
Conclusions:
Radical surgical resection is an efficient therapy for ACC, and hydrocortisone can be used to alleviate symptoms of secondary acute adrenal hypofunction.
... The phosphorylation of DNA damage response (DDR) proteins as well as ATM was demonstrated as a major mechanism of tumor evolution in various types of cancer [6,7]. Moreover, aberrant expression of ATM has been demonstrated in gastric, adenocortical, prostate, colorectal, and breast cancers [8][9][10][11][12]. Inefficiency of cells to repair DNA damage as a consequence of defects in DDR encoding genes was described as a possible reason for inducing brain tumor development [13]. ...
Ataxia telangiectasia mutated (ATM) is a key gene in DNA double-strand break (DSB), and therefore, most of its disabling genetic alterations play an important initiative role in many types of cancer. However, the exact role of ATM gene and its epigenetic alterations, especially promoter methylation in different grades of brain tumors, remains elusive. The current study was conducted to query possible correlations among methylation statue of ATM gene, ATM/ retinoblastoma (RB) protein expression, D1853N ATM polymorphism, telomere length (TL), and clinicopathological characteristics of various types of brain tumors. Isolated DNA from 30 fresh tissues was extracted from different types of brain tumors and two brain tissues from deceased normal healthy individuals. DNAs were treated with bisulfate sodium using DNA modification kit (Qiagen). Methylation-specific polymerase chain reaction (MSP-PCR) was implicated to determine the methylation status of treated DNA templates confirmed by promoter sequencing. Besides, the ATM and RB protein levels were determined by immunofluorescence (IF) assay using monoclonal mouse antihuman against ATM, P53, and RB proteins. To achieve an interactive correlation, the methylation data were statistically analyzed by considering TL and D1853N ATM polymorphism. More than 73 % of the brain tumors were methylated in ATM gene promoter. There was strong correlation between ATM promoter methylation and its protein expression (p < 0.001). As a triangle, meaningful correlation was also found between methylated ATM promoter and ATM protein expression with D1853N ATM polymorphism (p = 0.01). ATM protein expression was not in line with RB protein expression while it was found to be significantly correlated with ATM promoter methylation (p = 0.01). There was significant correlation between TL neither with ATM promoter methylation nor with ATM protein expression nor with D1853N polymorphism. However, TL has shown strong correlation with patient's age and tumor grade (p = 0.01). Given the important role of cell cycle checkpoint proteins as well as RB and ATM in TL and cancer evolution, further assessment is warranted to shed more light on the pathway linking the telomere instability to tumor progression. High ATM methylation rate in brain tumor patients could open a new avenue toward early screening and cancer therapy.
... The frequencies of these ATM variants in our series is higher than that expected in the general population [Single Nucleotide Polymorphism database (dbSNP; build 137); Supplemental Table 2]. A recent study demonstrated the ATM gene copy number is reduced in ACC compared with adrenal adenomas (35). We detected alterations of several key components of the cell cycle by CGH analysis: CDK4 amplification and CDKN2A and CDKN2B deletion. ...
Context:
Adrenocortical cancer (ACC) is a rare cancer with poor prognosis and scant treatment options. In ACC, no personalized approach has emerged but no extensive molecular screening has been performed to date.
Objective:
The objective of the study was to evaluate the presence of a large number of potentially targetable molecular events in a large cohort of advanced ACC.
Design, setting, and participants:
We used hot spot gene sequencing (Ion Torrent, 40 patients) and comparative genomic hybridization (CGH; 28 patients; a subset of the entire cohort) in adult stage III-IV ACC samples to screen for mutations and copy number abnormalities of potential interest for therapeutic use in 46 and 130 genes, respectively.
Results:
At least one copy number alteration or mutation was found in 19 patients (47.5%). The most frequent mutations were detected on TP53, ATM, and CTNNB1 [6 of 40 (15%), 5 of 40 (12.5%), and 4 of 40 (10%), respectively]. The most frequent copy number alterations identified were: amplification of the CDK4 oncogene (5 of 28; 17.9%) and deletion of the CDKN2A (4 of 28; 14.3%) and CDKN2B (3 of 28; 10.7%) tumor suppressor genes. Amplifications of FGFR1, FGF9, or FRS2 were discovered in three subjects (10.7%). Associated alterations were: deletions of CDKN2A, CDKN2B with ATM mutations, and TP53 mutations with CTNNB1 mutations.
Conclusions:
No simple targetable molecular event emerged. Drugs targeting the cell cycle could be the most relevant new therapeutic approach for patients with advanced ACC. Inhibitors of the fibroblast growth factor receptor pathway could also be a therapeutic option in a subset of patients, whereas other targeted therapies should be considered on a case-by-case basis.
Adrenocortical carcinoma (ACC) is an aggressive tumor with a short life expectancy and severe impact on quality of life. Due to the rarity of ACC, progress in the development of treatment options beyond surgery has been limited. Postoperative treatment with mitotane for adjuvant purposes is currently recommended in many expert centers for patients at elevated risk of recurrence. Lacking data from prospective trials, evidence in favor of adjuvant mitotane comes from retrospective studies that have shown a benefit in recurrence-free survival in treated patients. The management of patients with recurrent and metastatic disease is challenging, and the prognosis is often poor. Mitotane monotherapy may be used in patients with a low tumor burden and/or less aggressive disease, while cytotoxic chemotherapy is the treatment of choice for progressing tumors. No effective target therapy emerged from preclinical studies. At our center, we recommend adjuvant mitotane treatment in all patients bearing ACC at high risk of recurrence. Patients with low-risk tumors are encouraged to enter the ADIUVO trial. We use a monitored mitotane approach trying to reach and maintain a mitotane level of 14–20 mg/L. For treatment of advanced ACC, we stratify patients according to their prognostic factors and consequently apply mitotane or mitotane plus EDP. In a number of patients, who are elderly or present with impaired clinical conditions, we choose a simplified scheme with mitotane and cisplatin.
Introduction:
Adrenal tumor is a relatively common tumor. The discrimination between adrenal cortical adenoma (ACA) and adrenal cortical carcinoma (ACC) is crucial as these two diseases have distinct prognosis. ACA is a benign tumor curable by surgical excision, while the prognosis of ACC is extremely poor, with a 5-year mortality of 75-90%. Therefore, previous proteomic studies focused on markers allowing the differentiation between ACA and ACC.
Areas covered:
Several proteomic approaches based on the analysis of various samples such as human tissues, urine, and cell lines. In this review, we focused on proteomic studies performed to improve adrenal tumor diagnosis and identify ACC therapeutic targets. Expert commentary: The rapid development of cancer genomics provided a lot of information, which affects functional proteomics. In practice, differentially expressed proteins between ACA and ACC have been suggested in several proteomic studies and had a biologic implication in ACC.
All neoplasms have genetic abnormalities. Most neoplasms have visible nonrandom chromosome abnormalities. Interpretation of clonal chromosome anomalies and utilization of this knowledge can refine the histopathologic diagnosis of many tumors. The information gleaned from chromosome studies is in daily use by oncologists for patient care. The genetic data gathered by collaborative oncology groups advance chemotherapeutic regimens, which have, in turn, reduced mortality rates for many tumor types. The genetics of hematologic disorders has had a very significant clinical impact over the past several decades. The genetics of sarcomas, carcinomas, and central and peripheral nervous system tumors are used consistently today in pediatric diagnosis and management. There is an expanding influence of genetic information on the choice of therapeutic drugs for both adult and pediatric patients with solid tumors. Conventional cytogenetic analysis, fluorescence in situ hybridization (FISH) analysis, and now array CGH analysis of tumor tissues provide genetic data, which may improve the care and the therapeutics available for patients with benign and malignant neoplasms. This chapter discusses only those chromosomal abnormalities with a known impact on diagnosis, prognosis, or therapeutic response.
