Genomic signature of Fanconi anaemia DNA repair pathway deficiency in cancer.

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1-3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4-7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.

Oral cancer prediction by noninvasive genetic screening.

Oral squamous cell carcinomas (OSCCs) develop in genetically altered epithelium in the mucosal lining, also coined as fields, which are mostly not visible but occasionally present as white oral leukoplakia (OL) lesions. We developed a noninvasive genetic assay using next-generation sequencing (NGS) on brushed cells to detect the presence of genetically altered fields, including those that are not macroscopically visible. The assay demonstrated high accuracy in OL patients when brush samples were compared with biopsies as gold standard. In a cohort of Fanconi anemia patients, detection of mutations in prospectively collected oral brushes predicted oral cancer also when visible abnormalities were absent. We further provide insight in the molecular landscape of OL with frequent changes of TP53, FAT1 and NOTCH1. NGS analysis of noninvasively collected samples offers a highly accurate method to detect genetically altered fields in the oral cavity, and predicts development of OSCC in high-risk individuals. Noninvasive genetic screening can be employed to screen high-risk populations for cancer and precancer, map the extension of OL lesions beyond what is visible, map the oral cavity for precancerous changes even when visible abnormalities are absent, test accuracy of promising imaging modalities, monitor interventions and determine genetic progression as well as the natural history of the disease in the human patient.

Long-term Survival, Organ Function, and Malignancy after Hematopoietic Stem Cell Transplantation for Fanconi Anemia.

We report on long-term survival in 157 patients with Fanconi anemia (FA) who survived 2 years or longer after their first transplantation with a median follow-up of 9 years. Marrow failure (80%) was the most common indication for transplantation. There were 20 deaths beyond 2 years after transplantation, with 12 of the deaths occurring beyond 5 years after transplantation. Donor chimerism was available for 149 patients: 112 (76%) reported > 95% chimerism, 27 (18%) reported 90% to 95% chimerism, and 8 (5%) reported 20% to 89% donor chimerism. Two patients have < 20% donor chimerism. The 10- and 15-year probabilities of survival were 90% and 79%, respectively. Results of multivariate analysis showed higher mortality risks for transplantations before 2003 (hazard ratio [HR], 7.87; P = .001), chronic graft-versus-host disease (GVHD) (HR, 3.80; P = .004) and squamous cell carcinoma after transplantation (HR, 38.17; P < .0001). The predominant cause of late mortality was squamous cell carcinoma, with an incidence of 8% and 14% at 10 and 15 years after transplantation, respectively, and was more likely to occur in those with chronic GVHD. Other causes of late mortality included chronic GVHD, infection, graft failure, other cancers, and hemorrhage. Although most patients are disease free and functional long term, our data support aggressive surveillance for long periods to identify those at risk for late mortality.

A new physical interpretation of plant root capacitance.

Capacitance has been used as a non-destructive measure of root system size for 30 years. The equipment required is cheap and simple to apply in both field and laboratory. Good linear correlations have been reported between capacitance and root mass. A model by F. N. Dalton, predicting a linear relationship between these two variables, has become accepted widely. This model was tested for barley (Hordeum vulgare) grown hydroponically using treatments that included: raising roots out of solution, cutting roots at positions below the solution surface, and varying the distance between plant electrode and the solution surface. Although good linear correlations were found between capacitance and mass for whole root systems, when roots were raised out of solution capacitances were not linearly related to submerged root mass. Excision of roots in the solution had negligible effect on the measured capacitance. These latter observations conflict with Dalton's model. Capacitance correlated linearly with the sum of root cross-sectional areas at the solution surface and inversely with distance between plant electrode and solution surface. A new model for capacitance is proposed that is consistent with these observations.

A New Multi-Color FISH Assay for Brush Biopsy-Based Detection of Chromosomal Aneuploidy in Oral (Pre)Cancer in Patients with Fanconi Anemia.

