BIRC2-BIRC3 amplification: a potentially druggable feature of a subset of head and neck cancers in patients with Fanconi anemia.

Head-and-neck squamous cell carcinomas (HNSCCs) are relatively common in patients with Fanconi anemia (FA), a hereditary chromosomal instability disorder. Standard chemo-radiation therapy is not tolerated in FA due to an overall somatic hypersensitivity to such treatment. The question is how to find a suitable alternative treatment. We used whole-exome and whole genome mRNA sequencing to identify major genomic and transcriptomic events associated with FA-HNSCC. CRISPR-engineered FA-knockout models were used to validate a number of top hits that were likely to be druggable. We identified deletion of 18q21.2 and amplification of 11q22.2 as prevailing copy-number alterations in FA HNSCCs, the latter of which was associated with strong overexpression of the cancer-related genes YAP1, BIRC2, BIRC3 (at 11q22.1-2). We then found the drug AZD5582, a known small molecule inhibitor of BIRC2-3, to selectively kill FA tumor cells that overexpressed BIRC2-3. This occurred at drug concentrations that did not affect the viability of untransformed FA cells. Our data indicate that 11q22.2 amplifications are relatively common oncogenic events in FA-HNSCCs, as holds for non FA-HNSCC. Therefore, chemotherapeutic inhibition of overexpressed BIRC2-3 may provide the basis for an approach to develop a clinically realistic treatment of FA-HNSCCs that carry 11q22.2 amplifications.

Bone marrow transplant using fludarabine-based reduced intensity conditioning regimen with in vivo T cell depletion in patients with Fanconi anemia.

FA is the most common cause of inherited BMF syndromes. The only cure for BMF in FA remains HSCT. Due to DNA instability in FA, RIC has been used to decrease immediate and late complications of HSCT. Most FA conditioning regimens in mismatched and unrelated donor transplants rely on TBI, which increases the risk of secondary malignancies. Most of the non-TBI conditioning regimens use an ex vivo T-cell depletion approach, but this is not feasible at all pediatric stem cell transplant programs. To evaluate the success of HSCT in patients with FA using non-TBI conditioning regimens with in vivo T-cell depletion approach. HSCT using non-TBI based conditioning was performed on two siblings with FA. The first sibling underwent matched unrelated donor transplant with a BM graft using fludarabine, alemtuzumab, busulfan, and cyclophosphamide conditioning and cyclosporine and mycophenolate as GVHD prophylaxis. The second sibling underwent MSD transplant with UCB and BM grafts using similar approach, but without busulfan and mycophenolate. Both siblings had engraftment without signs of acute or chronic GVHD. Acute post-transplant complications included brief viral reactivations. At last follow-up, both siblings continued to have full immune reconstitution with stable chimerism. Conditioning regimens without radiation and inclusion of alemtuzumab can lead to successful engraftment without development of GVHD and reduce risk of developing secondary neoplasms, even with unrelated donor transplants.

FANCA, TP53, and del(5q)/RPS14 alterations in a patient with T-cell non-Hodgkin lymphoma and concomitant Fanconi anemia and Li-Fraumeni syndrome.

We traced the neoplastic history (from 5 to 11 years of age) of a child with concomitant Fanconi anemia and Li-Fraumeni syndrome. Interestingly, the patient developed a highly malignant T-cell non-Hodgkin lymphoma (NHL), which does not represent the typical tumor type in the two aforementioned syndromes, presumably due to the underlying genomic instability. By using a combination of molecular and immunohistochemical approaches, we characterized the accumulation of multiple genetic alterations in a single patient, with both germline (parentally inherited biallelic FANCA variants and a likely de novo nonsense variant in TP53) and somatic (TP53 loss of heterozygosity and 5q interstitial deletion) contributions. Our findings support the interplay of TP53 and FANC genes in DNA damage response pathways and further highlight the genetic heterogeneity of lymphomas as well as the contribution of genomic instability to lymphomagenesis.