MicroRNAs (miRNAs) are a group of small, non-protein-coding RNAs that inhibit gene expressions through binding their 3'-UTR regions. Each miRNA can regulate a number of target genes and play crucial roles in a lot of biological processes including organogenesis, hematopoiesis, cell development, proliferation, and invasion. Deregulated expression of miRNAs has been found to be associated with initiation and development of tumors. Increasing evidences showed that miRNAs play a crucial role in adrenocortical carcinomas (ACCs). Aberrant miRNA expression may contribute to ACC carcinogenesis, and it can act as tumor-suppressive or oncogenic miRNAs. In this review, we reviewed the recent studies available on ACC-associated miRNAs. We try to summarize the contribution of miRNAs to the initiation and development of ACCs.
Whenever adrenal cancer (ACC) is completely removed we should face the dilemma to treat by means of adjuvant therapy or not. In our opinion, adjuvant mitotane is the preferable approach in most cases because the majority of patients following radical removal of an ACC have an elevated risk of recurrence. A better understanding of factors that influence prognosis and response to treatment will help in stratifying patients according to their probability of benefiting from adjuvant mitotane, with the aim of sparing unnecessary toxicity to patients who are likely unresponsive. However, until significant advancements take place, we have to deal with uncertainty using our best clinical judgement and personal experience in the clinical decision process. In the present paper, we present the current evidence on adjuvant mitotane treatment and describe the management strategies of patients with ACC after complete surgical resection. We acknowledge the limit that most recommendations are based on personal experience rather than solid evidence.
p53, apoptosis, and senescence are frequently activated in preneoplastic lesions and are barriers to progression to malignancy. These barriers have been suggested to result from an ATM-mediated DNA damage response (DDR), which may follow oncogene-induced hyperproliferation and ensuing DNA replication stress. To elucidate the currently untested role of DDR in breast cancer initiation, we examined the effect of oncogene expression in several murine models of breast cancer. We did not observe a detectable DDR in early hyperplastic lesions arising in transgenic mice expressing several different oncogenes. However, DDR signaling was strongly induced in preneoplastic lesions arising from individual mammary cells transduced in vivo by retroviruses expressing either PyMT or ErbB2. Thus, activation of an oncogene after normal tissue development causes a DDR. Furthermore, in this somatic ErbB2 tumor model, ATM, and thus DDR, is required for p53 stabilization, apoptosis, and senescence. In palpable tumors in this model, p53 stabilization and apoptosis are lost, but unexpectedly senescence remains in many tumor cells. Thus, this murine model fully recapitulates early DDR signaling; the eventual suppression of its endpoints in tumorigenesis provides compelling evidence that ErbB2-induced aberrant mammary cell proliferation leads to an ATM-mediated DDR that activates apoptosis and senescence, and at least the former must be overcome to progress to malignancy. This in vivo study also uncovers an unexpected effect of ErbB2 activation previously known for its prosurvival roles, and suggests that protection of the ATM-mediated DDR-p53 signaling pathway may be important in breast cancer prevention.
The American Association of Clinical Endocrinologists Medical Guidelines for Growth Hormone use in Growth Hormone Deficient Adults and Transition Patients – 2009 Update are systematically developed statements to assist health care providers in medical decision making for specific clinical conditions. Most of the content herein is based on literature reviews. In areas of uncertainty, professional judgment was applied. These guidelines are a working document that reflects the state of the field at the time of publication. Because rapid changes in this area are expected, periodic revisions are inevitable. We encourage medical professionals to use this information in conjunction with their best clinical judgment. The presented recommendations may not be appropriate in all situations. Any decision by practitioners to apply these guidelines must be made in light of local resources and individual circumstances.
� The American Association of Clinical Endocrinologists and American Association of Endocrine Surgeons Medical Guidelines for the Management of Adrenal Incidentalomas are systematically developed statements to assist health care providers in medical decision making for specific clinical conditions. Most of the content herein is based on literature reviews. In areas of uncertainty, professional judgment was applied. These guidelines are a working document that reflects the state of the field at the time of publication. Because rapid changes in this area are expected, periodic revisions are inevitable. We encourage medical professionals to use this information in conjunction with their best clinical judgment. The presented recommendations may not be appropriate in all situations. Any decision by practitioners to apply these guidelines must be made in light of local resources and indi- vidual circumstances.
Hereditary breast cancer accounts for up to 5-10% of all breast carcinomas. Recent studies have demonstrated that mutations in two high-penetrance genes, namely BRCA1 and BRCA2, are responsible for about 16% of the familial risk of breast cancer. Even though subsequent studies have failed to find another high-penetrance breast cancer susceptibility gene, several genes that confer a moderate to low risk of breast cancer development have been identified; moreover, hereditary breast cancer can be part of multiple cancer syndromes. In this review we will focus on the hereditary breast carcinomas caused by mutations in BRCA1, BRCA2, Fanconi anaemia (FANC) genes, CHK2 and ATM tumour suppressor genes. We describe the hallmark histological features of these carcinomas compared with non-hereditary breast cancers and show how an accurate histopathological diagnosis may help improve the identification of patients to be screened for mutations. Finally, novel therapeutic approaches to treat patients with BRCA1 and BRCA2 germ line mutations, including cross-linking agents and PARP inhibitors, are discussed.
A gene, ATM, that is mutated in the autosomal recessive disorder ataxia telangiectasia (AT) was identified by positional cloning
on chromosome 11q22-23. AT is characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, cancer
predisposition, radiation sensitivity, and cell cycle abnormalities. The disease is genetically heterogeneous, with four complementation
groups that have been suspected to represent different genes. ATM, which has a transcript of 12 kilobases, was found to be
mutated in AT patients from all complementation groups, indicating that it is probably the sole gene responsible for this
disorder. A partial ATM complementary DNA clone of 5.9 kilobases encoded a putative protein that is similar to several yeast
and mammalian phosphatidylinositol-3' kinases that are involved in mitogenic signal transduction, meiotic recombination, and
cell cycle control. The discovery of ATM should enhance understanding of AT and related syndromes and may allow the identification
of AT heterozygotes, who are at increased risk of cancer.
Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity, and cancer predisposition. The genomic organization of the A-T gene, designated ATM, was established recently. To date, more than 100 A-T-associated mutations have been reported in the ATM gene that do not support the existence of one or several mutational hotspots. To allow genotype/phenotype correlations it will be important to find additional ATM mutations. The nature and location of the mutations will also provide insights into the molecular processes that underly the disease. To facilitate the search for ATM mutations and to establish the basis for the identification of transcriptional regulatory elements, we have sequenced and report here 184,490 bp of genomic sequence from the human 11q22-23 chromosomal region containing the entire ATM gene, spanning 146 kb, and 10 kb of the 5'-region of an adjacent gene named E14/NPAT. The latter shares a bidirectional promoter with ATM and is transcribed in the opposite direction. The entire region is transcribed to approximately 85% and translated to 5%. Genome-wide repeats were found to constitute 37.2%, with LINE (17.1%) and Alu (14.6%) being the main repetitive elements. The high representation of LINE repeats is attributable to the presence of three full-length LINE-1s, inserted in the same orientation in introns 18 and 63 as well as downstream of the ATM gene. Homology searches suggest that ATM exon 2 could have derived from a mammalian interspersed repeat (MIR). Promoter recognition algorithms identified divergent promoter elements within the CpG island, which lies between the ATM and E14/NPAT genes, and provide evidence for a putative second ATM promoter located within intron 3, immediately upstream of the first coding exon. The low G+C level (38.1%) of the ATM locus is reflected in a strongly biased codon and amino acid usage of the gene.
