Constitutional mismatch repair deficiency and childhood leukemia/lymphoma – report on a novel biallelic MSH6 mutation

Abstract

Biallelic mutations of mismatch repair genes cause constitutional mismatch repair deficiency associated with an increased risk for childhood leukemia/lymphoma. We report on a case with constitutional mismatch repair deficiency caused by a novel MSH6 mutation leading to a T-cell lymphoma and colonic adenocarcinoma at six and 13 years of age, respectively. A review of the literature on hematologic malignancies in constitutional mismatch repair deficiency showed that in almost half of the 47 known constitutional mismatch repair deficiency families, at least one individual is affected by a hematologic malignancy, predominantly T-cell lymphomas. However, diagnosing constitutional mismatch repair deficiency may be difficult when the first child is affected by leukemia/lymphoma, but identification of the causative germline mutation is of vital importance: (i) to identify relatives at risk and exclude an increased risk in non-mutation carriers; (ii) to prevent hematopoietic stem cell transplantation from sibling donors also carrying a biallelic germline mutation; and (iii) to implement effective surveillance programs for mutation carriers, that may reduce constitutional mismatch repair deficiency-associated mortality.

Introduction

Following DNA replication and DNA damage, the DNA mismatch repair (MMR) machinery recognizes and directs repair of base-base mismatches and insertion/deletion loops.1 Monoallelic germline mutations in the MMR genes MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MSH6, or post-meiotic segregation increased 2 (PMS2), are known to cause Lynch syndrome (LS), characterized by an early onset of non-polyposis colorectal cancer and extra colonic malignancies, e.g. carcinomas of the endometrium, stomach, or upper uroepithelial tract, while leukemias or lymphomas are not common in LS.2,3 In contrast, biallelic germline mutations lead to a more severe disease designated as constitutional mismatch repair deficiency (CMMRD).4,5 CMMRD is characterized by: (i) childhood onset of leukemia/lymphoma, brain tumors, and other rare malignancies, e.g. rhabdomyosarcoma;6 (ii) early onset of colorectal cancer or other LS-associated malignancies; and (iii) phenotypic features reminiscent of neurofibromatosis type 1 (NF1), mainly café-au-lait spots (CLS).5,7,8

Here we report on a novel biallelic germline mutation in MSH6 causing CMMRD with childhood T-cell non-Hodgkin’s lymphoma (T-NHL) and metastasized colorectal cancer by the age of 13. In addition, we review CMMRD families with known MMR gene mutations with at least one affected individual with a hematologic malignancy.

Design and Methods

Immunohistochemistry was performed in accordance with standard protocols. For the analysis of microsatellite instability (MSI), ten markers (Bethesda panel and five additional markers9) were investigated.

Mutation analysis of MSH6 (NM_000179) was performed by PCR amplification and direct sequencing. Mutation nomenclature follows the recommendation of the Human Genome Variation Society.

Written informed consent was obtained from all subjects before immunohistochemistry, microsatellite analysis, and blood or tissue sample collection for mutation analysis.

Clinical case

A non-resectable mediastinal T-NHL was diagnosed in our female patient at six years of age (V:10, Figure 1). Six months after her initial chemotherapy following the NHL BFM 95 protocol arm MR, she relapsed. The second regimen followed the ALL REZ BFM 2002 pilot 02 protocol arm S2, and she received hematopoietic stem cell transplantation (HSCT) from a matched sibling donor as consolidation.

At the age of 13, the patient presented with episodes of rectal bleeding and abdominal colic. In a colonoscopy, a bifocal ulcerative carcinoma was observed in the transversal colon and numerous tubulovillous polyps with high-grade intraepithelial atypia were seen throughout the colon. Colectomy with end ileostomy was performed. Histopathological investigations showed a bifocal colonic adenocarcinoma with local lymph node metastases. Adjuvant chemotherapy was performed according to the FOLFOX4 regimen. One year later, a rectal adenoma with low grade intraepithelial atypia was detected. Proctectomy with endoanal mucosectomy and ileal pouch-anal anastomosis was performed.