Comparison of the clinical phenotype and haematological course of siblings with Fanconi anaemia.

Fanconi anaemia (FA) is a genetic disorder due to mutations in any of the 22 FANC genes (FANCA-FANCW) and has high phenotypic variation. Siblings may have similar clinical outcome because they share the same variants; however, such association has not been reported. We present the detailed phenotype and clinical course of 25 sibling sets with FA from two institutions. Haematological progression significantly correlated between siblings, which was confirmed in an additional 55 sibling pairs from the International Fanconi Anemia Registry. Constitutional abnormalities were not concordant, except for a moderate degree of concordance in kidney abnormalities and microcephaly.

Impact of Epigenetics on Complications of Fanconi Anemia: The Role of Vitamin D-Modulated Immunity.

Fanconi anemia (FA) is a rare disorder with the clinical characteristics of (i) specific malformations at birth, (ii) progressive bone marrow failure already during early childhood and (iii) dramatically increased risk of developing cancer in early age, such as acute myeloid leukemia and squamous cell carcinoma. Patients with FA show DNA fragility due to a defect in the DNA repair machinery based on predominately recessive mutations in 23 genes. Interestingly, patients originating from the same family and sharing an identical mutation, frequently show significant differences in their clinical presentation. This implies that epigenetics plays an important role in the manifestation of the disease. The biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 controls cellular growth, differentiation and apoptosis via the modulation of the immune system. The nuclear hormone activates the transcription factor vitamin D receptor that affects, via fine-tuning of the epigenome, the transcription of >1000 human genes. In this review, we discuss that changes in the epigenome, in particular in immune cells, may be central for the clinical manifestation of FA. These epigenetic changes can be modulated by vitamin D suggesting that the individual FA patient's vitamin D status and responsiveness are of critical importance for disease progression.

Natural Killer Cell Degranulation Defect: A Cause for Impaired NK-Cell Cytotoxicity and Hyperinflammation in Fanconi Anemia Patients.

Fanconi anemia (FA) is a rare inherited syndrome characterized by progressive bone marrow failure (BMF), abnormal skin pigmentation, short stature, and increased cancer risk. BMF in FA is multifactorial and largely results from the death of hematopoietic stem cells due to genomic instability. Also, inflammatory pathology in FA has been previously reported, however the mechanism is still not clear. In literature, decreased NK-cell count and/or impaired NK-cell activity, along with other immunological abnormalities have been described in FA-patients (1). However, to the best of our knowledge, this is the first report showing a defective degranulation mechanism leading to abnormal NK-cell cytotoxicity in FA-patients, which may explain the development of a hyperinflammatory response in these patients. This may predispose some patients to develop Hemophagocytic lymphohistiocytosis (HLH) which manifests with prolonged fever, progressive cytopenias and organomegaly. Early diagnosis and initiation of immunosuppressive therapy in these patients will help to better manage these patients. We also propose FA genes to be listed as a cause of familial HLH.

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.

Myelodysplastic Syndrome, Acute Myeloid Leukemia, and Cancer Surveillance in Fanconi Anemia.

Fanconi anemia (FA) is a DNA repair disorder associated with a high risk of cancer and bone marrow failure. Patients with FA may present with certain dysmorphic features, such as radial ray abnormalities, short stature, typical facies, bone marrow failure, or certain solid malignancies. Some patients may be recognized due to exquisite sensitivity after exposure to cancer therapy. FA is diagnosed by increased chromosomal breakage after exposure to clastogenic agents. It follows autosomal recessive and X-linked inheritance depending on the underlying genomic alterations. Recognizing patients with FA is important for therapeutic decisions, genetic counseling, and optimal clinical management.

Antibody response to human papillomavirus vaccination and natural exposure in individuals with Fanconi Anemia.