Ataxia telangiectasia (AT) is a rare multisystem, autosomal, recessive disease characterised by neuronal degeneration, genome instability, and an increased risk of cancer. Approximately 10% of AT homozygotes develop cancer, mostly of the lymphoid system. Lymphoid malignancies in patients with AT are of both B cell and T cell origin, and include Hodgkin's lymphoma, non-Hodgkin's lymphoma, and several forms of leukaemia. The AT locus was mapped to the chromosomal region 11q22-23 using genetic linkage analysis in the late 1980s and the causative gene was identified by positional cloning several years later. The ATM gene encodes a large protein that belongs to a family of kinases possessing a highly conserved C-terminal kinase domain related to the phosphatidylinositol 3-kinase domain. Members of this kinase family have been shown to function in DNA repair and cell cycle checkpoint control following DNA damage. Recent studies indicate that ATM is activated primarily in response to double strand breaks and may be considered a caretaker of the genome. Most mutations in ATM result in truncation and destabilisation of the protein, but certain missense and splicing errors have been shown to produce a less severe phenotype. AT heterozygotes have a slightly increased risk of breast cancer. Atm deficient mice exhibit many of the symptoms found in patients with AT and have a high frequency of thymic lymphoma. The association between mutation of the ATM gene and a high incidence of lymphoid malignancy in patients with AT, together with the development of lymphoma in Atm deficient mice, supports the proposal that inactivation of the ATM gene may be of importance in the pathogenesis of sporadic lymphoid malignancy. Loss of heterozygosity at 11q22-23 (the location of the ATM gene) is a common event in lymphoid malignancy. Frequent inactivating mutations of the ATM gene have been reported in patients with rare sporadic T cell prolymphocytic leukaemia (T-PLL), B cell chronic lymphocytic leukaemia (B-CLL), and most recently, mantle cell lymphoma (MCL). In contrast to the ATM mutation pattern in AT, the most frequent nucleotide changes in these sporadic lymphoid malignancies were missense mutations. The presence of inactivating mutations, together with the deletion of the normal copy of the ATM gene in some patients with T-PLL, B-CLL, and MCL, establishes somatic inactivation of the ATM gene in the pathogenesis of lymphoid malignancies, and strongly suggests that ATM functions as a tumour suppressor. The presence of missense mutations in the germline of patients with B-CLL has been reported, suggesting that some patients with B-CLL may be constitutional AT heterozygotes. The putative hereditary predisposition of B-CLL, although intriguing, warrants further investigation.
Modern imaging modalities lead to frequent detection of adrenal masses, most of them incidental findings. Although the majority of adrenocortical and adrenomedullary tumors are benign, there are no reliable clinical and laboratory markers to distinguish most of them from malignant neoplasms. The molecular mechanisms underlying the pathogenesis of these tumors have recently begun to be unraveled. A fruitful avenue for the elucidation of tumorigenesis has been the study of adrenal tumors that are manifestations of hereditary or postzygotic genetic syndromes, because one knows the "first hit", i.e. the primary gene defect. In contrast, in sporadic adrenal tumors the first hit, possibly a somatic mutation of a tumor-related gene, is unknown, and therefore the sequence of genetic alterations is difficult to establish. In this article we review in addition to our own work the literature on molecular aspects of adrenocortical and adrenomedullary tumorigenesis.
We applied a novel method to detect single or multiple exon deletions and amplifications in the BRCA1 gene. The test, called multiplex ligation-dependent probe amplification (MLPA), uses probes designed to hybridize adjacently to the target sequence. After ligation, the joined probes are amplified and quantified. Our two diagnostic laboratories have tested in the recent years 805 families by conventional PCR-based techniques, and found 116 BRCA1 and 28 BRCA2 mutation-positive families. Using MLPA, we have tested the remaining 661 noninformative breast cancer families and identified five distinct BRCA1 germ-line mutations in five families: a deletion of exon 8, a deletion of exons 20-22, a duplication of exon 13 and exons 21-23, respectively, and a triplication, encompassing exons 17-19. Genomic deletions of BRCA1 constitute a substantial fraction of mutations in Dutch breast cancer families. If MLPA had been included in our initial BRCA1 testing, 33 families with a deletion or duplication would have been identified, representing 27% of the total 121 BRCA1 mutation-positive families. The MLPA test for BRCA1 ensures a sensitive and comprehensive high-throughput screening test for genomic rearrangement and can easily be implemented in the molecular analysis of BRCA1.
Early diagnosis of oral squamous cell carcinoma (OSCC) may have a major impact on survival and quality of life. Recent studies have shown that the majority of OSCC is preceded by precursor lesions characterized by genetic alterations. The aim of this study was to develop and evaluate a noninvasive screening test for oral preneoplastic lesions, based on genetic alterations as marker. Various methods to obtain a high yield of cells by brushing a small area of the oral mucosa were compared. A novel genetic assay, multiplex ligation-dependent probe amplification (MLPA), was applied that enables the measurement of gains and losses at 40 different chromosomal locations in one PCR reaction using 150 ng DNA. MLPA was performed on DNA of normal and dysplastic oral mucosa as well as of OSCC with the intention to select a specific probe set for accurate detection of precursor lesions in the oral cavity. The assay was correlated to loss of heterozygosity analysis using microsatellite markers, and evaluated on noncancer subjects and patients with oral leukoplakia. A noninvasive sampling method was developed with DNA yields ranging from 150 to 600 ng. Using 120 probes, we could detect large differences with MLPA in the number of alterations between normal vs dysplastic and dysplastic vs tumor tissue with P-values <0.001. A significant correlation was found between the number of alterations as detected by MLPA and the analysis for allelic loss. The available data enabled the selection of a set of 42 MLPA probes, which had the power to optimally discriminate between normal and dysplastic tissue. Our data show that MLPA is a sensitive, reliable, high-throughput and easy-to-perform technique, enabling the detection of genetic alterations on small noninvasive samples and can be considered a promising method for population-based screening of preneoplastic lesions in the oral cavity.
Adrenocortical carcinoma (ACC) is a rare endocrine malignancy accounting for between 0.02% and 0.2% of all cancer deaths. Surgical removal offers the only current potential for cure. Unfortunately, ACC has undergone metastatic spread in 40% to 70% of patients at the time of diagnosis. Standard chemotherapy with mitotane is often ineffective with intolerable side effects. The modern molecular technology of comparative genomic hybridization allows the examination of DNA for chromosomal alterations, which can lend biological insight into cancer processes. Genomes of 25 ACC clinical samples were queried on the Agilent 44K Human Genome comparative genomic hybridization array detecting regions of chromosomal gain and loss within the tumor population. Commonly shared amplifications appearing in > or =50% of tumors at P < or = 10(-4) include regions within chromosomes 5, 7, 12, 16q, and 20. Deleted genomic regions within ACC include portions of chromosomes 1, 3p, 10q, 11, 14q, 15q, 17, and 22q. Genomic aberrations in regions associated with differential survival (P < or = 0.05) and presence in > or =20% of tumors include amplifications of 6q, 7q, 12q, and 19p. Deletions within stratified survival groups include localized regions within 3, 8, 10p, 16q, 17q, and 19q. Statistical analysis of this genetic landscape reveals a set of chromosomal aberrations strongly associated with survival in an accumulation-dependent fashion. These regions may hold prognostic indicators and offer therapeutic targets that can be used to develop novel treatments for aggressive tumors.