BACKGROUND: Fanconi anemia (FA) is a rare inherited DNA instability disorder with a remarkably elevated risk of oral squamous cell carcinoma. These cancers can be detected with oral brush biopsy-based cytology even at early stages. This study aims to determine the diagnostic accuracy of a new multi-color fluorescent in situ hybridization (FISH) assay consisting of probes for CCND1, TERC, MYC and centromere of chromosome 6, as well as a 9p21 FISH assay consisting of probes for CDKN2A and centromere of chromosome 9 for the detection of oral (pre) malignant lesions in FA. METHODS: (I) Cutoffs for the dichotomization of positive or negative multi-color FISH results are determined and (II) retrospectively validated by using archived oral brush biopsy specimens from individuals with Fanconi anemia. In addition, the specimens for cutoff determination were re-hybridized with the 9p21 FISH assay. RESULTS: A cutoff of six or more chromosomal aneuploid cells for a positive FISH result was determined in the cutoff study on 160 biopsy specimens. The validating of this cutoff on 152 specimens showed at best a sensitivity of 87% and a specificity of 82.9%. CONCLUSION: Multi-color FISH is a sufficient tool to detect chromosomal aneuploidy in oral (pre) malignant lesions of individuals with Fanconi anemia. However, some false positive results may hamper the application as an adjuvant method to oral brush biopsy-based cytology in an oral cancer surveillance program.

Detection of cytogenetic changes and chromosomal aneuploidy with fluorescent in situ hybridization in cytological specimens of oral cancers in Fanconi anemia-Proof of concept.

OBJECTIVES: Fanconi anemia (FA) is a rare inherited DNA instability disorder with a remarkably elevated risk of neoplasia compared with the general population, mainly leukemia and squamous cell carcinoma (SCC). Two thirds of the SCCs arise in the oral cavity and are typically preceded by visible lesions. These lesions can be classified with brush biopsy-based cytological methods regarding their risk of a malignant transformation. As a proof of concept, this study aims to investigate genetic changes and chromosomal aneuploidy using fluorescent in situ hybridization (FISH) on oral squamous cells derived from FA affected individuals. MATERIAL AND METHODS: Five FA oral SCC (OSCC) tumor cell lines, one FA OSCC cervical lymph node metastasis as well as tumor-negative and atypical smears from oral brush biopsies were analyzed with FISH probes covering 5p15.2, MYC, EGFR, TERC, 9q34.1, CCND1, 9p21 and centromeres of chromosomes 3, 6, 7, 9, 11, and 17. RESULTS: OSCC specimens showed gains of all analyzed chromosomal regions. Chromosomal aneuploidy was observed in five of the six OSCC specimens in two multicolor FISH assays with panels of four probes each. Five out of six OSCC specimens displayed a relative deletion of 9p21. Applied on atypical brush biopsy-based smears, chromosomal aneuploidy was detected in malignant lesions but not in the smear derived from a severe parodontitis. CONCLUSIONS: As proof of concept, FISH was able to detect genetic changes and chromosomal aneuploidy discriminating oral cancer from noncancerous lesions in individuals with FA. This supports its application on oral brush biopsy-based cytology.

Diagnostic accuracy of brush biopsy-based cytology for the early detection of oral cancer and precursors in Fanconi anemia.

