Thirty‐three years later: Two distinct cases of acute lymphoblastic leukemia in one patient
Authors: Marisa J.L. Aitken, Christopher B. Benton, Feng Wang, Jianhua Zhang, Shelley M. Herbrich, Koichi Takahashi, Carlos E. Bueso‐Ramos, Nicholas J. Short
Journal: American Journal of Hematology (2020)

Rare reports have described childhood acute lymphoblastic leukemia (ALL) cases relapsed more than 10 years after diagnosis.1, 2 In most cases, cytogenetic and/or molecular features supported a conclusion of relapsed disease. However, to our knowledge, no reports have identified emergence of a truly distinct ALL disease process. Here, we describe a case of a woman diagnosed with Philadelphia chromosome positive (Ph+) B-ALL 33 years after being presumably cured of childhood ALL. Comparison of genetic material from the two separate diagnoses strongly indicates these were unique disease processes. In 1982, a 9-year-old girl presented with fatigue, daily fevers, night sweats, and weight loss. White blood cell count (WBC) was 166 × 109/L with 95% circulating blasts. Bone marrow biopsy showed 94% blasts expressing terminal deoxynucleotidyl transferase (TdT), and not expressing peroxidase, periodic acid-Schiff, acid phosphatase, or esterase—indicative of ALL. Lumbar puncture disclosed no evidence of central nervous system (CNS) involvement. No mitotic cells were recovered from the bone marrow specimen, precluding cytogenetic analysis. The patient received induction with vincristine and prednisone followed by consolidation with L-asparaginase and cyclophosphamide as part of the Childrenʼs Oncology Group (COG)-ALinc13 protocol.3 Per protocol, she received intrathecal CNS prophylaxis with methotrexate, cytarabine, and hydrocortisone. By day 5, no circulating blasts were identified. Day 14 bone marrow demonstrated morphologic complete response. Maintenance included methotrexate, 6-mercaptopurine, and prednisone for 36 months. Over the next three decades, the patient had three uncomplicated pregnancies and no other notable medical history. In 2015, at age 42, she presented with a symptomatic pulmonary embolism. The WBC at this time was 30 × 109/L with 22% circulating blasts. Bone marrow biopsy showed 83% blasts that expressed CD19, CD20, CD22, CD79a, cytoplasmic IgM, CD10, TdT, CD25, CD66c, HLA-DR, CD38, and CD123 by flow cytometry. Blasts did not express CD13, CD33, CD2, CD5, CD3, CD7, CD117, CD41, CD36, CD64, CD15, CRLF2, or myeloperoxidase. Karyotype was complex and included t(9;22) and monosomies 7 and 17 (37∼44,XX,add (1)(p13),+6,del (6)(q13q23),−7,der(9;15)(q10;q10)t(9;22)(q34;q11.2),−17,der(22)t(9;22),+1∼ 4mar[cp5]/46,XX[15]). The BCR-ABL1 rearrangement was detected by FISH, and RT-PCR revealed p190 BCR-ABL1 to ABL1 transcript ratio of 71.80%. These findings confirmed Ph+ B-ALL. The patient received rituximab with hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone alternating with high-dose cytarabine and methotrexate (R-hyper-CVAD) plus dasatinib, along with intrathecal CNS prophylaxis. Day 14 bone marrow demonstrated morphologic, immunophenotypic, and cytogenetic complete remission, with BCR-ABL1 transcript detectable by RT-PCR at 0.01%. The patient completed eight cycles of R-hyper-CVAD plus dasatinib. Maintenance consisted of 24 cycles of ponatinib (switched from dasatinib due to persistent BCR-ABL1 positivity), vincristine, and prednisone with one intensification cycle of R-hyper-CVAD. She was offered consolidative hematopoietic stem cell transplant but declined. Therefore, she transitioned to blinatumomab plus ponatinib for six cycles, followed by ponatinib plus venetoclax. This approach was utilized to maximize and prolong the efficacy of non-transplant therapies for this patient. Indeed, she remains on ponatinib and venetoclax, now over 4 years post-diagnosis with no appreciable side effects. However, BCR-ABL1 levels have remained between 0.02% and 0.29% in bone marrow, and between 0.13% and 0.82% in peripheral blood. To investigate whether this was an ultra-late relapse, blasts from the 1982 diagnostic bone marrow slides were laser dissected, genomic DNA isolated, and whole exome sequencing performed. Raw sequencing data was aligned and filtered using the Burrows-Wheeler Aligner4 and analyzed for genomic rearrangements using SplitThreader.5 This method identifies variants representing novel adjacencies separated by more than 10 kb in a reference genome, and is suitable for detecting BCR-ABL1. Critically, no routinely assessed leukemia-associated rearrangements, including BCR-ABL1 were identified in this sample, indicating that the childhood ALL was Ph-negative (Figure 1A,B). This is