Little is known about the pathophysiology of sporadic adrenocortical tumors in adults. Because loss of heterozygosity at the 11p15 locus has been described in childhood tumors, particularly, in adrenocortical tumors, associated with the Beckwith-Wiedemann syndrome and because insulin-like growth factor-II (IGF-II) is a crucial regulator of fetal adrenal growth, we looked for structural analysis at the 11p15 locus and IGF-II gene expression in 23 sporadic adrenocortical adult tumors: 6 carcinomas (5 with Cushing’s syndrome and 1 nonsecreting) and 17 benign adenomas (13 with Cushing’s syndrome, 1 pure androgen secreting, and 3 nonsecreting). Twenty-one patients were informative at the 11p15 locus, and six (four carcinomas and two adenomas) of them (28.5%) exhibited 11p15 structural abnormalities in tumor DNA (five, an uniparental disomy and one, a mosaicism). In a single case that could be further studied, a paternal isodisomy was observed. Very high IGF-II mRNA contents were detected in seven tumors (30%; 5 of the 6 carcinomas and 2 of the 17 adenomas). They were particularly found in tumors with uniparental disomy at the 11p15 locus. Overall, a strong correlation existed between IGF-II mRNA contents and DNA demethylation at the IGF-II locus. These data show that genetic alterations involving the 11p15 locus were highly frequent in malignant tumors, but found only in rare adenomas. These results in combination with evidence for overexpression of IGF-II from the 11p15.5 locus suggest that abnormalities in structure and/or expression of the IGF-II gene play a role as a late event of a multistep process of tumorigenesis.
To clarify gene alterations in functional human adrenal tumors, we performed molecular analysis for p53 abnormalities in 23 cases with adrenal neoplasms. The immunohistochemical study with anti-p53 monoclonal antibody pAb1801 demonstrated that 10 of 23 (43.5%) cases overexpressed p53 protein in the tumor cells. Using a polymerase chain reaction-single strand conformation polymorphism study, 5 of 6 (83.3%) pheochromocytoma tissues (1 malignant and 5 benign) and 11 of 15 (73.3%) adrenocortical adenomas (2 with Cushing's syndrome and 13 with primary aldosteronism, all benign) showed an apparent electrophoretic mobility shift between the tumor and its paired adjacent normal adrenal tissue. Such differences were detected in exon 4 (12 cases), exon 5 (2 cases), and exon 7 (3 cases). The types of these mutations in exon 4 were a substitution from threonine (ACC) to isoleucine (ATC) at codon 102 in 5 cases, from glutamine (CAG) to histidine (CAC) at codon 104 in 1 case, from glycine (GGG) to alanine (CGG) at codo...
Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder characterized by developmental anomalies, tissue and organ hyperplasia, and an increased risk of embryonal tumors. BWS is a multigenic disorder with dysregulation of the expression of imprinted genes involved in growth and cell cycle control in the 11p15 chromosomal region. The molecular genetics appear to involve either mutations of imprinted genes or chromosomal alterations of a putative imprinting center that controls gene expression across a large chromosomal domain. As the molecular genetics underlying BWS are unraveled, our knowledge of this imprinting disorder and its relationship to neoplasia and developmental anomalies will be expanded. Currently available diagnostic testing and follow-up protocols will evolve as our understanding of the molecular basis of this disease progresses.
Distinction of adrenocortical carcinoma from benign adrenocortical lesions by standard criteria is often difficult. In order to search for additional diagnostic parameters, a series of 25 adrenocortical tumors, 8 adenomas, 14 primary carcinomas, 1 metastasis, and the 2 adrenocortical carcinoma cell lines SW13 and NCI-H295 were analyzed by the approach of comparative genomic hybridization (CGH). Except for the two smallest adenomas, all tumors showed chromosomal imbalances with a high incidence of chromosomal gains, most frequently involving chromosomes or chromosome arms 5, 7, 8, 9q, 11q, 12q, 14q, 16, 17q, 19, 20, and 22q. The only significant loss of material concerned the distal part of 9p. Furthermore, 21 high-level amplifications were identified in 15 different regions of the genome. The consensus regions of recurrent gains and the focal high-level amplifications allowed identification of a series of chromosomal subregions containing candidate proto-oncogenes of potential pathogenic function in adrenocortical tumors: 1p34.3–pter, 1q22–q25, 3p24–pter, 3q29, 7p11.2–p14, 9q34, 11q12–11q13, 12q13, 12q24.3, 13q34, 14q11.2–q12, 14q32, 16p, 17q24–q25, 19p13.3, 19q13.4, and 22q11.2–q12. A subset of the CGH data was independently confirmed by interphase cytogenetics. Interestingly, the adenomas larger than 4 cm contained gained material of regions also overrepresented in carcinomas. In addition, several chromosomal gains, in particular the high-level amplifications, were exclusive for the malignant status of the tumors. These data indicate that the larger adrenal lesions need to be carefully considered in the diagnosis of adrenocortical tumors, and that genetic aberrations might provide useful markers for a better diagnostic differentiation. Genes Chromosomes Cancer 28:145–152, 2000. © 2000 Wiley-Liss, Inc.
BACKGROUND
The clinical features and natural history of adrenocortical carcinoma are highly dependent on the type of center reporting their experience. Observations from oncology services suggest a high incidence of nonfunctioning tumors, whereas reports from endocrine clinics emphasize excessive corticoid and androgen production in the majority of tumors. The incidence rate and natural history of childhood adrenal carcinoma generally has been under emphasized.METHODS
Over the past 17 years, the authors have evaluated and treated 47 patients with adrenocortical carcinoma referred to the University of Sao Paulo, 22 of whom were children.RESULTSThere is a bimodal age incidence of adrenal carcinoma, with the disease peaking in the first and fourth decades of life. Childhood adrenal carcinoma is characterized by a high rate of incidence of virilization, marked overproduction of androgens, and a less aggressive clinical course, and appears to be more amenable to surgical and other therapeutic modalities. By contrast, adrenocortical carcinoma occurring in adults presents more commonly as a mixed Cushing and virilizing syndrome, with overproduction of corticoids and androgens and a far more aggressive clinical course, leading to rapid death within months or years. Nonfunctioning adrenocortical carcinoma is less common; it generally occurs in older adults and exhibits a rapid downhill course. Modern day imaging methods have improved the diagnosis and staging of adrenal carcinoma greatly. In the authors' experience, the histologic criteria of Weiss appeared to predict tumor prognosis most accurately, whereas immunologic markers, cytoskeletal markers, DNA ploidy, cell phase markers, and oncogenic probes have yielded inconsistent results to date. Surgical removal of a localized tumor remains the best hope for long term survival. Medical therapy with mitotane and its successors in patients with Stage III or IV (MacFarlane system as modified by Sullivan et al.) disease appear to have added little to longevity or quality of life.CONCLUSIONS
When diagnosed in children, adrenal carcinoma is associated with virilism and a less aggressive natural history; however, when it occurs in adults, the disease presents more commonly as a mixed Cushing-virilizing syndrome and has a virulent course. The Weiss histologic criteria appear to correlate best with disease prognosis, but other histochemical, cell cycle, and genetic markers have not, to date, aided in disease management. Cancer 2000;88:711–36. © 2000 American Cancer Society.
