Case report 1 (CR1)

Following a four-month period of pain, the 12-year-old girl was diagnosed with OS of the proximal femur and an extensive intravascular tumor thrombus extending into the external iliac and pulmonary veins (Fig. 1A). Treatment according to an osteosarcoma protocol (EURAMOS 1/COSS) with six courses of MAP chemotherapy (methotrexate, adriamycin, cisplatin) was administered. Tumor resection including MUTARS® endoprosthesis for reconstruction was performed after two cycles. Regression stage according to Salzer-Kuntschik was classified as grade 3. Surgical margins were deemed to be insufficient in the area of the deep iliac veins. Hence, external hemipelvectomy was performed subsequently. On suspicion of pulmonary tumor emboli in the upper and lower lobe, atypical resections were performed after completion of standard chemotherapy. Histological evaluation confirmed avital metastases of OS. At the age of 15 years, a single pulmonary metastasis was diagnosed (Fig. 1B). Following segmentectomy the patient was administered 36 weeks of immune stimulatory therapy with mifamurtide. At the age of 17 years, the patient presented with a large intramuscular metastasis in the erector spinae and cervical and nuchal lymph node metastases (Fig. 1C). Partial resection of the muscle including replacement and lateral neck dissection were performed. The patient remains in complete radiological remission three years later.

To identify therapeutic targets, tumor analysis of the second relapse was conducted within the framework of the INFORM project [8] including whole-exome, low-coverage whole-genome, RNA sequencing, and methylation as well as expression microarray analyses. Analyses of the nuchal metastasis displayed increased genomic instability with hypermutation (tumor mutational burden 16.8) and alternative lengthening of telomeres (ALT) (Table 1). Accordingly, PD1/PDL1 and PARP inhibitors constituted potential therapeutic options.

Based on the genetic analyses, suspicion of a mismatch repair defect was raised and immunohistochemical (IHC) staining of mismatch repair (MMR) proteins and microsatellite instability (MSI) analyses were prompted. Loss of PMS2 expression was seen in tumor tissue, while PMS2 expression in non-neoplastic cells was retained (Fig. 2A-C). A high-degree of MSI (MSI-H) tested by PCR was observed. In line with this, loss of heterozygosity (LOH) of PMS2 in tumor tissue was demonstrated by the INFORM analyses. To further elucidate genetic predisposition, peripheral blood of the patient was analyzed via NGS sequencing for single nucleotide variants (SNVs).

A heterozygous duplication c.1076dup p.(Leu359Phefs*6) in exon 10 of NM_000535.6: PMS2 was detected by SNV analysis and confirmed by Sanger sequencing. Based on the classification criteria developed by the InSIGHT Variant Interpretation Committee for MMR gene variants v2.4 (2018-06), the PMS2 variant was classified as pathogenic (class 5; see also Supplemental Information), confirming the patient´s diagnosis of LS.

Daily use of ASA was recommended to the patient and LS surveillance initiated.

The family history in the three preceding generations was remarkable for renal cancer in the grandmother (deceased at the age of about 60 years) and leukemia in a grandaunt (deceased at the age of 50 years) in the paternal line. Complementary analysis of the parents´ blood confirmed that the PMS2 variant was inherited by the thus far clinically unaffected father. LS surveillance was recommended to the father. The maternal line was unsuspicious in terms of cancer.

Case report 2 (CR2)

The one-year-old girl presented with torticollis and an anamnestic episode of a tonic-clonic convulsive seizure a few days before admission. Ophthalmological examination revealed evidence of Heimann-Bielschowsky phenomenon suspicious of trochlear nerve palsy. Magnetic resonance imaging (MRI) demonstrated a posterior fossa tumor encircling the brain stem, growing into the internal acoustic meatus inducing occlusive hydrocephalus (Fig. 1D). Metastatic disease of the craniospinal axis or the cerebrospinal fluid was excluded. Total tumor resection was performed and histological evaluation showed anaplastic ependymoma WHO°III/posterior fossa ependymoma methylation subgroup A. Concurring with current international data and considering the patient´s age, proton beam therapy at a dose of 54 Gy was administered. At the age of 2.7 years, local relapse was diagnosed (Fig. 1E). Following tumor resection, proton beam therapy with a dose of 54 Gy was administered. At the age of 4.3 years, follow-up MRI demonstrated 2nd local relapse. Once more a tumor resection was performed. Due to radiotherapy-related occlusion of both carotid arteries, the patient subsequently underwent angioplasty and since then is on acetylsalicylic acid (ASA) therapy. Three years later, the patient sustained a 3rd local relapse of ependymoma.

Fig. 1

Magnetic resonance imaging (MRI), computed tomography (CT) and positron-emission tomography (PET). CR1: A Coronal T2-weighted MRI of the femur and pelvic region demonstrating the tumor of the left proximal femur. B CT scan demonstrating a single pulmonary metastasis. C PET scan demonstrating a large metastasis at the back and multiple cervical und nuchal metastases. CR2: D Sagittal T2-weighted MRI demonstrating the tumor of the posterior fossa encircling the brain stem. E Axial T2-weighted MRI demonstrating first local relapse. F Axial T2-weighted-Fluid-Attenuated MRI demonstrating second local relapse

Analyses of the 2nd relapse within the INFORM project did not identify therapeutic targets. DNA methylation-based classification confirmed a group A posterior fossa ependymoma (PF-A). It further displayed evidence for ALT and low tumor mutational burden (0.6). Whole-genome sequencing (WGS) analysis, however, identified the heterozygous SNV c.1 A > T p.? in exon 1 of PMS2 in tumor and germline material and was further confirmed by Sanger sequencing in peripheral blood of the patient. According to the InSIGHT criteria this variant is classified as likely pathogenic (class 4; see supplement).

The family history was remarkable for acute myeloid leukemia (not further specified) in the mother in young adulthood and a cousin on the maternal side who deceased of sarcoma (not further specified) at the age of 5 years. The family history on the paternal side was unsuspicious. The parents as yet refused genetic testing of themselves.

To further elucidate the role of the PMS2 germline variant in tumor development, we initiated IHC and MSI analysis. In tumor material of the 2nd relapse, PMS2 expression was retained, and MSI was low (Fig. 2D-F). To test for a second pathogenic variant in PMS2 in terms of constitutional mismatch repair deficiency (CMMRD), multiplex ligation-dependent probe amplification (MLPA) was prompted and identified no additional PMS2 variant. In addition, germline MSI (gMSI) testing [9] did not reveal increased gMSI ratios in the patient´s lymphocytes and, hence, could not confirm CMMRD in the child.

Fig. 2

CR1: (A) Hematoxylin and eosin staining of the nuchal metastasis of the osteosarcoma. Immunohistochemical (IHC) staining of mismatch repair proteins demonstrating loss of PMS2 expression in (B) neoplastic cells and (C) retained nuclear expression of MSH6 in the tumor cells. CR2: (D) Hematoxylin and eosin staining of the 2nd relapse of ependymoma. IHC of mismatch repair proteins demonstrating nuclear expression of PMS2 (E) and MSH6 (F) in the tumor cells. A-B, D-F: 40x magnification. C: 100x magnification

Table 1 Details on patients´ characteristics and diagnostics