Case description: patients 1 and 2
Monozygotic twin sisters (XP2003VI and XP2004VI) were diagnosed with XP during their first year of life due to very high sun-sensitivity. Genetic testing revealed homozygous c.1643_1644delTG; p.Val548Alafs25 (called therefore delTG) mutation in the XPC gene, thus confirming the diagnosis (Table 1, Fig. 1a). The patients were not diagnosed with skin cancers but had actinic keratosis and freckles on their faces. The patients were from consanguineous family. For both 16 years old-patients, following hemorrhages a large cervical mass that was prolapsing through the vagina, was discovered following pelvic MRI with no local extension. Histological examination of the biopsies confirmed a vaginal embryonal rhabdomyosarcoma (vERMS). The tumoral cells were positive for Desmin and Myogenin expression. These tumors were classified Group C following the RMS 2005 protocol.
Patient 1: The treatment was performed according to the SFCE RMS 2005 study16, Group C with five cycles of Ifosamide-Vincristine-Actinomycin (IVA) followed by four cycles of Vincristine-Actinomycin (Standard of Care (SoC) therapy). Chemotherapy doses have been reduced between 30 and 50% because of their toxicity due to the patient DNA repair-deficiency17,18. Following the chemotherapy and after ovarian transposition, local treatment was performed with vaginal brachytherapy with a delivered dose of 24 Gy. The patient recovered completely and is followed for her XP disease regularly without any specific medical problems for total 10 years follow-up.
Patient 2: The same SoC treatments with chemotherapy, surgery and brachytherapy as her sister were administered to this patient at the same age (Table 1). At the age of 20, pancytopenia was discovered following standard exams for XP with 7% of blast cells in blood and 74% of bone marrow blast cells. Immunophenotyping revealed an acute myeloid leukemia type 2 (AML-2). Treatment with Adriamycin and Aracitidine for 4 months induced a complete remission but a year later the patient developed a total aplasia and died at 22.
Case description: patient 3
Patient 3 (XP694VI) is a 19-years old female diagnosed with XP-C at the age of three and had a history of skin carcinomas, ephelides and freckles on the sun-exposed body sites (Table 1). She was referred for an ACTH-independent Cushing’s syndrome leading to the discovery of a rapidly growing left ovarian tumor of 11 cm. A laparotomy was performed. On surgical exploration, a preoperative cyst rupture was found, without any suspicious lesion on the peritoneum. A left adnexectomy was decided. Expert pathologic examination concluded to a juvenile granulosa-cell tumor of the ovary (J-GCT), partially ruptured, with a negative peritoneal cytology but tumor cells on the tubal serosa. The patient was treated with the SoC therapy with slight modifications due to DNA repair deficiency. Adjuvant chemotherapy, despite being theoretically indicated for a FIGO stage IC2 J-GCT, was rejected because of an increased risk of adverse events in the context of XP-C. Restaging laparoscopy was decided and found no tumor localization on all biopsies. However, hypercortisolism was still persistent three months after surgery. A magnetic resonance imaging found a new right ovarian mass of 2.5 cm and a micronodular left adrenal gland. Right adnexectomy and left adrenalectomy were performed by laparoscopy and histological examination found a poorly differentiated stage IA Sertoli-Leydig cell tumor (SLCT) on the right ovary and a primary pigmented nodular adrenal dysplasia. Following the last operation, Cushing’s syndrome has improved and there have been no recurrences during 6 months follow-up. After nine months of follow-up, a magnetic resonance imaging found a bulky pelvic tumor. A subsequent laparotomy was performed, and the mass was completely removed, requiring a colorectal resection. Pathologic examination confirmed J-GCT recurrence. She received four injections of weekly paclitaxel, but adjuvant treatment had to be modified due to anxiety disorders. She continued receiving paclitaxel 175 mg/m2 every 3 weeks for three cycles without experiencing serious adverse events.