Adrenocortical carcinoma (ACC) is a rare and heterogeneous malignancy, and most of the diagnostic and therapeutic strategies are not fully established according to criteria of evidence-based medicine. However, recently collaborative efforts (e.g. International Consensus Conference 2003 and networks like the European Network for the Study of Adrenal Tumours (ENSAT)) have significantly advanced the field. This article summarizes current standards in the management of ACC. In patients with suspected ACC a thorough endocrine and imaging work-up is followed by complete (Ro) resection of the tumour by an expert surgeon and initiation of adjuvant mitotane. In advanced disease not amenable to radical resection, cytotoxic drugs will be added to mitotane. The most promising regimens (etoposide, doxorubicin, cisplatin plus mitotane and streptozotocin plus mitotane) are currently compared in an international phase-III trial. Several targeted therapies are under investigation (e.g. IGF-1 inhibitors, sunitinib, sorafenib) and may lead to new treatment options.
Adrenocortical carcinoma (ACC) is a rare malignancy, and it was only in 2004 that the International Union Against Cancer (UICC) defined TNM criteria and published the first staging classification. However, to date, the prognostic value of the proposed classification has not been evaluated.
The German ACC Registry comprising 492 patients was searched for patients who were diagnosed between 1986 and 2007 with detailed information on primary diagnosis and a minimum follow-up of 6 months. Patients were assigned to UICC tumor stage, and disease-specific survival (DSS) was assessed. In addition, the contribution of potential risk factors for DSS was evaluated.
In total, 416 patients with a mean follow-up of 36 months met the inclusion criteria (stage I, n=23 patients; stage II, n=176 patients; stage III, n=67 patients; stage IV, n=150 patients). Kaplan-Meier analysis revealed a stage-dependent DSS. However, DSS in patients with stage II ACC did not differ significantly from DSS in patients with stage III ACC (hazard ratio, 1.38; 95% confidence interval, 0.89-2.16). Furthermore, patients who had stage IV ACC without distant metastases had an improved DSS compared with patients who had metastatic disease (P=.004). An analysis of different potential risk factors for defining stage III ACC revealed important roles in DSS for tumor infiltration in surrounding tissue, venous tumor thrombus (VTT), and positive lymph nodes; whereas tumor invasion in adjacent organs carried a prognosis similar to that of infiltration in surrounding tissue only.
The 2004 UICC staging classification for ACC has significant limitations. On the basis of the current analysis, a revised classification with superior prognostic accuracy is proposed (the European Network for the Study of Adrenal Tumors classification). In this system, stage III ACC is defined by the presence of positive lymph nodes, infiltration of surrounding tissue, or VTT; and stage IV ACC is restricted to patients with distant metastasis.
There are currently no well-established pathologic prognostic factors helpful in distinguishing low versus high grade adrenocortical carcinomas. The effect of 11 pathologic parameters on survival was investigated in 42 cases of adrenocortical carcinoma. Only one variable, mitotic rate, had a strong statistical association with patient outcome. The 21 patients with carcinomas with greater than 20 mitoses per 50 high power fields (hpf) had a median survival of 14 months, whereas the 21 patients with carcinomas with less than or equal to 20 mitoses had a median survival of 58 months (p less than 0.02). The presence of atypical mitoses, capsular invasion, tumor weight greater than 250 g, and size greater than 10 cm each showed a marginal statistical association with poor survival (p less than 0.06), whereas other features assessed, such as nuclear grade, presence of necrosis or of venous or sinusoidal invasion, character of the tumor cell cytoplasm, or architectural pattern, showed no statistical significance in predicting survival. It is proposed that adrenal cortical carcinomas with greater than 20 mitoses be designated high grade, whereas tumors with less than or equal to 20 mitoses be designated low grade.
A series of 43 adrenocortical tumors was analyzed using nine histologic features. Mitotic activity, especially with atypical forms, and venous invasion correlated best with metastasizing or recurring tumors; however, no single criterion was useful alone. The combination of the following nine criteria was most useful in distinguishing malignant from benign tumors: nuclear grade III or IV; mitotic rate greater than 5/50 high-power fields; atypical mitoses; clear cells comprising 25% or less of the tumor; a diffuse architecture; microscopic necrosis; and invasion of venous, sinusoidal, and capsular structures. None of the 24 tumors with two or less of these criteria metastasized or recurred, while all but one of the 19 tumors with four or more of these criteria either recurred or metastasized.
Little is known about the pathophysiology of sporadic adrenocortical tumors in adults. Because loss of heterozygosity at the 11p15 locus has been described in childhood tumors, particularly, in adrenocortical tumors, associated with the Beckwith-Wiedemann syndrome and because insulin-like growth factor-II (IGF-II) is a crucial regulator of fetal adrenal growth, we looked for structural analysis at the 11p15 locus and IGF-II gene expression in 23 sporadic adrenocortical adult tumors: 6 carcinomas (5 with Cushing's syndrome and 1 nonsecreting) and 17 benign adenomas (13 with Cushing's syndrome, 1 pure androgen secreting, and 3 nonsecreting). Twenty-one patients were informative at the 11p15 locus, and six (four carcinomas and two adenomas) of them (28.5%) exhibited 11p15 structural abnormalities in tumor DNA (five, an uniparental disomy and one, a mosaicism). In a single case that could be further studied, a paternal isodisomy was observed. Very high IGF-II mRNA contents were detected in seven tumors (30%; 5 of the 6 carcinomas and 2 of the 17 adenomas). They were particularly found in tumors with uniparental disomy at the 11p15 locus. Overall, a strong correlation existed between IGF-II mRNA contents and DNA demethylation at the IGF-II locus. These data show that genetic alterations involving the 11p15 locus were highly frequent in malignant tumors, but found only in rare adenomas. These results in combination with evidence for overexpression of IGF-II from the 11p15.5 locus suggest that abnormalities in structure and/or expression of the IGF-II gene play a role as a late event of a multistep process of tumorigenesis.
To clarify gene alterations in functional human adrenal tumors, we performed molecular analysis for p53 abnormalities in 23 cases with adrenal neoplasms. The immunohistochemical study with anti-p53 monoclonal antibody pAb1801 demonstrated that 10 of 23 (43.5%) cases overexpressed p53 protein in the tumor cells. Using a polymerase chain reaction-single strand conformation polymorphism study, 5 of 6 (83.3%) pheochromocytoma tissues (1 malignant and 5 benign) and 11 of 15 (73.3%) adrenocortical adenomas (2 with Cushing's syndrome and 13 with primary aldosteronism, all benign) showed an apparent electrophoretic mobility shift between the tumor and its paired adjacent normal adrenal tissue. Such differences were detected in exon 4 (12 cases), exon 5 (2 cases), and exon 7 (3 cases). The types of these mutations in exon 4 were a substitution from threonine (ACC) to isoleucine (ATC) at codon 102 in 5 cases, from glutamine (CAG) to histidine (CAC) at codon 104 in 1 case, from glycine (GGG) to alanine (CGG) at codon 117 in 1 case, from glutamate (GAG) to glutamine (CAG) at codon 68 in 1 case, and single base changes resulting in a premature stop codon at codon 100 in 2 cases. A 2-basepair deletion at codon 175 in exon 5 resulting in a frame shift was identified in 1 case. A single point mutation was identified, resulting in the substitution of glutamine (CAG) for arginine (CGG) at codon 248 of exon 7 in 1 case. A single basepair deletion at codon 249 resulted in a frame shift in 2 cases. There was 1 case with malignant pheochromocytoma that combined a single point mutation in exon 4 and a single base deletion in exon 7. Only 2 of 23 cases showed a loss of a normal allele encoding in the p53 gene. Northern blot analysis with 1.8-kilobase p53 cDNA revealed that p53 mRNA was overexpressed in 6 cases. Our results indicate that high frequencies of p53 gene mutation, especially in exon 4, exist in functional adrenal tumors. As p53 protein is a regulator of guanine nucleotide synthesis, the loss of normal inhibitory regulation by the p53 mutation would serve to increase the availability of GTP for the transduction of signals essential for increased cell growth and hormone expression in the adrenal tumors. These findings suggest that the p53 gene mutation may play a role in the tumorigenesis of benign and functional human adrenal tumors.