BACKGROUND: Individuals with Fanconi anemia (FA) have a 500-fold to 700-fold elevated risk, much earlier onset, and limited therapeutic options for oral squamous cell carcinoma (SCC) compared with the general population. The early detection of SCC, or preferably its precursors, is mandatory to retain curative therapeutic options. Due to frequent synchronic and metachronic oral lesions, tissue biopsies, as usually recommended by guidelines, often are not feasible. In the current study, an alternative strategy for early detection using oral brush biopsy-based cytology was validated regarding its diagnostic accuracy. METHODS: Over a 12-year period, the oral cavities of a large cohort of 713 individuals with FA were inspected systematically and brush biopsy-based cytology of 1233 visible oral lesions was performed. In cases of inconclusive cytology, analysis of DNA ploidy was performed whenever possible. The results were correlated to a long-term clinicopathological follow-up reference standard. RESULTS: A total of 737 lesions were suitable for statistical analysis, including 86 lesions with at least high-grade oral epithelial dysplasia in 30 patients. For cytology, the sensitivity and specificity were 97.7% and 84.5%, respectively. Additional analysis of DNA ploidy increased the sensitivity and specificity to 100% and 92.2%, respectively. CONCLUSIONS: Careful inspection of the oral cavity of individuals with FA followed by brush biopsy-based cytology appears to identify visible oral, potentially malignant and malignant lesions that warrant treatment. Approximately 63% of SCC and precursor lesions are detected at a noninvasive or early stage. Negative cytology or a lack of DNA aneuploidy can exclude high-grade oral epithelial dysplasia or SCC with high accuracy and thus reduce the need for invasive diagnostic biopsies.

Development of a Non-invasive Methodology for the Assessment of Muscle Fibre Composition.

The percentage area of fast twitch fibres of a muscle is a major determinant of muscle mechanical power and, thus, an important biomarker for the evaluation of training processes. However, the invasive character of the assessment (muscle biopsy) limits the wide application of the biomarker in the training praxis. Our purpose was to develop a non-invasive method for the assessment of fast twitch fibre content in human soleus muscle. From a theoretical point of view, the maximum muscle mechanical power depends on the fibre composition, the muscle volume and muscle specific tension. Therefore, we hypothesised that the percentage area of type II fibres would show a correlation with the maximum muscle mechanical power normalised to muscle volume and specific muscle contractile strength (i.e., plantar flexion moment divided by muscle cross-sectional area). In 20 male adults, the percentage area of type II fibres, volume and maximum cross-sectional area of the soleus muscle as well as the maximum plantar flexion moment and the maximum mechanical power were measured using muscle biopsies, magnetic resonance imaging and dynamometry. The maximum mechanical power normalised to muscle volume and specific muscle contractile strength provided a significant relationship (r = 0.654, p = 0.002) with the percentage area of type II fibres. Although the proposed assessment parameter cannot fully replace histological measurements, the predictive power of 43% can provide a relevant contribution to performance diagnostics in the training praxis.

Limited detection of human polyomaviruses in Fanconi anemia related squamous cell carcinoma.

Fanconi anemia is a rare genome instability disorder with extreme susceptibility to squamous cell carcinoma of the head and neck and anogenital tract. In patients with this inherited disorder, the risk of head and neck cancer is 800-fold higher than in the general population, a finding which might suggest a viral etiology. Here, we analyzed the possible contribution of human polyomaviruses to FA-associated head and neck squamous cell carcinoma (HNSCC) by a pan-polyomavirus immunohistochemistry test which detects the T antigens of all known human polyomaviruses. We observed weak reactivity in 17% of the HNSCC samples suggesting that based on classical criteria, human polyomaviruses are not causally related to squamous cell carcinomas analyzed in this study.

Targeting PLK1 as a novel chemopreventive approach to eradicate preneoplastic mucosal changes in the head and neck.

Head and neck squamous cell carcinomas (HNSCC) and local relapses thereof develop in preneoplastic fields in the mucosal linings of the upper aerodigestive tract. These fields are characterized by tumor-associated genetic changes, are frequently dysplastic and occasionally macroscopically visible. Currently, no adequate treatment options exist to prevent tumor development. Array-based screening with a panel of tumor-lethal small interfering RNAs (siRNAs) identified Polo-like kinase 1 (PLK1) as essential for survival of preneoplastic cells. Inhibition of PLK1 caused cell death of preneoplastic and HNSCC cells, while primary cells were hardly affected. Both siRNAs and small molecule inhibitors caused a strong G2/M cell cycle arrest accompanied by formation of monopolar spindles. In a xenografted mouse model PLK1 caused a significant tumor growth delay and cures, while chemoradiation had no effect. Thus, PLK1 seems to be a promising target for chemopreventive treatment of preneoplastic cells, and could be applied to prevent HNSCC and local relapses.

