The child Long XX, male, 9 years old, was diagnosed with relapsed T-cell acute lymphoblastic leukemia (T-ALL) complicated with multiple extramedullary leukemia (testes and central nervous system), and 1 year and 3 months after related haploidentical hematopoietic stem cell transplantation.
In September 2016, the child developed bilateral multiple enlargement of cervical lymph nodes, about 1-2 cm in diameter, with no tenderness and no fever, and the enlargement of lymph nodes gradually disappeared after treatment with antibiotics in a local hospital. At the beginning of October 2016, the child developed enlargement of cervical lymph nodes again, which was gradually aggravated, with the maximum diameter of about 5-6 cm, complicated with fever, with the highest body temperature of 39°C. After anti-infective treatment in the local hospital, the lymph nodes were shrunk by about 2/3, and the body temperature dropped to normal. At the local hospital, blood routine test showed significantly elevated white blood cell count, so on October 19, 2016, the child was admitted to Peking University People’s Hospital. Blood routine test: white blood cell count 409.68×109/L, lymphocyte percentage 84.9%, hemoglobin count 116 g/L, platelet count 50×109/L; bone marrow: lymphoblast and prolymphocyte percentage 71%; immunophenotype: abnormal lymphocyte phenotype, P3 accounted for 68.38% , expressing CD7, CD38, CD8, cCD3, TdT, CD5, CD2 and CD45, some cells express mCD3 but do not express CD10, CD19, CD34, CD117, CD33, CD57, CD1a, CD15, CD4, CD56, CD16 and CD123; fusion gene: negative; IgH negative, TCRδ negative; chromosomes: 46, XY, t (11:14) (p13:q11)  / 46, XY , B ultrasound: hepatomegaly, diagnosed as T-ALL. The VDLP chemotherapy was given for induction, and on day 15, bone marrow: prolymphocyte percentage 4%; flow cytometry: cCD3+T cells accounted for 17.54%, phenotype cCD3+mCD3+CD5+TdT-CD99-, determined as mature T lymphocytes. Then consolidation chemotherapy with CAM, high-dose MTX, VDL and high-dose Ara-c was given for 9 cycles, no abnormalities were found by lumbar puncture for 24 times, and the bone marrow achieved complete remission.
On October 13, 2017, the child developed bilateral testicle swelling with pain (Fig. 1), B ultrasound: enlarged testicles, manifested as diffuse lesions, with abundant blood flow signals; testicular biopsy: diffuse infiltrating lymphoid cells were found in testicular tissue, the cells were in medium size, the nucleoli were unclear, and the chromatin was fine, considered as tumors originated in the lymphoid hematopoietic system. On October 18, bone marrow: grade III hyperplasia, mature lymphocyte percentage 5%; flow cytometry: MRD positive, diagnosis: extramedullary relapse of T-ALL. On October 21, ifosfamide + mitoxantrone + VP-16 + L-asp chemotherapy was given.
Fig. 1: bilateral testicle swelling at relapse before transplantation
On November 13, 2017, the child was transferred to Beijing Boren Hospital, and the conditions were evaluated on admission, bone marrow: remission; lumbar puncture and intrathecal injection were given, and no malignant immature cells were found in cerebrospinal fluid. On November 18, the child began to receive HDAra-c + IDA chemotherapy, and on December 13, B ultrasound found a 0.58×0.69×0.47 cm mass in the left testis, and it was planned to perform related haploidentical hematopoietic stem cell transplantation after local testicular radiotherapy. On December 21, the child started 7 days of testicular radiotherapy, with a total amount of 1400 cGy. On January 01, 2018, the child developed varicella and received antiviral treatment, during which, white blood cell count was progressively elevated and rapidly multiplied, up to 130x109/L, and primitive cells were found in the peripheral blood, suggesting relapsed leukemia. VP-16 + Ara-C chemotherapy was given to reduce the load of leukemia during varicella. On January 12, the child received Flu + Ara-C + Acla + G-CSF chemotherapy. Bone marrow gene mutation: NRAS, FBXW7, NOTCH1, PTEN, PTPN11 (Fig. 2).