Due to her medical history of cancer, the patient was referred for genetic counseling. She presented with several CLS, and lateral conjunctival melanosis of the left eye. Her consanguineous parents (2 degree cousins; IV:8 and IV:9) reported a colorectal cancer in a paternal uncle (IV:7) married to a sister of the mother (IV:10). At the age of 42, he was diagnosed with synchronous adenocarcinomas in the sigmoid and descending colon.

Results and Discussion

A novel mutation in MSH6

A medical history of a T-NHL at the age of six, a relapse at the age of eight, and the diagnosis of colorectal cancer at the age of 13 in a young girl is rare. The combination of: (i) cancer history; (ii) CLS; (iii) consanguinity of the parents; and (iv) early onset of synchronous colorectal cancer in a paternal uncle led us to speculate that an MMR defect might be the reason for the family history of cancer.

In our patient, MSI analysis and immunohistochemical analysis of MMR proteins displayed high-grade MSI and a loss of MSH6 in non-malignant and malignant cells of the colonic mucosa. In contrast, following HSCT, reactive lymphocytes infiltrating the colonic mucosa displayed nuclear staining for MSH6 (Figure 2A). In addition, MSI analysis and immunohistochemistry of colorectal cancer specimens from the diseased paternal uncle were performed. Both specimens displayed high-grade MSI. However, in contrast to our patient, loss of MSH6 was seen only in malignant cells.

Figure 1.Pedigree of the family investigated. Roman numerals (I–V), generations; circles, females; squares, males; numbers above the symbol, individual identifier; numbers below the symbol, age at diagnosis of colorectal cancer (diseased individuals) or age (healthy individuals); black symbol, patient with childhood T-NHL at the age of six, T-NHL relapse at the age of eight, and colorectal cancer at the age of 13; gray symbol, diseased uncle with synchronous colorectal cancer; +/+, wild-type; +/−, heterozygous carrier, −/−, homozygous carrier of the identified frameshift mutation in MSH6 (c.691delG, p.Val231TyrfsX15).

Figure 2.Immunohistochemistry and molecular analyses in family members. (A) Immunohistochemical analysis of colorectal cancer of our patient (V:10). While MSH2, MLH1 and PMS2 staining were inconspicuous (data not shown), a loss of MSH6 was seen in malignant and non-malignant colonic mucosa in comparison to a control. In contrast, following HSCT, lymphocytes infiltrating the colonic mucosa show nuclear staining for MSH6 (NBT/BCIP staining, 100x magnification). (B) Direct sequencing of MSH6 (NM_000179) identified a novel frameshift mutation in exon 4: c.691delG, p.Val231TyrfsX15. The electropherograms represent an example of two independent sequence analyses. Black arrows indicate the G in the wild-type sequence that is deleted in one or both alleles in the case of a monoallelic or biallelic mutation, respectively. (C) Allele-specific PCR. Using a specific forward primer for the wild-type or mutant allele, results of direct sequencing were confirmed by allele-specific PCR. DNA was extracted from peripheral blood cells of the parents (IV:9, IV:8) and their siblings (IV:7, IV:10). To access DNA of our patient prior to HSCT, DNA was extracted from a formalin-fixed paraffin-embedded lymph node without any evidence of malignancy. WT, wild-type control; L, molecular marker.

Sequence analysis of MSH6 in the index patient displayed a homozygous single nucleotide deletion in exon 4 (c.691delG) leading to a frameshift and premature termination (p.Val231TyrfsX15) (Figure 2B). Subsequently, the diseased uncle, the father, and the mother were shown to be heterozygous carriers of the identified mutation in MSH6. The maternal aunt married to the diseased uncle does not carry the familial mutation. Sequencing results were confirmed by allele-specific PCRs (Figure 2C).

In summary, the molecular findings of a highly microsatellite unstable colorectal cancer with a loss of MSH6 confirm the consideration of an MMR defect. While the diseased uncle carries one mutated allele, our patient suffering from a more severe cancer phenotype has a biallelic mutation leading to a constitutional deficiency of MSH6.