Fanconi anemia (FA) is a rare genetic disorder associated with predisposition to head and neck and gynecological squamous cell cancers. In the general population, these cancers are commonly linked to human papillomavirus (HPV) infection. Antibodies to natural HPV infection and HPV vaccination were evaluated in 63 individuals with FA while considering host immune factors. Approximately 30% of reportedly unvaccinated participants were seropositive (HPV6-38%, HPV11-25%, HPV16-26%, and HPV18-26%). Seropositivity was significantly associated with having had sex regardless of age (p=.007). Most participants showed seropositivity after HPV vaccination (HPV6-100%, HPV11-100%, HPV16-100% and HPV18-92%). Interestingly, titers for all 4 subtypes were significantly lower in the post-hematopoietic stem cell transplant (HSCT) participants compared to those who received the vaccine, but had not undergone HSCT (HPV6-p=.030, HPV11-p=.003, HPV16-p=.018, HPV18-p=<.001). It is unclear if these titers sufficiently protect from new infection since protective serologic cut offs have not yet been defined for the HPV vaccine. Individual immune functions were not associated with HPV seropositivity, however, underlying heterogeneous immune deficiency may explain higher rates of seropositivity in our younger unvaccinated participants (age 4-13 years). To better measure the efficacy of HPV vaccination in those with FA and other immune-compromised or cancer-prone disorders, future well-controlled vaccine studies are required.

Cerebral toxoplasmosis after haematopoietic stem cell transplantation.

Toxoplasmosis is an opportunistic infection caused by the parasite Toxoplasma gondii. The infection is severe and difficult to diagnose in patients receiving allogeneic haematopoietic stem cell transplantation (HSCT). It frequently involves the central nervous system. The case is presented of cerebral toxoplasmosis in a 17-year-old youth with Fanconi anaemia treated with haematopoietic stem cell transplantation (HSCT).

Impaired immune function in children and adults with Fanconi anemia.

BACKGROUND: Fanconi anemia (FA) is a rare genetic disorder characterized by genome instability, bone marrow failure, and cancer predisposition. Previously, small studies have reported heterogeneous immune dysfunction in FA. PROCEDURE: We performed a detailed immunologic assessment in a large FA cohort who have not undergone bone marrow transplantation or developed malignancies. Comprehensive quantitative and functional immunologic assessment of 29 FA individuals was compared to healthy age-matched controls. RESULTS: Compared to non-FA persons of similar ages, FA individuals showed lower absolute total B cells (P < 0.001), lower memory B cells (P < 0.001), and decreased IgM (P < 0.001) but normal IgG. NK cells (P < 0.001) and NK cytotoxicity (P < 0.001) were decreased. CD4+ T cells were decreased (P = 0.022), while CD8+ T cell and absolute T-cell numbers were comparable. Cytotoxic T cells (P < 0.003), and antigen proliferation response to tetanus (P = 0.019) and candida (P = 0.019), were diminished in FA. Phytohemagglutinin responses and plasma cytokines were normal. Within FA subjects, adults and older children (≥10 years) exhibited higher CD8+ T cells than younger children (P = 0.004). Documented atypical infections were infrequent, although oral human papilloma virus (HPV) prevalence was higher (31% positive) in FA. CONCLUSIONS: Overall, these results demonstrate a high rate of significant humoral and cellular immune dysfunction. Continued longitudinal study of immune function is critical to understand evolution with age, bone marrow failure, and cancer development.

Late Effects Screening Guidelines after Hematopoietic Cell Transplantation for Inherited Bone Marrow Failure Syndromes: Consensus Statement From the Second Pediatric Blood and Marrow Transplant Consortium International Conference on Late Effects After Pediatric HCT.