consistent with the patientʼs favorable childhood course, as children with Ph+ ALL enrolled in the ALinc13 study were at very high risk for disease progression and/or relapse. This was likely due largely to the absence of available tyrosine kinase inhibitor therapies in this era.3 At the time of her second ALL diagnosis, routine clinical targeted sequencing of 28 leukemia-associated genes did not identify any mutations. Remarkably, we retrospectively identified 14 unique leukemia-associated somatic mutations in the 1982 sample, seven of which had a variant allele frequency (VAF) >5%. Notably, four mutations had a VAF >10% (EZH2 42.9%, PTPN11 26.7%, KRAS 20%, WT1 13.9%; Figure 1C). Since no such mutations were detected by targeted sequencing of the 2015 sample in our clinical laboratory, we PCR-amplified relevant portions of EZH2, PTPN11, and KRAS and performed Sanger sequencing. This did not reveal any mutations, corroborating the clinical sequencing results (Figure 1D). The disparate genetic findings between these two cases strongly suggest that they are indeed separate disease processes—not an ultra-late relapse of childhood ALL. To our knowledge, this is the longest time from childhood ALL to development of B-ALL as an adult. While several reports describe late “relapses” of childhood ALL, most cases lack genomic or molecular data from the original disease, making it difficult to distinguish late relapses from de novo disease. It is therefore conceivable that some of these prior reports of supposed relapses actually describe a second distinct leukemia. Access to the 1982 diagnostic bone marrow from our patient allowed us to gather information unavailable at diagnosis, and determine whether the adult disease was a subclonal outgrowth. Notably, none of the leukemia-associated mutations in the 1982 sample were found in the 2015 disease, making emergence of a subclonal disease highly unlikely. Lending credence to this assertion, Ph+ ALL arising from previously Ph-negative ALL has not been described to our knowledge. Secondary leukemias following childhood ALL treatment are well-described. While most therapy-related malignancies are myeloid in origin, 10%-15% of therapy-related leukemias are ALL (t-ALL). Compared with de novo ALL, t-ALL associates with poor-risk cytogenetic features, including 11q23 rearrangements, hypodiploidy, monosomy 5, 7, and/or 17 (myelodysplastic-like karyotypic features), and/or BCR-ABL1 rearrangementl.6, 7 Interestingly, one study reported that patients with Ph+ t-ALL had longer latency periods between initial cancer diagnosis and ALL diagnosis.6 Indeed, presence of BCR-ABL1 in a complex cytogenetic background plus the long period between diagnoses suggests the possibility of t-ALL in this case. In contrast, the extremely long latency period in this case argues against t-ALL, as median time to t-ALL after cytotoxic chemotherapy is approximately 5 years.7 Another explanation could be the existence of a dormant leukemic stem cell (LSC) present in this individual. While extremely challenging to adequately evaluate this scenario, the possibility of such an LSC would further encourage long-term treatment, including transplant. This case highlights that genomic studies can potentially distinguish ultra-late relapse from emergence of a second, distinct leukemia. We propose these distinctions be made such that therapy can be tailored to address underlying genetic predisposition to malignancy (in the case of development of a second, distinct leukemia). Or, take particular care in designing maintenance strategies or encouraging transplant (in the case of ultra-late relapse). Taken together, we have described a patient in which two independent cases of ALL developed—separated by 33 years. These were distinct biological entities based on the difference in Ph-status and starkly different mutational landscapes. To our knowledge, this is the longest period between ALL diagnoses in a single patient. In addition, we believe this to be the first report in which two distinct cases of ALL have been reported in an individual. We thank Xiaorui Zhang for helpful comments on this manuscript. Marisa Aitken is the recipient of a Dr. John J. Kopchick Fellowship. The authors report no conflicts of interest. Marisa J.L. Aitken collected and analyzed data and wrote the manuscript; Christopher B. Benton collected data; Feng Wang, Jianhua Zhang, and Shelley M. Herbrich collected and analyzed data; Koichi Takahashi collected and analyzed data; Carlos E. Bueso-Ramos collected and analyzed data and performed pathologic interpretation; Nicholas J. Short provided support and critically revised the manuscript.