CMMRD and hematologic malignancies – predominance of T-cell lymphomas

CMMRD has been reported in 47 families affecting 77 individuals (Online Supplementary Table S1). In almost half of these families (20 out of 47, 43%), at least one individual was affected by a hematologic malignancy. Approximately one-third of the affected individuals (26 of 77, 34%) suffered from hematologic malignancies, namely non-Hodgkin’s lymphoma (NHL; n=17), acute lymphoblastic leukemia (ALL; n=6), acute myeloid leukemia (AML; n=3), acute leukemia (n=1), or atypical chronic myelogenous leukemia (n=1). While the disease-causing mutation can be found in MLH1 and MSH2 in up to 90% of patients with LS,10 CMMRD is mainly caused by biallelic mutations in PMS2 and MSH6 (Figure 3).5 Biallelic mutations of MSH6, either homozygous or compound heterozygous, were found in 10 of the 47 families (21%) comprising 14 affected individuals (Online Supplementary Figure S1). Even though hematologic malignancies tend to be more frequent in families with biallelic mutations in MLH1 or MSH2,5 we report here on the second CMMRD family with an MSH6 mutation and a lymphoma as the primary malignancy. In general, a predominance of NHL, especially T-NHL, can be seen in CMMRD patients with hematologic malignancies (Online Supplementary Table S1). Lymphomas reported so far frequently occurred as a sole malignancy (n=8) or in combination with colorectal cancer (n=7). Notably, the combination of an early onset of lymphomas, mainly T-NHL, followed by gastrointestinal adenomas and adenocarcinomas completely reflects the phenotype seen in biallelic MMR gene knock-out mice that may serve as a good model for CMMRD.11

Acute myeloid leukemia in CMMRD

Despite the clear predominance of lymphoid malignancies, 3 patients with CMMRD developed AML (Online Supplementary Table S1). Even though relatively rare, there are two interesting aspects concerning AML and CMMRD:

• 2 out of 3 AMLs were secondary AMLs following treatment of medulloblastomas, leading to the hypothesis that these AML cases might be therapy related.12 In contrast, the third case of AML occurred as a primary neoplasm one year before the diagnosis of medulloblastoma, indicating a potential link between medulloblastoma and AML that are both rather infrequent malignancies in CMMRD;5
• CMMRD patients have phenotypic similarities with NF1 (Online Supplementary Table S1).5 NF1, an autosomal dominant disorder caused by germline NF1 mutations occurring de novo in up to 50% of the cases, predisposes to a variety of benign and malignant neoplasms in childhood,13 including juvenile myelomonocytic leukemia.14–16 Generally, NF1 is diagnosed on the basis of clinical criteria.17 Mutation analysis of NF1 is not usually performed. Like other disorders, CMMRD is an important differential diagnosis of NF1,17 and should be considered in the presence of medulloblastoma and AML in children or adolescents, as well as in the case of LS-associated malignancies.5 In contrast to extensive molecular analysis of the very large NF1 gene, CMMRD can be investigated by a relatively easy screening for MMR deficiency using immunohistochemistry. If there are any doubts in the diagnosis of NF1, CMMRD deficiency should be ruled out.5

Chemotherapy in CMMRD

Little is known about the efficiency of standard protocols used to treat childhood lymphoma and leukemia in CMMRD. In vitro analyses showed that certain drugs, e.g. monofunctional methylating agents, are less effective in MMR-deficient cell lines.1,10,18 One can speculate that treatment response might differ between lymphomas with acquired MMR deficiency surrounded by MMR-competent cells, including a competent immune system, and lymphomas in CMMRD patients with MMR deficiency in all cells. Further investigations are needed to assess the frequency of MSI and/or MMR gene mutations in hematologic malignancies and to test their influence on the outcome.

Figure 3.Molecular cause of LS and CMMRD. Relative distribution of causative mutations in MLH1, MSH2, MSH6 and PMS2 in LS10 and CMMRD5 indicating the predominance of mutations MLH1 and MSH2, and MSH6 and PMS2 in LS and CMMRD, respectively.