Case description: patient 4
Patient 4 (XP2020VI) is a 13-years old female with XP-C diagnosed at the age of two. As usual in XP, she had a history of melanocytic hyperplasia (Table 1). At the age of 11, before puberty onset, the patient suffered from recurrent abdominal pain, abdominal enlargement and slight vaginal bleeding. On MRI, a large cystic and solid abdominal mass (larger axis equal to 20 cm) was observed suggesting an ovarian tumor. AFP was slightly increased (64.8 ug/l) whereas HCG, Inhibin B and AMH were normal. A left adnexectomy with per-operative cystic rupture was performed with peritoneal washing and peritoneal examination. Final diagnosis was ovarian FIGO stage IC1 SLCT with predominantly retiform pattern and heterologous components, inhibin positive. Peritoneal washing was cytologically negative and no distant lesion was observed. AFP level normalized after surgery. Because of the great sensitivity of XP patients to chemotherapy and especially to cisplatin17, the main chemotherapy agent used in this kind of tumor, and contrary to actual national guidelines recommending systematic adjuvant chemotherapy with three cycles of BEP (Bleomycin-Etoposide-Cisplatin), it was decided to not give adjuvant chemotherapy to this young XP child in agreement with her parents. Other treatments were performed according to the SoC. The patient is currently still followed every 3 months with abdominal and pelvic MRI and biological markers (Inhibin B and AFP). During the 20 months-long follow-up no recurrences have been detected.
For patients 1–4 we obtained tumor and/or normal DNA samples and performed Whole Genome or Exome Sequencing (WGS or WES). Patient 1: WGS of tumor (vERMS – AS2005) and germline DNA2, Patient 2: WES of germline DNA (this study), Patient 3: WGS of 2 tumors (SLCTs – SA015T1, and J-GCT – SA015T2) and germline DNA (this study), Patient 4: WGS of tumor (SLCTs – SA014T1) and germline DNA (this study).
Germinal DNA analysis
Analysis of germinal DNA of patients revealed abundance of runs of homozygosity (RoH) in patients 1–3 (27–65 Mbp per individual) including RoH overlapping XPC, and a causative founder homozygous germline mutation typical for the studied Northern African XP-C population (delTG, XPC p.V548fs). Patient 4 had a compound heterozygous mutation in XPC gene (p.E284X and p.V548fs) and only 12 Mbp in RoHs across the genome (Fig. 1a). We did not identify any cancer-predisposing germline variants except in the XPC gene in the studied patients.
In twin sisters (patients 1 and 2) with young-onset vERMS we identified a region of homozygosity on chromosome 19 (7.5–13.2 Mbp) which overlaps SMARCA4. Mutations in this gene are known to be associated with Rhabdoid Tumor Predisposition Syndrome 219 (RTPS2). However, we did not identify any mutations in SMARCA4 in our patients. Moreover, the IHC staining for SMARCA4 revealed normal level of protein expression. These results did not confirm RTPS2 in patients 1 and 2.
Analysis of somatic mutations in tumors
To understand genetic basis of gynecological tumors in XP-C patients we compared WGS data from those 4 cancers to other 7 internal cancers from XP-C patients2, and to a panel (n = 45) of diverse OGCT11. Genomic mutation analysis revealed a mutator phenotype in XP-C gynecological cancers with a mean of 27216 mutations per genome. Mutational density in XP-C gynecological tumors was 4.4-fold increased (P = 0.00046, Mann–Whitney U test, two-sided) in comparison with sporadic OGCT for SBS and 1.7-fold increase for indels (P = 0.041, Mann–Whitney U test, two-sided), but was not significantly different from the other types of internal XP-C tumors of hematological origins (Fig. 1b).
To better understand if contribution of XPC deficiency to the mutational processes is confined to cancer or also impacts normal tissues we combined our WGS cancer dataset with WGS of iPSCs from n = 20 XP samples and n = 20 controls20. We compared mutational profiles between individual genomes using cosine similarity distance and revealed clear separate grouping of XP-C gynecological tumors with XP-C leukemias, XP-C breast sarcoma and with XP-C iPSCs on the MDS plot (Fig. 1c). In line with previous observation in XP-C leukemia, in XP-C gynecological tumors we revealed strong transcriptional bias across 6 main substitution groups (C > A/T/G,T > A/C/G; Fig. 1d). We further refitted known COSMIC mutational signatures to our dataset and revealed an enrichment of the SBS8 signature in XP-C ovarian tumors and rhabdomyosarcoma (67–77% of mutations explained; mean = 71%) and in 3/5 XP-C iPSCs (Fig. 1e). In sporadic cohort of OGCT tumors SBS8 explained only 0-38% (mean = 23%) of mutations and dominating mutational signature was SBS5 usually widespread across different tumor types and normal human tissues21.
Analysis of potential oncogenic events in the four studied tumors revealed oncogenic or likely oncogenic mutations in DICER1 gene in all cases, with biallelic deactivation in 2 tumors. TP53 was mutated in vERMS and in SLCT tumor from patient 3. Genomes of J-GCT and SLCTs were relatively stable without polyploidization, while the genome of vERMS was polyploid and unstable with focal amplifications of RICTOR (16 copies) and GLI2 (14 copies) genes (Fig. 1f).