An increasing body of evidence suggests that insulin-like growth factors (IGFs) are important in the development of some tumors. In the present study, we investigated the gene expression of IGF-I, IGF-II, and their receptors in different adrenal tumors and hyperplasias. Four adrenocortical carcinomas, 15 adenomas, 4 pheochromocytomas, 5 nodular hyperplasias, and hyperplastic adrenals from 2 patients with pituitary Cushing's disease were analyzed and compared to normal adrenals. Northern blots, dot blots, and reverse transcription polymerase chain reaction analyses were used for mRNA detection, and immunohistochemistry was used for IGF-II peptide localization. The IGF-I mRNA content was low in normal, hyperplastic, and neoplastic adrenals. IGF-II mRNA levels were more than 10-fold higher in hormonally active adrenocortical carcinomas than in normal adult adrenals, and increased IGF-II-like immunoreactivity was detectable in these carcinomas. A moderate elevation of the IGF-II mRNA content was also noted in one nonfunctioning carcinoma. The IGF-II mRNA content was high in pheochromocytomas, as reported previously. Furthermore, Cushing's and Conn's adenomas expressed IGF-II mRNA levels similar to those in the normal adrenal. In nodular adrenocortical hyperplasia, we found variable IGF-II mRNA content (40-400% of normal adrenal expression). All of the normal adrenal and tumor tissues studied contained both the type I and type II IGF receptor mRNAs. The findings in the present study show that both IGF-I and IGF-II and their receptor mRNAs are expressed in various adrenocortical tumors. Moreover, the high IGF-II expression in functional adrenocortical carcinomas suggests that the IGFs may be involved in the auto/paracrine regulation of certain adrenocortical tumors.
Non-familial human adrenocortical adenomas and carcinomas were screened for mutations in exons 5-8 of the p53 tumor suppressor gene by single-strand-conformation-polymorphism (SSCP) analysis, followed by direct sequencing of PCR-amplified DNA. Point mutations in codons 12, 13 and 61 in H-ras, K-ras and N-ras proto-oncogenes were similarly assessed by direct DNA sequencing. Three out of 15 primary adrenocortical carcinomas (20%) contained a mis-sense point mutation in the conserved regions (exons 5 and 8) of the p53 gene. Mutations were located in codon 157 (GTC-->TTC; Val-->Phe), codon 163 (TAC-->AAC; Tyr-->Asn), and codon 273 (CGT-->TGT; Arg-->Cys). The mutation in codon 157 was detected in the primary tumor as well as in brain and lymph-node metastases. Among 18 adrenocortical adenomas, there was only a single non-miscoding mutation in codon 295 (CCT-->CCC; Pro-->Pro). These data suggest that mutational inactivation of the p53 gene occurs in a minority (20%) of sporadic adrenocortical carcinomas and that these mutations constitute a late event in the multi-step process of malignant transformation. No ras mutations were detected in any of these tumors, suggesting that these genes are not involved in the development of tumors originating from the adrenal cortex.
Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, radiation
sensitivity, and cancer predisposition. A-T heterozygotes are moderately cancer prone. The A-T gene, designated ATM, was recently
identified in our laboratory by positional cloning, and a partial cDNA clone was found to encode a polypeptide with a PI-3
kinase domain. We report here the molecular cloning of a cDNA contig spanning the complete open reading frame of the ATM gene.
The predicted protein of 3056 amino acids shows significant sequence similarities to several large proteins in yeast, Drosophila and mammals, all of which share the PI-3 kinase domain. Many of these proteins are involved in the detection of DNA damage
and the control of cell cycle progression. Mutations in their genes confer a variety of phenotypes with features similar to
those observed in human A-T cells. The complete sequence of the ATM gene product provides useful clues to the function of
this protein, and furthers understanding of the pleiotropic nature of the A-T mutations.
Mutations in the p53 tumor suppressor gene are frequently present in human cancers but have rarely been described in benign tumors. We previously reported mutations in the "hot spots" between exons 5-8 of the p53 gene in adrenocortical carcinomas but not in adenomas. Recently, a previously unknown hot spot in exon 4 of the p53 gene was described in adrenal adenomas and pheochromocytomas of Taiwanese patients. We, therefore, investigated whether these mutations are also present in Caucasian patients from the U.S. and Europe. We analyzed tumor tissue of 12 aldosterone-producing adenomas, 7 cortisol-producing adenomas, and 6 pheochromocytomas. Overexpression of the p53 protein was investigated by immunohistochemistry. Point mutations within exon 4 were identified by polymerase chain reaction (PCR) amplification and direct sequencing of the PCR product. The pYNZ22 microsatellite located on chromosome 17p, close to the p53 gene, was used to screen for allelic loss (LOH) of the p53 gene. Overexpression of p53 was not identified in any of the adenomas and pheochromocytomas. Point mutations within exon 4 were found in 0/25 tumors. LOH was present in 1/13 informative adenomas and 0/2 informative pheochromocytomas. We conclude that p53 mutations do not play a major role in the tumorigenesis of adrenal adenomas and pheochromocytomas of Caucasian patients. Thus, ethnic and environmental factors may be responsible for the mutational spectrum found in Taiwanese patients.
About 1.4% of the general population are heterozygous carriers of the gene for ataxiatelangiectasia (A-T), an autosomal recessive progressive neurologic syndrome in which cancer incidence of homozygotes is approximately 100-fold greater than the general population's rates. The hypothesis that A-T heterozygotes are predisposed to breast cancer was tested by the unbiased statistically powerful index-test method based on molecular genotyping. The A-T gene carrier status of 775 blood relatives in 99 A-T families was determined by tracing the A-T gene in each family through tightly linked flanking DNA markers. There were 33 women with breast cancer who could be genotyped; 25 of these were A-T heterozygotes, compared to an expected 14.9 (odds ratio 3.8, 95% confidence limits 1.7-8.4, one-sided p = .0001). This demonstrates that the A-T gene predisposes heterozygotes to breast cancer. For the 21 breast cancers with onset before age 60, the odds ratio was 2.9 (1.1-7.6, p = .009) and for the 12 cases with onset at age 60 or older, the odds ratio was 6.4 (1.4-28.8, p = .002). Thus the breast cancer risk for A-T heterozygous women is not limited to young women but appears even higher at older ages. Of all breast cancers in the United States, 6.6% may occur in women who are A-T heterozygotes. This proportion is several fold greater than the estimated proportion of carriers of BRCA1 mutations in breast cancer cases with onset at any age.