Patients with inherited bone marrow failure syndromes (IBMFS), such as Fanconi anemia (FA), dyskeratosis congenita (DC), or Diamond Blackfan anemia (DBA), can have hematologic manifestations cured through hematopoietic cell transplantation (HCT). Subsequent late effects seen in these patients arise from a combination of the underlying disease, the pre-HCT therapy, and the HCT process. During the international consensus conference sponsored by the Pediatric Blood and Marrow Transplant Consortium on late effects screening and recommendations following allogeneic hematopoietic cell transplantation for immune deficiency and nonmalignant hematologic diseases held in Minneapolis, Minnesota in May 2016, a half-day session was focused specifically on the unmet needs for these patients with IBMFS. This multidisciplinary group of experts in rare diseases and transplantation late effects has already published on the state of the science in this area, along with discussion of an agenda for future research. This companion article outlines consensus disease-specific long-term follow-up screening guidelines for patients with IMBFS.

Common Variable Immunodeficiency Caused by FANC Mutations.

Common variable immunodeficiency (CVID) is the most common adult-onset primary antibody deficiency disease due to various causative genes. Several genes, which are known to be the cause of different diseases, have recently been reported as the cause of CVID in patients by performing whole exome sequencing (WES) analysis. Here, we found FANC gene mutations as a cause of adult-onset CVID in two patients. B cells were absent and CD4+ T cells were skewed toward CD45RO+ memory T cells. T-cell receptor excision circles (TRECs) and signal joint kappa-deleting recombination excision circles (sjKRECs) were undetectable in both patients. Both patients had no anemia, neutropenia, or thrombocytopenia. Using WES, we identified compound heterozygous mutations of FANCE in one patient and homozygous mutation of FANCA in another patient. The impaired function of FANC protein complex was confirmed by a monoubiquitination assay and by chromosome fragility test. We then performed several immunological evaluations including quantitative lymphocyte analysis and TRECs/sjKRECs analysis for 32 individuals with Fanconi anemia (FA). In total, 22 FA patients (68.8%) were found to have immunological abnormalities, suggesting that such immunological findings may be common in FA patients. These data indicate that FANC mutations are involved in impaired lymphogenesis probably by the accumulation of DNA replication stress, leading to CVID. It is important to diagnose FA because it drastically changes clinical management. We propose that FANC mutations can cause isolated immunodeficiency in addition to bone marrow failure and malignancy.

Radiation-free, alternative-donor HCT for Fanconi anemia patients: results from a prospective multi-institutional study.

Fanconi anemia (FA) is an inherited bone marrow failure syndrome characterized by chromosomal fragility, progressive marrow failure, and cancer predisposition. Hematopoietic cell transplantation (HCT) is curative for FA-related marrow failure or leukemia, but both radiation exposure during transplant and graft-versus-host disease (GVHD) may increase risk of later malignancies of the head and neck and anogenital area. In this study, we tested a radiation-free conditioning regimen with a T-cell-depleted graft to eliminate radiation exposure and minimize early and late toxicities of transplant. Forty-five patients (median age, 8.2 years; range 4.3-44) with FA underwent HCT between June 2009 and May 2014. The preparative regimen included busulfan, cyclophosphamide, fludarabine, and rabbit anti-thymocyte globulin. Busulfan levels were monitored to avoid excess toxicity. All grafts were CD34-selected/T-cell-depleted using the CliniMacs CD34 columns (Miltenyi). Thirty-four patients (75.6%) with marrow failure and 11 (24.4%) with myelodysplastic syndrome underwent HCT using matched unrelated (n = 25, 55.5%), mismatched unrelated (n = 14, 31.1%), or mismatched related donors (n = 6, 13.4%). One year probabilities of overall and disease-free survival for the entire cohort, including patients with myeloid malignancy and those receiving mismatched related/haploidentical grafts, were 80% (±6%) and 77.7% (±6.2%), respectively (median follow-up 41 months). All young children (<10 years of age) undergoing HCT for marrow failure using low-dose busulfan-containing regimen survived. No patients developed acute grade 3-4 GVHD. Sequential reduction of busulfan dose was successfully achieved per study design. Our results show excellent outcomes in patients with FA undergoing alternative donor HCT without radiation exposure. The study is registered to www.clinicaltrials.gov as #NCT01082133.