I. To be Detected
1. High-throughput DNA sequencing technology was used for targeted capture sequencing of the sample DNA to detect the mutation of blood system disease-related genes and targeted drug-related genes (see Appendix 1 for the list of genes for detection), with the coverage of >99%;
2. Detection range: single nucleotide variation (SNV) and small fragment insertion/deletion (inDel) in the coding sequence;
3. Analysis content: detection of mutation of genes related to blood tumor targeting drugs approved by the FDA and recommended by the NCCN guidelines; detection of mutation of genes related to the diagnosis, prognosis and relapse of blood tumors.
II. Detection results and conclusion
1. The sample submitted for detection was within the scope of detection, and the following gene mutations were detected:
NRAS p.G125 mutation;
FBXW7 p.R465C mutation;
NOTCH1 p.R1598P mutation;
PTEN p.P246_L247insEPFDYL mutation;
III. Result annotation
Mutations closely related to clinical significance
Fig. 2: NGS deep sequencing for gene mutations in January 2018 (hematological relapse before transplantation)
On January 17, the child received VLD chemotherapy, as well as sunitinib and chidamide. On January 29, bone marrow test: hyperplasia was reduced, lymphoblast and prolymphocyte percentage<5%, flow cytometry: T-ALL cells accounted for 4%. The relevant examinations were completed for hematopoietic stem cell transplantation, but on February 11, the child developed sudden right facial paralysis, cerebrospinal fluid routine test: white blood cell count 3,838×106/L, mononuclear cell percentage 99.7%; cerebrospinal fluid biochemistry test: protein 0.61 g/L, glucose 2.51 mmol/L, considered as central nervous system leukemia, so lumbar puncture and intrathecal injection were given. On February 14, the child began to receive HBDTX, DEX and Pegaspargase chemotherapy. On February 22, bone marrow puncture was performed, bone marrow: lymphoblast and prolymphocyte percentage 73.5%, flow cytometry: 54.36% of the cells were phenotypically abnormal naive T cells.
On February 26, salvage haploidentical hematopoietic stem cell transplantation (father to son, HLA matching 4/6, blood type B+ for B+) was started, pretreatment regimen: TBI+FLU+VP16+Me-CCNU+ATG. On March 12 and 13, the donor's hematopoietic stem cells were back transfused, ANC 21.08×108/kg, CD34+ cells 14.34×106/kg. After transplantation, short-term methotrexate, cyclosporine and Cellcept were given to prevent GVHD, and +15 days later, white blood cells and platelets were survived. In the first month after transplantation, leukemia was relieved, residual leukemia was 0, and the chimeric rate of CD3+ cells in bone marrow and peripheral blood was completely as the donor type. After transplantation, the child’s facial paralysis and testicle swelling were gradually relieved (Fig. 3).
Fig. 3: testicle swelling disappeared after transplantation
The child had refractory T-ALL and high leukemia load and rapid multiplication of cells before transplantation, complicated with extramedullary leukemia, and underwent salvage transplantation. Due to the above-mentioned high-risk factors, it was expected that relapse might occur early after transplantation. In order to prevent relapse, we selected the targeted drug, trametinib, for maintenance treatment after transplantation based on the presence of NRAS mutation in the child. At the end of June 2018 (+3 months after transplantation), the child started to orally take trametinib 0.5 mg/d up to now. In mid-July 2018, no NRAS mutation was found in bone marrow test (Fig. 4).
Clinical diagnosis: T-cell acute lymphoblastic leukemia; sample receiving time: July 11, 2018
After related haploidentical hematopoietic stem cell transplantation.
The mutations of totally 185 genes related to the diagnosis, prognosis, relapse and treatment of blood tumors were detected, including the mutations of genes related to blood tumor targeting drugs approved by the FDA and recommended by the NCCN guidelines.
