Treatment of Relapsed and Refractory ALK-Positive Anaplastic Large Cell Lymphoma With ALK-Specific Tyrosine Kinase Inhibitor in Children: A Case Series.

Relapsed and refractory ALK-positive anaplastic large cell lymphoma (ALCL) has a poor prognosis. In this report, we present 3 relapsed/refractory pediatric ALCL patients, 1 of these with central nervous system involvement. All 3 patients were treated with ALK inhibitor and achieved complete response. Both crizotinib and alectinib have shown significant activity in pediatric patients with refractory ALK-positive ALCL.

Coexpression of MCT1 and MCT4 in ALK-positive Anaplastic Large Cell Lymphoma: Diagnostic and Therapeutic Implications.

In solid tumors, glycolytic cancer or stromal cells export lactates through monocarboxylate transporter (MCT) 4, while oxidative cancer or stromal cells take up lactates as metabolic fuels or signaling molecules through MCT1. CD147 acts as a chaperone of MCT1 or MCT4. Unlike solid tumors, malignant lymphomas have a peculiar tumor microenvironment. To investigate the metabolic phenotype of malignant lymphoma associated with lactate transport, we analyzed immunohistochemical expressions of MCT1, MCT4, and CD147 in 247 cases of various malignant lymphomas. Surprisingly, both MCT1 and MCT4 were diffusely expressed on tumor cell membranes in all cases (11/11, 100%) of anaplastic lymphoma kinase (ALK) (+) anaplastic large cell lymphoma (ALCL). In contrast, only MCT1 was diffusely expressed in tumor cells of ALK(-) ALCL, as well as in B-cell, natural killer/T-cell, T-cell, and classic Hodgkin lymphomas. In these lymphomas, MCT4 expression was mostly localized to adjacent stromal cells. The pattern of diffuse membranous MCT1 and partial MCT4 expressions in tumor cells was observed in 1 case each of peripheral T-cell lymphoma (1/15, 6.7%) and multiple myeloma (1/34, 2.9%). CD147 was diffusely expressed in all types of lymphoma tumor and/or stromal cells. In conclusion, ALK(+) ALCL has a unique metabolism showing high coexpression of MCT1 and MCT4 in tumor cells. Because only ALK(+) ALCL overexpresses MCT4, immunostaining for MCT4 together with ALK is very useful for differential diagnosis from ALK(-) ALCL or peripheral T-cell lymphoma. Moreover, dual targeting against MCT1 and MCT4 would be an appropriate therapeutic approach for ALK(+) ALCL.

Diagnosis of anaplastic lymphoma kinase-negative anaplastic large cell lymphoma on pleural fluid cytology.

ALK-negative anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin T-cell lymphoma. Compared to ALK-positive ALCL, patients with ALK-negative ALCL typically are older, present with nodal disease, and have lower survival rates. We report a unique presentation of ALK-negative ALCL in a pleural fluid. Cell block preparation enabled both confirmation of the diagnosis via immunohistochemical staining and gene rearrangement testing for prognostic information.

JAK2 Rearrangements Are a Recurrent Alteration in CD30+ Systemic T-Cell Lymphomas With Anaplastic Morphology.

Peripheral T-cell lymphoma (PTCL) comprises a heterogenous group of rare mature T-cell neoplasms. While some PTCL subtypes are well-characterized by histology, immunophenotype, and recurrent molecular alterations, others remain incompletely defined. In particular, the distinction between CD30+ PTCL, not otherwise specified and anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma can be subject to disagreement. We describe a series of 6 JAK2 rearrangements occurring in a cohort of 97 CD30+ ALK- PTCL (6%), assembled after identifying an index case of a novel PABPC1-JAK2 fusion in a case of ALK- anaplastic large cell lymphoma with unusual classic Hodgkin lymphoma (CHL)-like features. Fusions were identified using a comprehensive next-generation sequencing based assay performed between 2013 and 2020. Five of 6 cases (83%) showed JAK2 rearrangements with 4 novel partners: TFG, PABPC1, ILF3, and MAP7, and 1 case demonstrated a previously described PCM1-JAK2 fusion. By morphology, all cases showed anaplastic large cells and multinucleated Reed-Sternberg-like cells within a polymorphous inflammatory background with frequent eosinophilia reminiscent of CHL. By immunohistochemistry, atypical large cells expressed CD30 with coexpression of at least 1 T-cell marker, aberrant loss of at least 1 T-cell marker and, in 4 of 5 cases stained (80%), unusual CD15 coexpression. These findings suggest that a subset of CD30+ ALK- systemic PTCL with anaplastic morphology carry JAK2 rearrangements, some of which appear to show CHL-like morphologic features. The presence of JAK2 rearrangements in cases of CD30+ PTCL augments current classification and may provide a therapeutic target via JAK2 inhibition.