V(D)J recombination process and the Pre-B to immature B-cells transition are altered in Fanca-/- mice.

B-lymphocytes in the bone marrow (BM) must generate a functional B-cell receptor and overcome the negative selection induced by reactivity with autoantigens. Two rounds of DNA recombination are required for the production of functional immunoglobulin heavy (Ig-HCs) and light (LCs) chains necessary for the continuation of B-lymphocyte development in the BM. Both rounds depend on the joint action of recombination activating gene-1 (RAG-1) and RAG-2 endonucleases with the DNA non-homologous end-joining pathway. Loss of the FANC gene leads to the chromosome breakage and cancer predisposition syndrome Fanconi anemia. Because the FANC proteins are involved in certain aspects of the recombination process, we sought to determine the impact of the FANC pathway on the Ig diversification process using Fanca-/- mice. In this work we demonstrated that Fanca-/- animals have a mild B-cell differentiation defect characterized by a specific alteration of the IgM- to IgM+ transition of the B220low B-cell population. Pre-B cells from Fanca-/- mice show evidence of impaired kLC rearrangement at the level of the Vk-Jk junction. Furthermore, Fanca-/- mice showed a skewed Vκ gene usage during formation of the LCs Vk-Jk junctions. Therefore, the Fanca protein appears as a yet unidentified factor involved in the primary diversification of Ig.

Upregulated LINE-1 Activity in the Fanconi Anemia Cancer Susceptibility Syndrome Leads to Spontaneous Pro-inflammatory Cytokine Production.

Fanconi Anemia (FA) is a genetic disorder characterized by elevated cancer susceptibility and pro-inflammatory cytokine production. Using SLX4(FANCP) deficiency as a working model, we questioned the trigger for chronic inflammation in FA. We found that absence of SLX4 caused cytoplasmic DNA accumulation, including sequences deriving from active Long INterspersed Element-1 (LINE-1), triggering the cGAS-STING pathway to elicit interferon (IFN) expression. In agreement, absence of SLX4 leads to upregulated LINE-1 retrotransposition. Importantly, similar results were obtained with the FANCD2 upstream activator of SLX4. Furthermore, treatment of FA cells with the Tenofovir reverse transcriptase inhibitor (RTi), that prevents endogenous retrotransposition, decreased both accumulation of cytoplasmic DNA and pro-inflammatory signaling. Collectively, our data suggest a contribution of endogenous RT activities to the generation of immunogenic cytoplasmic nucleic acids responsible for inflammation in FA. The additional observation that RTi decreased pro-inflammatory cytokine production induced by DNA replication stress-inducing drugs further demonstrates the contribution of endogenous RTs to sustaining chronic inflammation. Altogether, our data open perspectives in the prevention of adverse effects of chronic inflammation in tumorigenesis.

Cytokine overproduction and crosslinker hypersensitivity are unlinked in Fanconi anemia macrophages.

The Fanconi anemia proteins participate in a canonical pathway that repairs cross-linking agent-induced DNA damage. Cells with inactivated Fanconi anemia genes are universally hypersensitive to such agents. Fanconi anemia-deficient hematopoietic stem cells are also hypersensitive to inflammatory cytokines, and, as importantly, Fanconi anemia macrophages overproduce such cytokines in response to TLR4 and TLR7/8 agonists. We questioned whether TLR-induced DNA damage is the primary cause of aberrantly regulated cytokine production in Fanconi anemia macrophages by quantifying TLR agonist-induced TNF-α production, DNA strand breaks, crosslinker-induced chromosomal breakage, and Fanconi anemia core complex function in Fanconi anemia complementation group C-deficient human and murine macrophages. Although both M1 and M2 polarized Fanconi anemia cells were predictably hypersensitive to mitomycin C, only M1 macrophages overproduced TNF-α in response to TLR-activating signals. DNA damaging agents alone did not induce TNF-α production in the absence of TLR agonists in wild-type or Fanconi anemia macrophages, and mitomycin C did not enhance TLR responses in either normal or Fanconi anemia cells. TLR4 and TLR7/8 activation induced cytokine overproduction in Fanconi anemia macrophages. Also, although TLR4 activation was associated with induced double strand breaks, TLR7/8 activation was not. That DNA strand breaks and chromosome breaks are neither necessary nor sufficient to account for the overproduction of inflammatory cytokines by Fanconi anemia cells suggests that noncanonical anti-inflammatory functions of Fanconi anemia complementation group C contribute to the aberrant macrophage phenotype and suggests that suppression of macrophage/TLR hyperreactivity might prevent cytokine-induced stem cell attrition in Fanconi anemia.

