Chromosomal instability associated with adverse outcome: a case report of patient with Nijmegen breakage syndrome and rapidly developed T-NHL with complex karyotype

Nijmegen breakage syndrome (NBS) is a rare genetic disorder inherited in an autosomal recessive pattern associated with an increased risk of developing lymphoproliferative disorders, mainly non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL). NBS patients are 50 times more likely to develop malignancy than healthy controls. Moreover, in NBS, mortality rate from cancers, mainly lymphomas, is the highest among all diseases associated with excessive fragility of chromosomes. This work presents a patient previously diagnosed with Nijmegen breakage syndrome who rapidly developed T-NHL despite of constant medical supervision. Cytogenetic karyotype and microarray tests revealed complex aberrations, indicating enhanced chromosomal instability. Despite initial steroid therapy, the patient passed away due to multiorgan failure. The lack of well-established diagnostic procedures in NBS patients make it difficult to determine any therapeutic target or predictive marker. Moreover, anticancer treatment is the biggest challenge in NBS patients due to therapy-related toxicity and immunodeficiency. Our case indicates the importance of identifying parameters useful in prognosis of disease outcome, as main risk factor affecting overall survival in NBS patients is an extremely high incidence of malignancy development.


Introduction
Nijmegen breakage syndrome (NBS) is a rare genetic disorder inherited in an autosomal recessive pattern associated with an increased risk of developing lymphoproliferative disorders, mainly non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL) [1,2].
Moreover, NBS patients are 50 times more likely to develop malignancy than healthy controls [3]. The disease is caused by mutations in NBS1 gene located on chromosome 8q21. The most commonly observed NBS mutation, affecting approximately 90% of all NBS cases, is 657_661del5 in exon 6 of NBN gene [1,4]. Furthermore, most of NBS patients are of Slavic origin, thus this particular alteration is called Slavic mutation [4]. NBS seems to occur worldwide, but the majority of cases were reported among Central European and Eastern European populations (Poland, Czech Republic, Ukraine) with relatively high (1/177) carrier frequency [1,[5][6][7].
Characteristic cellular features of the NBS include increased telomere loss and sensitivity to ionizing radiation, and chromosomal instability resulting from spontaneous chromosome aberrations, usually affecting locus on chromosomes 7 (T-cell receptor (TCR) gene cluster) and 14 (immunoglobulin heavy chain gene cluster) [4]. Among the clinical features of NBS are progressive microcephaly, dysmorphic facial features (including sloping forehead, prominent nose, small mandible, long philtrum), dysmorphic ears, mild growth retardation and immunodeficiency [5,7]. In NBS, mortality rate from cancers, mainly lymphomas, is the highest among all diseases associated with excessive fragility of chromosomes [8,9].

Case report
A 4-year-old boy was admitted to Department of Genetic Diagnostics due to microcephaly, mild growth retardation and dysmorphic facial features, such as sloping forehead, large ears and prominent nose. There were no comorbidities, as well as no significant findings in the patient's family history. To assess the somatic karyotype of patient culture of blood samples was performed under standard conditions of 37°C and 5% CO 2 in PB MAX Karyotyping Medium (Thermo Fisher Scientific, Waltham, MA, USA). To stop cell division at mitosis, a mitotic inhibitor (Colchicyne Solution 10 μg/μl in NBSS, Thermo Fischer Scientific, Waltham, MA, USA) was added to the cell culture. Then, Carnoy's solution (3:1 methanol:acetic acid) was used to fixation of cells. GTG band staining was performed and the karyotype of patient was assessed using Axio Imager.Z2 microscope (Zeiss, Oberkochen, Germany) and Applied Spectral Imaging (Carlsbad, CA, USA) software. The karyotype was described according to The International System for Human Cytogenetic Nomenclature (ISCN). Cytogenetic analysis revealed normal karyotype, including no cytogenetic abnormalities involving chromosomes 7 and 14.
No chromosomal instability was found in any of the chromosomes, thus further analysis was performed using molecular techniques (Sanger method, ABI 3130, Applied Biosystem, MA, USA). The patient was diagnosed with Nijmegen breakage syndrome as genetic test confirmed homozygotic deletion c.657_661delACAAA in the NBN gene.
NBN mutation results in the fragmentation of nibrin into two nonfunctional parts: the 26 kDa N-terminal fragment and the 70 kDa fragment, which retains the residual nibrin function [4]. Homozygous carrier of this mutation is associated with very early incidence of lymphomas, sarcomas and gliomas [4,12,13]. However, in Slavic populations, heterozygous carriers of the 657del5 mutation or the molecular variant R215W of the NBN gene are often observed [1]. Population studies revealed that heterozygous carriers of the NBN mutation are also at increased risk of developing lymphoproliferative cancers [1,14].
Early diagnosis of NBS is crucial as it prevents from severe recurrent infections and unnecessary exposure to radiation during diagnostics procedures [4,7]. Due to the evolution of monoclonal gammopathy towards lymphoproliferative disorders in immunocompromised patients, monitoring of this parameter may be useful in determining the risk of developing malignancies in NBS patients [4]. Nevertheless, an improvement of immune system is needed to avoid further malignancies in patients with NBS and NHL.
From the moment of diagnosis, the patient was under constant medical supervision, and yet he developed advanced NHL as the consequence of extremely high chromosomal instability. Predisposition to malignancies, including lymphoid malignancies, is associated with chromosomal instability, as NBS patients have 250-fold risk of developing lymphomas [1,4]. Several non-specific symptoms, such as nodal enlargement and fever are thought to be connected with infection disease in NBS patients. Therefore, in NBS cases, advanced stages of lymphomas with multiorgan involvement are commonly observed [14,15]. High incidence of lymphoma relapse, reduced treatment tolerance and delayed diagnosis of lymphoproliferative disorders in NBS patients are the cause of poor prognosis [15,16]. The distribution of B and T cell lymphoma in NBS patients was described in several studies to date [17]. We present for the first time a case of patient with NBS who developed T-NHL in relatively short time despite medical geneticists' supervision.
Chromosomal instability is associated with development of complex genetic markers in pre-cancer cells. Moreover, simultaneous acquisition of structural chromosomal aberrations and mutation enables tumor evolution, thus leading to poor outcome [18]. Despite the karyotype of NBS patients is generally normal, a lot of abnormalities in the form of aneuploidies, structural rearrangements and marker chromosomes may be observed in 10-60% of cells [4].
As NBN mutations affects maturation and function of T and B cells, NBS patients are high susceptible to infections, mostly involving respiratory system [4]. Moreover, due to bone marrow failure, severe infections, cardio-  and nephrotoxicity, some forms of chemotherapy (including anthracyclines methotrexate and alkylating agents) and radiotherapy should be limited in the treatment of patients with NBS [4,19]. Hematopoietic stem cell transplantation seems to be a last treatment option in NBS patients in whom standard chemotherapy protocols have failed [19].
The lack of well-established diagnostic procedure in NBS patients make it difficult to determine any therapeutic target or predictive marker [19]. Furthermore, anticancer treatment is the biggest challenge in NBS patients due to therapy-related toxicity and immunodeficiency.
The main risk factor affecting overall survival in NBS patients is an extremely high incidence of malignancy development. Most of NBS patients die in first decade of life due to unsuccessful cancer treatment, thus novel therapeutic intervention development is of great clinical importance [4,19]. Therefore, our case indicates the necessity of identifying parameters useful in the prognosis of NBS patients.