ALK-negative anaplastic large cell lymphoma, null type with aberrant expression of PAX5 and CD138: A diagnostic pitfall.

A 72-year-old man was found to have generalized lymphadenopathy, splenomegaly, and elevated serum lactate dehydrogenase. Fine-needle aspiration and core needle biopsy of a cervical lymph node revealed a large lymphoid cell proliferation with features suggestive of anaplastic large cell lymphoma (ALCL). However, immunophenotypically, the neoplastic cells expressed PAX5 and CD138 in addition to CD30, CD45, MUM-1 and were negative for T-cell markers, B-cell markers, CD15, ALK-1, HHV-8, EBER, kappa, lambda, and pancytokeratin. The ambiguous phenotype triggered further workup. Subsequent molecular studies demonstrated T-cell receptor gene rearrangement and lack of immunoglobulin gene rearrangement. Based on these findings, a diagnosis of ALK-negative ALCL, null type with aberrant expression of PAX5 and CD138, was rendered. The patient received palliative care due to his poor condition and died of the disease. This case presents a diagnostic pitfall and highlights the importance of cytological evaluation and complete workup in the diagnosis of unconventional lymphomas.

Second allogeneic transplantation using umbilical cord blood for a patient with relapsed ALK+ anaplastic large cell lymphoma after allogeneic bone marrow transplantation in the era of ALK inhibitors: A case report.

RATIONALE: Anaplastic lymphoma kinase (ALK) + anaplastic large cell lymphoma (ALCL) is considered as a good prognosis lymphoma. However, in an extremely rare subset of patients, ALK+ ALCL with leukemic presentations is known to be chemotherapy-resistant. Although several novel therapies have been tested, the standard therapy for relapsed/refractory ALK+ ALCL has not been established yet. PATIENT CONCERNS: An 18-year-old female patient who had conventional chemotherapy- and Brentuximab Vedotin (BV)-resistant ALK+ ALCL with leukemic presentation. She was successfully treated with an ALK inhibitor, crizotinib. Crizotinib induced complete remission (CR) and bridged to allogeneic bone marrow transplantation (BMT). DIAGNOSIS: However, her ALCL relapsed on day 60 after BMT and she developed high grade fever and lymphadenopathy. INTERVENTION: Although crizotinib was given to the patient immediately after relapse, she developed grade 3 nausea and could not continue to take it. Then, we gave alectinib to the patient, which promptly induced sustained CR without any further chemotherapy. The patient received second stem cell transplantation using umbilical cord blood with myeloablative regimen in 2nd CR. OUTCOMES: The patient has been in CR under maintenance therapy of alectinib for more than 16 months. LESSONS: Both ALK inhibitors demonstrated drastic efficacy for our patient who had chemotherapy- and BV-resistant ALK+ ALCL with leukemic presentation. Alectinib showed less gastro-intestinal toxicity than crizotinib and the patient was able to take it even at the relatively early phase of stem cell transplantation.

Brentuximab vedotin in combination with chemotherapy for pediatric patients with ALK+ ALCL: results of COG trial ANHL12P1.

