A rare PALB2 germline variant causing G2/M cell cycle arrest is associated with isolated myelosarcoma in infancy

Abstract Background Isolated myelosarcoma of infancy is a rare presentation of acute myelogenous leukaemia (AML). Because of its rarity and early onset in infancy underlying genetic predisposition is potentially relevant in disease initiation. Methods and Results We report an oncologic emergency in an infant with thoracic and intraspinal aleukaemic myeloid sarcoma causing acute myelon compression and lower leg palsy. Whole‐exome sequencing of the patient's germline DNA identified a rare PALB2 (OMIM 610355) variant (p.A1079S), which is located in a domain critical for the gene's proper function within the homology‐directed repair pathway. In line with potential DNA damage repair defects mediated by the PALB2 deregulation, the patient's fibroblasts showed increased sensitivity towards radiation and DNA intercalating agents. Conclusion Therefore, we suggest PALB2 p.A1079S as a pathogenic variant potentially contributing to the here observed patient phenotype.


| INTRODUCTION
PALB2 is a tumour suppressor that plays a critical role in homologous recombination repair (HR). Mechanistically, it mediates the recruitment of BRCA2 and RAD51 to sites of DNA damage and functions downstream of BRCA1 (Hanenberg & Andreassen, 2018;Xia et al., 2006). Apart from HR, PALB2 regulates S and G2 DNA damage checkpoints and suppresses genome instability by protecting transcriptionally active genes from genotoxic stress (Hanenberg & Andreassen, 2018;Menzel et al., 2011).
Bi-allelic inactivating germline mutations in PALB2 that at least partly truncate the BRCA2-binding C-terminal WD40 domain, result in the Fanconi anaemia (FA) subtype N. These patients display a severe FA phenotype with a high cancer incidence, including acute myeloid leukaemia (AML) before the age of 5 years (Reid et al., 2007;Xia et al., 2007).
Here, we report an infant with an extended atypic localization of myeloid sarcoma as isolated manifestation of an AML with a paternally inherited rare PALB2 germline variation.

| Case description
A 7-month old male infant was transferred to our clinic in deteriorating clinical condition over the last 2 days, with massive tachydyspnea. The physical examination on admission was remarkable for a silent chest, reduced percussion sound on the right side and a very mild partial paralysis of both lower limbs, while laboratory results were unremarkable (Table S1). The ultrasound and chest x-ray revealed a large right-sided pleural effusion ( Figure S1) with monoblastic cells, aberrant expression of CD7 and in part CD56 ( Figure  1a). Consequently, we performed a CT ( Figure S2) and MRI of the chest (Figure 1b), which showed a tumour involving the right pleura with infiltration of the thoracic wall, the mediastinum and further ranging through the neural foramina to extra-axial intraspinal and growing intra-abdominal along the blood vessels. The intraspinal proportion led to a compression of the myelon causing partial palsy of the legs.
Bone marrow morphology revealed normal erythropoiesis, myelopoiesis and megakaryopoiesis with 7.5% of myelomonocytic blasts. No increased proportion of myeloid associated blasts (0.6%) was identified by flow cytometry. Cytogenetics verified a chromosome 6q deletion. The transthoracic tumour biopsy revealed a tumour cell population with positivity for CD117, partially for CD14 and focally for CD45. The Ki67 proliferation index was 80% (Figure 1c). In synopsis with the radiological, laboratory, flow cytometric and histopathological findings, we finally diagnosed an isolated extramedullary manifestation of a myelomonocytic sarcoma. The family history was unremarkable for neoplasia except for a great-grandfather with skin cancer.
The patient was initially stratified into the intermediate risk group of the treatment protocol AML-BFM 2012 and therapy was started in an emergency because of myelon compression. The palsy of the lower limbs declined during the pre-phase with cytarabine and MRI confirmed the rapid regression of the spinal cord compression. After the first induction cycle, 16% blasts were detectable in the bone marrow cytology, therefore the patient was re-stratified into the high-risk group with an indication for allogeneic stem cell transplantation (SCT) in the first complete remission. After the fourth cycle, he underwent SCT and received PBSCs from a matched unrelated donor. As an early complication, he developed acute Graft-versus-Host-Disease of the skin grade I on day +9 and an early CMV reactivation on day +12. He was discharged on day +40, the donor chimerism on day +100 was 100% and following MRIs did not show any residual signs of the EML now 17 months post-SCT.