Wiedemann-Beckwith syndrome (WBS) is a heterogeneous overgrowth syndrome associated with malformations and an elevated risk of developing embryonal tumors. WBS is a multigenic disorder caused by dysregulation of imprinted growth regulatory genes within the 11p15 region. Elucidation of the genetic cause of WBS will provide important insights into the molecular and epigenetic changes associated with loss of normal growth control and cancer in this syndrome. Currently available protocols for diagnostic testing, patient monitoring and genetic counselling will evolve as our understanding of the molecular basis of WBS progresses.
The clinical features and natural history of adrenocortical carcinoma are highly dependent on the type of center reporting their experience. Observations from oncology services suggest a high incidence of nonfunctioning tumors, whereas reports from endocrine clinics emphasize excessive corticoid and androgen production in the majority of tumors. The incidence rate and natural history of childhood adrenal carcinoma generally has been under emphasized.
Over the past 17 years, the authors have evaluated and treated 47 patients with adrenocortical carcinoma referred to the University of Sao Paulo, 22 of whom were children.
There is a bimodal age incidence of adrenal carcinoma, with the disease peaking in the first and fourth decades of life. Childhood adrenal carcinoma is characterized by a high rate of incidence of virilization, marked overproduction of androgens, and a less aggressive clinical course, and appears to be more amenable to surgical and other therapeutic modalities. By contrast, adrenocortical carcinoma occurring in adults presents more commonly as a mixed Cushing and virilizing syndrome, with overproduction of corticoids and androgens and a far more aggressive clinical course, leading to rapid death within months or years. Nonfunctioning adrenocortical carcinoma is less common; it generally occurs in older adults and exhibits a rapid downhill course. Modern day imaging methods have improved the diagnosis and staging of adrenal carcinoma greatly. In the authors' experience, the histologic criteria of Weiss appeared to predict tumor prognosis most accurately, whereas immunologic markers, cytoskeletal markers, DNA ploidy, cell phase markers, and oncogenic probes have yielded inconsistent results to date. Surgical removal of a localized tumor remains the best hope for long term survival. Medical therapy with mitotane and its successors in patients with Stage III or IV (MacFarlane system as modified by Sullivan et al.) disease appear to have added little to longevity or quality of life.
When diagnosed in children, adrenal carcinoma is associated with virilism and a less aggressive natural history; however, when it occurs in adults, the disease presents more commonly as a mixed Cushing-virilizing syndrome and has a virulent course. The Weiss histologic criteria appear to correlate best with disease prognosis, but other histochemical, cell cycle, and genetic markers have not, to date, aided in disease management.
Distinction of adrenocortical carcinoma from benign adrenocortical lesions by standard criteria is often difficult. In order to search for additional diagnostic parameters, a series of 25 adrenocortical tumors, 8 adenomas, 14 primary carcinomas, 1 metastasis, and the 2 adrenocortical carcinoma cell lines SW13 and NCI-H295 were analyzed by the approach of comparative genomic hybridization (CGH). Except for the two smallest adenomas, all tumors showed chromosomal imbalances with a high incidence of chromosomal gains, most frequently involving chromosomes or chromosome arms 5, 7, 8, 9q, 11q, 12q, 14q, 16, 17q, 19, 20, and 22q. The only significant loss of material concerned the distal part of 9p. Furthermore, 21 high-level amplifications were identified in 15 different regions of the genome. The consensus regions of recurrent gains and the focal high-level amplifications allowed identification of a series of chromosomal subregions containing candidate proto-oncogenes of potential pathogenic function in adrenocortical tumors: 1p34.3-pter, 1q22-q25, 3p24-pter, 3q29, 7p11.2-p14, 9q34, 11q12-11q13, 12q13, 12q24.3, 13q34, 14q11.2-q12, 14q32, 16p, 17q24-q25, 19p13.3, 19q13.4, and 22q11.2-q12. A subset of the CGH data was independently confirmed by interphase cytogenetics. Interestingly, the adenomas larger than 4 cm contained gained material of regions also overrepresented in carcinomas. In addition, several chromosomal gains, in particular the high-level amplifications, were exclusive for the malignant status of the tumors. These data indicate that the larger adrenal lesions need to be carefully considered in the diagnosis of adrenocortical tumors, and that genetic aberrations might provide useful markers for a better diagnostic differentiation.
As its name suggests, the ATM ? 'ataxia-telangiectasia, mutated'
? gene is responsible for the rare disorder ataxia-telangiectasia. Patients
show various abnormalities, mainly in their responses to DNA damage, but also
in other cellular processes. Although it is hard to understand how a single
gene product is involved in so many physiological processes, a clear picture
is starting to emerge.
Adrenocortical cancer (ACC) is an uncommon disorder that remains a challenge to the surgeon and oncologist. When the disease is localized to the adrenal gland and readily amenable to surgical resection, reasonable 5-year survival rates are possible. Locally invasive disease carries a poorer prognosis, and metastatic disease is uniformly fatal within 1 year. In this review, we summarize the current knowledge regarding the clinical management of ACC and the molecular mechanisms underlying the disease.
The clinical manifestations, staging, and current treatment for ACC has been well documented. Surgery is still the mainstay of treatment, but identifying molecular targets for chemotherapeutic agents or monoclonal antibodies would be a great advance. At present, our understanding of pathogenic mechanisms is crude; however, the molecular events regulating this aggressive disease are beginning to emerge, especially in the last few years. The advent of laparoscopic adrenalectomy has also created its own dilemmas regarding the appropriate surgical approach to the large, potentially malignant adrenal mass.
The challenge in the management of this disease lies in understanding the molecular mechanisms that underlie the development of ACC with the diagnostic and therapeutic benefits that would ensue.
Flat adenomas are flat or slightly elevated dysplastic lesions of the colorectal mucosa, mostly with a tubular architecture. Compared with polypoid adenomas of similar size, flat adenomas show a higher frequency of high-grade dysplasia and rapid submucosal invasion. The aim of this study was to survey whether flat colorectal lesions differ in their pattern of chromosomal aberrations from their polypoid counterparts. Six flat adenomas and 12 flat carcinomas were analysed by comparative genomic hybridization (CGH) and the pattern of chromosomal aberrations was compared with a previously published series of 112 polypoid adenomas and 82 polypoid carcinomas. In addition, multiplex ligation-dependent probe amplification (MLPA) for identifying DNA copy number changes of 25 individual genes on chromosome 20 was performed on 14 flat and 15 polypoid tumours. With CGH, flat adenomas showed on average 1.8 gains (range 1-4) and 3.2 losses (range 0-4), and the flat carcinomas 4.5 gains (range 0-8) and 3.5 losses (range 1-6). In both adenomas and carcinomas, high frequencies of 20q gain (83% and 92%, respectively) and 18q loss (83% and 92%, respectively) were found. This correlation between 20q gain and 18q loss had previously been observed in a subgroup of polypoid colorectal tumours. Both flat and polypoid colorectal tumours with 20q gains by CGH showed similar patterns of copy number ratios for the individual genes tested. TOP1, BCL2L1, and E2F1 had median copy number ratios of 2 or higher, while ZNF217 had a ratio around 3. In conclusion, flat adenomas and carcinomas of the large intestine show a similar pattern of chromosomal aberrations to that observed in a specific subgroup of polypoid lesions. The transcription factor ZNF217 is an important candidate for driving the 20q gain.