Approximately 30% of pediatric patients with anaplastic large cell lymphoma (ALCL) relapse. Although brentuximab vedotin has demonstrated excellent activity in ALCL, it has not been used for newly diagnosed patients. Children's Oncology Group (COG) trial ANHL12P1 determined the toxicity and efficacy of brentuximab vedotin with chemotherapy in children with newly diagnosed nonlocalized anaplastic large cell lymphoma kinase (ALK)+/CD30+ ALCL. From 2013 to 2017, 68 children with ALK+ ALCL were enrolled and received brentuximab vedotin. All patients received 5-day prophase, followed by 6 cycles of chemotherapy. Brentuximab vedotin was given on day 1 of each of the 6 cycles. Of the 67 patients eligible for toxicity evaluation, 66 completed all 6 cycles of chemotherapy, resulting in 399 evaluable cycles. There were no toxic deaths, no case of progressive multifocal leukoencephalopathy syndrome, and no case of grade 3 or 4 neuropathy. The 2-year event-free survival (EFS) was 79.1% (95% confidence interval [CI], 67.2-87.1). The 2-year overall survival (OS) was 97.0% (95% CI, 88.1-99.2). Fourteen patients relapsed. Eleven of 14 (79%) relapses occurred within 10 months of diagnosis; only 1 patient (1.5%) relapsed during therapy. Quantitative reverse transcription polymerase chain reaction for NPM-ALK at baseline (minimal disseminated disease) demonstrated prognostic value for EFS (P = .0004). Overall, the addition of brentuximab vedotin to standard chemotherapy does not add significant toxicity or alter the desired interval between cycles. The addition of brentuximab vedotin prevented relapses during therapy, and the OS and EFS estimates compare favorably with results obtained using conventional chemotherapy. This trial was registered at www.clinicaltrials.gov as #NCT01979536.

Minimal Residual Disease Monitoring Using a 3'ALK Universal Probe Assay in ALK-Positive Anaplastic Large-Cell Lymphoma: ddPCR, an Attractive Alternative Method to Real-Time Quantitative PCR.

In ALK-positive anaplastic large-cell lymphomas, positive qualitative PCR for NPM1-anaplastic lymphoma kinase (ALK) in peripheral blood and/or bone marrow at diagnosis and during treatment are associated with a higher risk of treatment failure. Real-time quantitative PCR allows identification of very high risk patients. However, this latter technique initially designed for patients with lymphomas carrying the most frequent NPM1-ALK translocation necessitates calibration curves, limiting interlaboratory reproducibility. An ALK universal quantitative PCR based on 3'ALK transcript amplification was designed to allow the detection of all ALK fusion transcripts. The absolute concordance of 3'ALK quantitative PCR results were validated with the routine NPM1-ALK qualitative and quantitative PCR on 46 samples. The universality of ALK fusion transcript detection also was validated on TPM3-, ALO17-, and ATIC-ALK-positive samples, and the EML4-ALK-positive cell line. Digital droplet PCR using the 3'ALK universal probe showed highly concordant results with 3'ALK universal quantitative PCR. A major benefit of digital droplet PCR is a reduced experimental set-up compared with quantitative PCR, without generation of standard curves, leading to a reliable protocol for multilaboratory validation in multicenter clinical trials essential for this rare pathology. Our ALK universal method could be used for the screening of ALK fusion transcripts in liquid biopsy specimens of other ALK-positive tumors, including non-small cell lung carcinomas.

Striking Association of Lymphoid Enhancing Factor (LEF1) Overexpression and DUSP22 Rearrangements in Anaplastic Large Cell Lymphoma.