Immune Thrombocytopenia in Two Unrelated Fanconi Anemia Patients - A Mere Coincidence?

Thrombocytopenia and pancytopenia, occurring in patients with Fanconi anemia (FA), are interpreted either as progression to bone marrow failure or as developing myelodysplasia. On the other hand, immune thrombocytopenia (ITP) represents an acquired and often self-limiting benign hematologic disorder, associated with peripheral, immune-mediated, platelet destruction requiring different management modalities than those used in congenital bone marrow failure syndromes, including FA. Here, we describe the clinical course of two independent FA patients with atypical - namely immune - thrombocytopenia. While in one patient belonging to complementation group FA-A, the ITP started at 17 months of age and showed a chronically persisting course with severe purpura, responding well to intravenous immunoglobulins (IVIG) and later also danazol, a synthetic androgen, the other patient (of complementation group FA-D2) had a self-limiting course that resolved after one administration of IVIG. No cytogenetic aberrations or bone marrow abnormalities other than FA-typical mild dysplasia were detected. Our data show that acute and chronic ITP may occur in FA patients and impose individual diagnostic and therapeutic challenges in this rare congenital bone marrow failure/tumor predisposition syndrome. The management and a potential context of immune pathogenesis with the underlying marrow disorder are discussed.

Noninvasive molecular screening for oral precancer in Fanconi anemia patients.

LOH at chromosome arms 3p, 9p, 11q, and 17p are well-established oncogenetic aberrations in oral precancerous lesions and promising biomarkers to monitor the development of oral cancer. Noninvasive LOH screening of brushed oral cells is a preferable method for precancer detection in patients at increased risk for head and neck squamous cell carcinoma (HNSCC), such as patients with Fanconi anemia. We determined the prevalence of LOH in brushed samples of the oral epithelium of 141 patients with Fanconi anemia and 144 aged subjects, and studied the association between LOH and HNSCC. LOH was present in 14 (9.9%) nontransplanted patients with Fanconi anemia, whereas LOH was not detected in a low-risk group (n = 50, >58 years, nonsmoking/nonalcohol history) and a group with somewhat increased HNSCC risk (n = 94, >58 years, heavy smoking/excessive alcohol use); Fisher exact test, P = 0.023 and P = 0.001, respectively. Most frequent genetic alteration was LOH at 9p. Age was a significant predictor of LOH (OR, 1.13, P = 0.001). Five patients with Fanconi anemia developed HNSCC during the study at a median age of 39.6 years (range, 24.8-53.7). LOH was significantly associated with HNSCC (Fisher exact test, P = 0.000). Unexpectedly, the LOH assay could not be used for transplanted patients with Fanconi anemia because donor DNA in brushed oral epithelium, most likely from donor leukocytes present in the oral cavity, disturbed the analysis. Noninvasive screening using a LOH assay on brushed samples of the oral epithelium has a promising outlook in patients with Fanconi anemia. However, assays need to be adapted in case of stem cell transplantation, because of contaminating donor DNA.

Defects in the Fanconi Anemia Pathway and Chromatid Cohesion in Head and Neck Cancer.