PALB2 variant (p.A1079S)
Despite a neoplasia unremarkable family history, the combination of a rare presentation of AML and early onset in infancy points towards a potential germline predisposition as an initiator of the disease. Therefore, TRIO sequencing, encompassing whole-exome sequencing of the patient as well as his parents, was carried out. By applying the CPSR engine (Nakken et al., 2019), no proven pathogenic or likely pathogenic variants were found ( Figure 2a). Nevertheless, a variety of possibly damaging variants of unknown significance (VUS) were identified (Table S2). Taking minor allele frequency (MAF) as well as potential functional consequences into account, the heterozygous germline variant PALB2 c.3235G>T, which was validated in the boy as well as his father (Figure 2b), was chosen for further analysis. While the missense variant is extremely rare (MAF < 0.01) it leads to exchange from alanine to serine in Exon 12 (p.A1079S), which is located within the fifth WD40-repeats region of the protein. This domain was shown to interact with RAD51, BRCA2 and POLH and is required for POLH DNA synthesis stimulation ( mutation is predicted to be damaging with a CADD score of 22.9. To assess the pathogenicity of the variant further, we correlated mutational frequencies of each PALB2 nucleotide by comparing its germline variation in healthy individuals (Gnom-AD v2.1.1 non-cancer n = 118.479) and somatic mutations in cancer (COSMIC v91 n = 37,221). As expected, PALB2 c.3235G>T and its surrounding nucleotides are rarely mutated in the germline of healthy individuals, while they show a high frequency within cancer (Figure 2c).

| PALB2 p.A1079S fibroblasts show increased sensitivity towards DNA damage
It has previously been shown that due to its essential function in HR, PALB2 loss confers hypersensitivity to ionizing radiation and to DNA interstrand crosslinking agents like Mitomycin C (Park et al., 2014;Xia et al., 2006). To assess whether PALB2 p.A1079S hampers the cells' capacity for DNA double-strand break-induced HR, we subjected primary fibroblasts of the patient, of healthy control and a patient with a known heterozygous pathogenic BRCA2 mutation (p.Met1?; rs80358650) as a positive control to 6 Gy of ionizing irradiation. After 48 h, the cells' response was measured using cell cycle analysis. Consistent with a functional effect of the identified PALB2 variant, fibroblasts carrying p.A1079S showed a significantly higher percentage of cells arrested in G2/M phase compared to the healthy control ( Figure 3a). Likewise, Mitomycin C treatment showed an increased sensitivity in cells harbouring the identified PALB2 variant compared to the healthy control, with patient cells being arrested at S/G2/M to similar levels like fibroblasts carrying the pathogenic BRCA2 start lost mutation (Figure 3b).

| DISCUSSION
The diagnosis of a primarily extramedullary manifestation of an AML can be very complex and, dependent on   the localization of the myeloid sarcoma, can even lead to an oncological emergency. To our knowledge, this case is the first description of an infant with a vast isolated extramedullary medio-thoraco-spinal tumour infiltration as the first manifestation of an AML. The rarity of presentation and the early onset suggest an underlying genetic predisposition.
Carriers of heterozygous germline variants within the HR pathway have an increased lifetime risk to develop breast, ovarian and other cancers (Antoniou et al., 2014;Roy et al., 2011). The identified PALB2 p.A1079S variant is located within a highly relevant functional domain, in close proximity to the breast cancer-associated functional missense mutations p.L939W, p.T1030I and p.L1143P and likewise shows increased cellular sensitivity to ionizing radiation (Park et al., 2014). Moreover, the identified variant was previously reported in a breast cancer patient (Damiola et al., 2015), further supporting its functional consequence in tumour pathogenesis. Nevertheless, since PALB2 p.A1079S was inherited from the healthy father, the variant either displays incomplete penetrance or requires additional genetic or environmental stressors to facilitate malignant transformation. In case of additional contributing variants, the likely benign, maternally inherited POLQ p.(L1720F) variant (MAF = 0.002; Table S2) could potentially potentiate the effect of deregulated PALB2, as POLQ is an important part of the microhomology-mediated endjoining (MMEJ), a back-up double-strand-break repair mechanism used in cells with a defective HR pathway (Sfeir & Symington, 2015). Likewise, the more commonly found maternally inherited FANCC VUS p.(PS26F) (MAF = 0.004; Table S2) could act as a synergistic double hit in the patient.
Taken together, our case explicates the complexities involved in diagnosing a primarily EML and showcases the oncological emergency and complications that can be entailed. The identified PALB2 variant and its functional consequence in G2/M arrest provide pathogenetic evidence for the early onset and rare presentation of the here described extramedullary AML.