Molecular analysis on formalin-fixed paraffin-embedded tissue is of increasing importance in diagnostic histopathology and tumor research. Multiplex ligation-dependent probe amplification (MLPA) is a technique that can be used for detection of copy number alterations of up to 45 different DNA sequences in one experiment. It can be performed on partially degraded DNA, which makes this technique very suitable for analysis of formalin-fixed lesions. We tested the reliability of MLPA by analyzing DNA isolated from formalin-fixed melanomas that were previously characterized by comparative genomic hybridization (CGH), and additionally the applicability of MLPA was tested by analyzing 29 routinely processed melanocytic lesions. MLPA appears to be a reliable and efficient method to evaluate DNA copy number changes as 86% of the loci tested revealed concordant CGH results. Discordance mainly involved alterations that were detected by MLPA and not by CGH probably due to a combination of lower resolution of CGH and occasionally false positive MLPA results. For application of MLPA in a diagnostic setting, different probes on a specific region of interest should be used to prevent false positive MLPA results. In a research setting as well as in a diagnostic setting, MLPA is a fast technique to screen large numbers of formalin-fixed lesions for DNA gains and losses.
Adrenal masses can be detected in up to 4% of the population, and are mostly of adrenocortical origin. Adrenocortical tumours (ACTs) may be responsible for excess steroid production and, in the case of adrenocortical cancers, for morbidity or mortality due to tumour growth. Our understanding of the pathogenesis of ACTs is more limited than that for other tumours. However, studies of the genetics of ACTs have led to major advances in this field in the last decade. The identification of germline molecular defects in the hereditary syndrome responsible for ACTs has facilitated progress. Indeed, similar molecular defects have since been identified as somatic alterations in sporadic tumours. The familial diseases concerned are Li-Fraumeni syndrome, which may be due to germline mutation of the tumour-suppressor gene TP53 and Beckwith-Wiedemann syndrome, which is caused by dys-regulation of the imprinted IGF-II locus at 11p15. ACTs also occur in type 1 multiple endocrine neoplasia (MEN 1), which is characterized by a germline mutation of the menin gene. Cushing's syndrome due to primary pigmented nodular adrenocortical disease (PPNAD) has been observed in Carney complex patients presenting inactivating germline PRKAR1A mutations. Interestingly, allelic losses at 17p13 and 11p15 have been demonstrated in sporadic adrenocortical cancer and somatic PRKAR1A mutations have been found in secreting adrenocortical adenomas. More rarely, mutations in Gs protein (gsp) and the gene for ACTH receptor have been observed in ACTs. The genetics of another group of adrenal diseases that can lead to adrenal nodular hyperplasia -- congenital adrenal hyperplasia (CAH) and glucocorticoid-remediable aldosteronism (GRA) -- have also been studied extensively. This review summarizes recent advances in the genetics of ACTs, highlighting both improvements in our understanding of the pathophysiology and the diagnosis of these tumours.
DNA damage response (DDR), the guardian of genomic integrity, emerges as an oncogene-inducible biological barrier against progression of cancer beyond its early stages. Recent evidence from both cell culture and animal models as well as analyses of clinical specimens show that activation of numerous oncogenes and loss of some tumour suppressors result in DNA replication stress and DNA damage that alarm the cellular DDR machinery, a multifaceted response orchestrated by the ATR-Chk1 and ATM-Chk2 kinase signalling pathways. Such activation of the DDR network leads to cellular senescence or death of oncogene-transformed cells, resulting in delay or prevention of tumorigenesis. At the same time, the ongoing chronic DDR activation creates selective pressure that eventually favours outgrowth of malignant clones with genetic or epigenetic defects in the genome maintenance machinery, such as aberrations in the ATM-Chk2-p53 cascade and other DDR components. Furthermore, the executive DDR machinery is shared by at least two anticancer barriers, as both the oncogene-induced DNA replication stress and telomere shortening impact the cell fate decisions through convergence on DNA damage signalling. In this study, we highlight recent advances in this rapidly evolving area of cancer research, with particular emphasis on mechanistic insights, emerging issues of special conceptual significance and discussion of major remaining challenges and implications of the concept of DDR as a tumorigenesis barrier for experimental and clinical oncology.
The DNA damage response (DDR), through the action of sensors, transducers, and effectors, orchestrates the appropriate repair of DNA damage and resolution of DNA replication problems, coordinating these processes with ongoing cellular physiology. In the past decade, we have witnessed an explosion in understanding of DNA damage sensing, signaling, and the complex interplay between protein phosphorylation and the ubiquitin pathway employed by the DDR network to execute the response to DNA damage. These findings have important implications for aging and cancer.
Adrenocortical carcinoma (ACC) is a rare neoplasm with poor prognosis. Patients present with signs of steroid hormone excess (e.g. Cushing's syndrome, virilization) or an abdominal mass. Tumour size at presentation (mean diameter at diagnosis > 10 cm) is the most important indicator of malignancy. In addition, computed tomography (CT) typically demonstrates an inhomogeneous adrenal lesion with irregular margins and variable enhancement of solid components after intravenous contrast media. Magnetic resonance imaging (MRI) is equally effective as CT and is particularly helpful to visualize invasion into large vessels. Complete tumour removal (R0 resection) offers by far the best chance for long-term survival and therefore surgery is the treatment of choice in stage I-III ACC. Despite tumour resection for cure most patients will eventually develop local recurrence or distant metastases. Thus adjuvant treatment options need to be evaluated in high-risk patients (e.g. radiation therapy of the tumour bed and/or chemotherapy). In tumour recurrence re-operation should always be considered. In metastatic disease (stage IV ACC) not amenable to surgery mitotane (o,p'DDD) remains the first-line therapy. Drug monitoring is needed for effective treatment aiming at concentrations between 14 and 20 mg/l. Patients not responding to mitotane may benefit from cytotoxic chemotherapy (23% partial remissions, 4% complete remissions). Only large prospective multicentre trials comparing different treatment options will allow to make systematic progress in the management of ACC.
Significant progress has been made in recent years in elucidating the molecular controls of cellular responses to DNA damage in mammalian cells. Much of our understanding of the mechanisms involved in cellular DNA damage response pathways has come from studies of human cancer susceptibility syndromes that are altered in DNA damage responses. Ataxia-telangiectasia mutated (ATM), the gene mutated in the disorder ataxia-telangiectasia, codes for a protein kinase that is a central mediator of responses to DNA double-strand breaks (DSB) in cells. Once activated, ATM phosphorylates numerous substrates in the cell that modulate the response of the cell to the DNA damage. We recently developed a novel system to create DNA DSBs at defined endogenous sites in the human genome and used this system to detect protein recruitment and loss at and around these breaks by chromatin immunoprecipitation. Results from this system showed the functional importance of ATM kinase activity and phosphorylation in the response to DSBs and supported a model in which ordered chromatin structure changes that occur after DNA breakage and that depend on functional NBS1 and ATM facilitate DNA DSB repair. Insights about these pathways provide us with opportunities to develop new approaches to benefit patients. Examples and opportunities for developing inhibitors that act as sensitizers to chemotherapy or radiation therapy or activators that could improve responses to cellular stresses, such as oxidative damage, are discussed. Relevant to the latter, we have shown benefits of an ATM activator in disease settings ranging from metabolic syndrome to cancer prevention.
Pathologic features of prognostic significance in adrenocortical carcinoma
- L M Weiss
- L J Medeiros
- A L Vickery
- LM Weiss
Pals, Large genomic deletions and duplications in the BRCA1 gene identified by a novel quantitative method
- F B Hogervorst
- P M Nederlof
- J J Gille
- C J Mcelgunn
- M Grippeling
- R Pruntel
- R Regnerus
- T Van Welsem
- R Van Spaendonk
- F H Menko
- I Kluijt
- C Dommering
- S Verhoef
- J P Schouten
- L J Van
- FB Hogervorst