Failure to repair DNA damage or defective sister chromatid cohesion, a process essential for correct chromosome segregation, can be causative of chromosomal instability (CIN), which is a hallmark of many types of cancers. We investigated how frequent this occurs in head and neck squamous cell carcinoma (HNSCC) and whether specific mechanisms or genes could be linked to these phenotypes. The genomic instability syndrome Fanconi anemia is caused by mutations in any of at least 16 genes regulating DNA interstrand crosslink (ICL) repair. Since patients with Fanconi anemia have a high risk to develop HNSCC, we investigated whether and to which extent Fanconi anemia pathway inactivation underlies CIN in HNSCC of non-Fanconi anemia individuals. We observed ICL-induced chromosomal breakage in 9 of 17 (53%) HNSCC cell lines derived from patients without Fanconi anemia. In addition, defective sister chromatid cohesion was observed in five HNSCC cell lines. Inactivation of FANCM was responsible for chromosomal breakage in one cell line, whereas in two other cell lines, somatic mutations in PDS5A or STAG2 resulted in inadequate sister chromatid cohesion. In addition, FANCF methylation was found in one cell line by screening an additional panel of 39 HNSCC cell lines. Our data demonstrate that CIN in terms of ICL-induced chromosomal breakage and defective chromatid cohesion is frequently observed in HNSCC. Inactivation of known Fanconi anemia and chromatid cohesion genes does explain CIN in the minority of cases. These findings point to phenotypes that may be highly relevant in treatment response of HNSCC.

A novel Fanconi anaemia subtype associated with a dominant-negative mutation in RAD51.

Fanconi anaemia (FA) is a hereditary disease featuring hypersensitivity to DNA cross-linker-induced chromosomal instability in association with developmental abnormalities, bone marrow failure and a strong predisposition to cancer. A total of 17 FA disease genes have been reported, all of which act in a recessive mode of inheritance. Here we report on a de novo g.41022153G>A; p.Ala293Thr (NM_002875) missense mutation in one allele of the homologous recombination DNA repair gene RAD51 in an FA-like patient. This heterozygous mutation causes a novel FA subtype, 'FA-R', which appears to be the first subtype of FA caused by a dominant-negative mutation. The patient, who features microcephaly and mental retardation, has reached adulthood without the typical bone marrow failure and paediatric cancers. Together with the recent reports on RAD51-associated congenital mirror movement disorders, our results point to an important role for RAD51-mediated homologous recombination in neurodevelopment, in addition to DNA repair and cancer susceptibility.

Fanconi anemia: young patients at high risk for squamous cell carcinoma.

BACKGROUND: Fanconi anemia is one of the best studied inherited cancer-prone diseases. Greatly improved protocols for hematopoietic stem cell transplantation increasingly save the lives of these young patients. However, in both transplanted and not transplanted patients, the emergence of aggressive squamous cell carcinoma represents a major medical challenge. CONCLUSIONS: This mini review summarizes current knowledge about the pathogenesis of squamous cell carcinoma (SCC) in the special context of Fanconi anemia.

Mycoplasma salivarium as a dominant coloniser of Fanconi anaemia associated oral carcinoma.

Mycoplasma salivarium belongs to the class of the smallest self-replicating Tenericutes and is predominantly found in the oral cavity of humans. In general it is considered as a non-pathogenic commensal. However, some reports point to an association with human diseases. M. salivarium was found e.g. as causative agent of a submasseteric abscess, in necrotic dental pulp, in brain abscess and clogged biliary stent. Here we describe the detection of M. salivarium on the surface of a squamous cell carcinoma of the tongue of a patient with Fanconi anaemia (FA). FA is an inherited bone marrow failure syndrome based on defective DNA-repair that increases the risk of carcinomas especially oral squamous cell carcinoma. Employing high coverage, massive parallel Roche/454-next-generation-sequencing of 16S rRNA gene amplicons we analysed the oral microbiome of this FA patient in comparison to that of an FA patient with a benign leukoplakia and five healthy individuals. The microbiota of the FA patient with leukoplakia correlated well with that of the healthy controls. A dominance of Streptococcus, Veillonella and Neisseria species was typically observed. In contrast, the microbiome of the cancer bearing FA patient was dominated by Pseudomonas aeruginosa at the healthy sites, which changed to a predominance of 98% M. salivarium on the tumour surface. Quantification of the mycoplasma load in five healthy, two tumour- and two leukoplakia-FA patients by TaqMan-PCR confirmed the prevalence of M. salivarium at the tumour sites. These new findings suggest that this mycoplasma species with its reduced coding capacity found ideal breeding grounds at the tumour sites. Interestingly, the oral cavity of all FA patients and especially samples at the tumour sites were in addition positive for Candida albicans. It remains to be elucidated in further studies whether M. salivarium can be used as a predictive biomarker for tumour development in these patients.