Recognizing CMMRD

In CMMRD, the median age of onset is 4.5 years (range 0.42–14) for T-NHL, six years (range 2–15) for ALL and six years (range 0.4–17) for hematologic malignancies in general. In comparison to brain tumors (median 8 years, range 2–35 years),5 LS-associated malignancies (median 16 years, range 8–41 years), and other malignancies (median 13 years, range 1–65 years) in CMMRD,5 hematologic malignancies have the earliest median age of onset. This early disease onset may render the diagnosis of CMMRD difficult since, in 15 out of the 20 families (75%), no other family members were affected by LS-associated malignancies when the first child became affected (Online Supplementary Table S1). Moreover, lymphoblastic lymphomas and ALL are frequent hematologic malignancies in childhood. Obviously, no germline mutations will be found in most of them, but awareness of CMMRD in children with NHL, ALL and other rare tumors in CMMRD may allow for an earlier diagnosis, at least in some affected families. This clearly requires careful evaluation and re-evaluation of the family history of cancer at the time of diagnosis and during follow-up of the patient.

In the family reported here, the mediastinal T-NHL was the primary neoplasm diagnosed. While at that point, no family history indicative of an MMR deficiency was present, synchronous colorectal cancer in a 42-year old paternal uncle (IV:7, Figure 1) was diagnosed one year later, i.e. one year prior to the lymphoma relapse and six years before the diagnosis of metastasized colorectal cancer in our patient (V:10). Due to the diagnosis of synchronous colorectal cancer before the age of 50, revised Bethesda guidelines19 were fulfilled and LS could have been considered in the paternal uncle, opening up the possibility of identifying the underlying germline mutation.19

Conclusions

Diagnosing CMMRD has important implications for the entire family. Healthy parents, as in our case, will learn that they carry a monoallelic mutation in an MMR gene and may thus suffer from LS. In addition, they have to cope with the knowledge that their other children have an a priori risk of 25% and 50% to carry a biallelic and monoallelic mutation, respectively. Healthy siblings of the parents are confronted with a 50% a priori risk of being a carrier of a heterozygous mutation that can be inherited by their own children. Considering the wide-reaching clinical relevance of this rare syndrome, genetic counseling should be offered to all families at risk. Identification of the causative germline mutation would have three main clinical consequences: (i) following genetic counseling of individuals at risk, molecular genetic testing allows the identification of those carrying the mutation and exclusion of an increased risk in non-mutation carriers; (ii) prevention of HSCT from sibling donors also carrying a biallelic germline mutation, that may have an adverse clinical outcome, as was recently reported in another cancer syndrome caused by a heterozygous runt-related transcription factor 1 (RUNX1) germline mutation;20 and (iii) surveillance programs for mutation carriers, e.g. regular colonoscopy, enabling early diagnosis and treatment of LS and CMMRD-associated neoplasms. Besides the improvements required for the therapy of diseased CMMRD patients, efficient surveillance programs have to be developed to reduce mortality, as has been demonstrated for regular colonoscopy21 in patients with LS.

Acknowledgments

the authors would like to thank the study subjects and their attending doctors for their participation in this study, Bernd Haermeyer for his expert technical assistance, and Gillian Teicke for her help in editing the manuscript.

Footnotes

• Funding: TR was supported by a grant from Hannover Biomedical Research School, Graduate School of Excellence, PhD Program Molecular Medicine, Hannover Medical School, Hannover, Germany, and by the EU COST initiative EUGESMA.
• The Online version of this article has a Supplementary Appendix.
• Authorship and Disclosures TR and BS co-ordinated the study. TR, CB, and KWS recruited the patients. NR participated in the evaluation of the family. TR, CLB, UL, and HHK performed the histopathological and molecular genetic analyses. TR and BS wrote the paper.
• The authors reported no potential conflicts of interest.

• Received August 10, 2009.
• Revision received October 9, 2009.
• Accepted October 13, 2009.