Immune status of patients with inherited bone marrow failure syndromes.

Immune function abnormalities have been reported in patients with Fanconi anemia (FA), dyskeratosis congenita (DC) and, rarely, in Shwachman-Diamond syndrome (SDS), and Diamond-Blackfan anemia (DBA), but large systematic studies are lacking. We assessed immunological parameters in 118 patients with these syndromes and 202 unaffected relatives. We compared the results in patients with reference values, and with values in relatives after adjusting for age, sex, corticosteroid treatment, and severe bone marrow failure (BMF). Adult patients (≥18 years) with FA had significantly lower immunoglobulins (IgG, IgA and IgM), total lymphocytes, and CD4 T cells than reference values or adult relatives (P < 0.001); children with FA had normal values. Both children and adults with FA had lower B- and NK cells (P < 0.01) than relatives or reference values. Patients with DC had essentially normal immunoglobulins but lower total lymphocytes than reference values or relatives, and lower T-, B-, and NK-cells; these changes were more marked in children than adults (P < 0.01). Most patients with DBA and SDS had normal immunoglobulins and lymphocytes. Lymphoproliferative responses, serum cytokine levels, including tumor necrosis factor-α and interferon-γ, and cytokine levels in supernatants from phytohemagglutinin-stimulated cultures were similar across patient groups and relatives. Only patients with severe BMF, particularly those with FA and DC, had higher serum G-CSF and Flt3-ligand and lower RANTES levels compared with all other groups or relatives (P < 0.05). Overall, immune function abnormalities were seen mainly in adult patients with FA, which likely reflects their disease-related progression, and in children with DC, which may be a feature of early-onset severe disease phenotype.

Impaired immune response to Candida albicans in cells from Fanconi anemia patients.

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and cancer predisposition. Several studies show alterations of the immunological status of FA patients including defects in peripheral blood lymphocyte subsets, serum immunoglobulin levels, and inflammatory cytokines. However scanty information is available on the response of FA cells to specific infectious antigens. In this work we examined the response of FA cells to different immunological stimuli and found a defective response of IL-1ß, TNF-α and IL-17 to Candida albicans stimulation thus pointing to a potentially impaired response to fungal infections of FA patients.

Brca2 deficiency leads to T cell loss and immune dysfunction.

Germline mutations in the breast cancer type 2 susceptibility gene (BRCA2) are linked to familial breast cancer and the progressive bone marrow failure syndrome Fanconi anaemia. Established Brca2 mouse knockout models show embryonic lethality, but those with a truncating mutation at the C-terminus survive to birth and develop thymic lymphoma at an early age. To overcome early lethality and investigate the function of BRCA2, we used T cell-specific conditional Brca2 knockout mice, which were previously shown to develop thymic lymphoma at a low penetrance. In the current study we showed that the number of peripheral T cells, particularly naïve pools, drastically declined with age. This decline was primarily ascribed to improper peripheral maintenance. Furthermore, heterozygous mice with one wild-type Brca2 allele manifested reduced T cell numbers, suggesting that Brca2 haploinsufficiency might also result in T cell loss. Our study reveals molecular events occurring in Brca2-deficient T cells and suggests that both heterozygous and homozygous Brca2 mutation may lead to dysfunction in T cell populations.