Young and old adults prioritize dynamic stability control following gait perturbations when performing a concurrent cognitive task.

There are conflicting results regarding the effect of aging on postural prioritization. The present study investigated balance recovery performance of young and old adults following unexpected gait perturbations in a dual task condition. Thirty-two young and 30 elderly male subjects were assigned to either control or dual task group. After baseline assessment, an unexpected gait perturbation was induced by a sudden change of surface rigidity. The dual task groups performed a mental arithmetic task. Dynamic stability was quantified based on the 'extrapolated center of mass' concept. The margin of stability decreased significantly at touchdown of the recovery leg following the unexpected perturbation (P) compared with baseline (base), yet irrespective of cognitive load (base: -4.63cm; P: -13.32cm; p<0.05). The number of errors in the cognitive task increased significantly (base: 0.13; P: 0.48; p<0.05) in both age groups. Since the stability performance was unaffected by additional cognitive load, whereas the cognitive task performance declined following the perturbation in both groups, it is concluded that postural prioritization occurs independent of age in response to unexpected gait perturbations.

Cognitive demand and predictive adaptational responses in dynamic stability control.

We studied the effects of a concurrent cognitive task on predictive motor control, a feedforward mechanism of dynamic stability control, during disturbed gait in young and old adults. Thirty-two young and 27 elderly male healthy subjects participated and were randomly assigned to either control or dual task groups. By means of a covered exchangeable element the surface condition on a gangway could be altered to induce gait perturbations. The experimental protocol included a baseline on hard surface and an adaptation phase with twelve trials on soft surface. After the first, sixth and last soft surface trial, the surface condition was changed to hard (H1-3), to examine after-effects and, thus, to quantify predictive motor control. Dynamic stability was assessed using the 'margin of stability (MoS)' as a criterion for the stability state of the human body (extrapolated center of mass concept). In H1-3 the young participants significantly increased the MoS at touchdown of the disturbed leg compared to baseline. The magnitude and the rate of these after-effects were unaffected by the dual task condition. The old participants presented a trend to after-effects (i.e., increase of MoS) in H3 but only under the dual task condition.In conclusion, the additional cognitive demand did not compromise predictive motor control during disturbed walking in the young and old participants. In contrast to the control group, the old dual task group featured a trend to predictive motor adjustments, which may be a result of a higher state of attention or arousal due to the dual task paradigm.

Clinical and molecular characteristics of squamous cell carcinomas from Fanconi anemia patients.

Fanconi anemia is a recessively inherited disease that is characterized by congenital abnormalities, bone marrow failure, and a predisposition to develop cancer, particularly squamous cell carcinomas (SCCs) in the head and neck and anogenital regions. Previous studies of Fanconi anemia SCCs, mainly from US patients, revealed the presence of high-risk human papillomavirus (HPV) DNA in 21 (84%) of 25 tumors analyzed. We examined a panel of 21 SCCs mainly from European Fanconi anemia patients (n = 19 FA patients; 16 head and neck squamous cell carcinomas [HNSCCs], 2 esophageal SCCs, and 3 anogenital SCCs) for their clinical and molecular characteristics, including patterns of allelic loss, TP53 mutations, and the presence of HPV DNA by GP5+/6+ polymerase chain reaction. HPV DNA was detected in only two (10%) of 21 tumors (both anogenital SCCs) but in none of the 16 HNSCCs. Of the 18 tumors analyzed, 10 contained a TP53 mutation. The patterns of allelic loss were comparable to those generally found in sporadic SCCs. Our data show that HPV does not play a major role in squamous cell carcinogenesis in this cohort of Fanconi anemia patients and that the Fanconi anemia SCCs are genetically similar to sporadic SCCs despite having a different etiology.