I. Detection results and conclusion
1. The sample submitted for detection was within the scope of detection, and no mutations closely related to clinical significance were detected.
2. No targeted drugs related to the patient's gene mutation sites were detected.
3. The following gene mutations were detected in the sample submitted for detection:
TET2 p.P29R mutation;
KMT2C p.G8385 mutation;
DNAH2 p.P1500L mutation.
The functional effects of the above-mentioned gene mutation sites have not been clearly studied and reported, but the software analysis predicts that they were harmful mutations. Please combine with the results of other examinations and clinical phenotypes for comprehensive judgment.
Fig. 4: NRAS mutation was not detected by NGS deep sequencing at +4 months after transplantation
After transplantation, the child developed mild chronic GVHD, mainly manifested as a small amount of scattered rash and slightly elevated liver transaminase. The concentration of ciclosporin was maintained at a lower level, and it was discontinued at +8 months after transplantation. Up to now, it was 1 year and 3 months after hematopoietic stem cell transplantation, the leukemia achieved persistent complete remission, and the residual leukemia was maintained at 0, without extramedullary leukemia.
Acute lymphocytic leukemia is the most common tumor in children, in which, T-ALL accounts for 10-15%. Currently, the 5-year event-free survival (EFS) and overall survival (OS) of children with B-ALL are more than 80% and 90%, respectively, but the 5-year EFS and OS of children with T-ALL are worse than those of children with B-ALL, especially the 5-year survival of children with relapsed T-ALL is only about 20%. During the treatment, the child developed extramedullary relapse first, manifested as testicular leukemia, then hematological relapse and finally central nervous system leukemia, and leukemia cells multiplied rapidly, and the chemotherapy achieved poor response, so the child had refractory T-ALL and extremely poor prognosis.
After hematological relapse, NRAS, FBXW7, NOTCH1, PTEN and PTPN11 mutations were detected by second-generation sequencing, and the frequency of NRAS mutation was 37.12%. The NRAS gene belongs to the proto-oncogene RAS family and is responsible for encoding the N-Ras protein. The N-Ras protein is involved in the RAS-RAF-MEK-ERK pathway, regulates gene transcription and cell cycle, and is closely related to cell proliferation. When the NRAS gene is mutated, its encoded N-Ras protein will be continuously activated, resulting in uncontrolled cell proliferation, thereby forming the tumors. NRAS mutation is more common in melanoma, acute myeloid leukemia and colorectal cancer, and can also be found in T-ALL.
The child underwent salvage transplantation, and the leukemia cells had a high load and were multiplied rapidly, so it was easy to relapse early after transplantation. To prevent relapse, we took the following measures: 1. reduce and discontinue the anti-GVHD drugs as soon as possible: control the concentration of immunosuppressant cyclosporin at a lower level, and reduce and discontinue it as soon as possible (discontinue at +8 months); 2. maintenance treatment with targeted drug: due to the presence of NRAS mutation in the child, the MEK inhibitor trametinib was used as maintenance therapy after transplantation. The child orally took trametinib from +3 months, and the NARS mutation was not detected in the bone marrow test at +4 months, proving that the allogeneic transplantation cleared the leukemia cells with NRAS mutation, and the child continued taking trametinib for maintenance treatment up to now. and it was 1 year and 3 months after transplantation already, the leukemia achieved persistent remission, and no obvious transplantation-related adverse events occurred.
This case suggests that allogeneic hematopoietic stem cell transplantation is not the ultimate therapy for high-risk relapsed leukemia. If a patient has gene mutation and targeted drugs are available, targeted drugs can be used for maintenance treatment after a donor hematopoiesis and immune system platform is established, so as to continuously remove possible residual leukemia cells, thereby reducing the relapse rate and improving the cure rate. Second-generation sequencing technology, especially deep sequencing, can detect more gene mutations and help to screen out corresponding targeted drugs, and some gene mutations can be used as markers for monitoring residual leukemia, so as to determine the treatment response.