Anaplastic large cell lymphomas (ALCLs) are broadly classified into ALK-positive and ALK-negative. ALK-negative ALCL is composed of DUSP22-rearranged, TP63-rearranged, and triple-negative cases. While lymphoid enhancer-binding factor (LEF1) plays a crucial role in T-cell maturation, limited data exist on its expression in T-cell lymphomas, including ALCL. We characterized the expression of LEF1 in ALCL by immunohistochemistry. LEF1 nuclear expression in the neoplastic cells was graded as negative (0), weak (1+), intermediate (2+), or strong (3+), with the percentage of LEF1-positive neoplastic cells recorded. A total of 45 ALCL cases were evaluated, of which 16 were DUSP22-rearranged. About 93.8% (15/16) DUSP22-rearranged cases showed strong expression of LEF1 in >75% tumor cells, compared with 3.4% (1/29) non-DUSP22-rearranged ALCL (P<0.0001). The striking association of LEF1 protein overexpression with DUPS22 rearrangement in ALCL was further confirmed by a gene expression profiling study which revealed significantly higher LEF1 expression in DUSP22-rearranged ALCL compared with other ALCL subtypes (P=0.0001). Although LEF1 is a nuclear mediator of the Wnt/ß-catenin pathway, CTNNB1 RNA and protein levels were not overexpressed in LEF1-positive cases, suggesting the LEF1 overexpression in ALCL may not be involved in the Wnt/ß-catenin pathway. The strong and uniform LEF1 expression pattern has a high positive predictive value (93.8%) and high negative predictive value (96%) for DUSP22 rearrangement in ALK-negative ALCL. The combination of characteristic morphologic and molecular features of DUSP22-rearranged cases with the high LEF1 expression further emphasizes that DUSP22-rearranged ALCL represents a distinct clinicopathologic subset of ALCL.

Discovery of CJ-2360 as a Potent and Orally Active Inhibitor of Anaplastic Lymphoma Kinase Capable of Achieving Complete Tumor Regression.

We report herein the discovery of a class of potent small-molecule inhibitors of anaplastic lymphoma kinase (ALK) containing a fused indoloquinoline scaffold. The most promising compound CJ-2360 has an IC50 value of 2.2 nM against wild-type ALK and low-nanomolar potency against several clinically reported ALK mutants. This compound is capable of achieving complete tumor regression in the ALK-positive KARPAS-299 xenograft model with oral administration in mice. CJ-2360 represents a promising ALK inhibitor for advanced preclinical development.

ALK-transformed mature T lymphocytes restore early thymus progenitor features.

Anaplastic large cell lymphoma (ALCL) is a mature T cell neoplasm that often expresses the CD4+ T cell surface marker. It usually harbors the t(2;5) (p23;q35) translocation, leading to the ectopic expression of NPM-ALK, a chimeric tyrosine kinase. We demonstrated that in vitro transduction of normal human CD4+ T lymphocytes with NPM-ALK results in their immortalization and malignant transformation. The tumor cells displayed morphological and immunophenotypical characteristics of primary patient-derived anaplastic large cell lymphomas. Cell growth, proliferation, and survival were strictly dependent on NPM-ALK activity and include activation of the key factors STAT3 and DNMT1 and expression of CD30 (the hallmark of anaplastic large-cell lymphoma). Implantation of NPM-ALK-transformed CD4+ T lymphocytes into immunodeficient mice resulted in the formation of tumors indistinguishable from patients' anaplastic large cell lymphomas. Integration of "Omic" data revealed that NPM-ALK-transformed CD4+ T lymphocytes and primary NPM-ALK+ ALCL biopsies share similarities with early T cell precursors. Of note, these NPM-ALK+ lymphoma cells overexpress stem cell regulators (OCT4, SOX2, and NANOG) and HIF2A, which is known to affect hematopoietic precursor differentiation and NPM-ALK+ cell growth. Altogether, for the first time our findings suggest that NPM-ALK could restore progenitor-like features in mature CD30+ peripheral CD4+ T cells, in keeping with a thymic progenitor-like pattern.

Alectinib for relapsed or refractory anaplastic lymphoma kinase-positive anaplastic large cell lymphoma: An open-label phase II trial.

Anaplastic lymphoma kinase (ALK) inhibition is expected to be a promising therapeutic strategy for ALK-positive malignancies. We aimed to examine the efficacy and safety of alectinib, a second-generation ALK inhibitor, in patients with relapsed or refractory ALK-positive anaplastic large cell lymphoma (ALCL). This open-label, phase II trial included patients (aged 6 years or older) with relapsed or refractory ALK-positive ALCL. Alectinib 300 mg was given orally twice a day (600 mg/d) for 16 cycles, and the duration of each cycle was 21 days. Patients who weighed less than 35 kg were given a reduced dose of alectinib of 150 mg twice a day (300 mg/d). Ten patients were enrolled, and the median age was 19.5 years (range, 6-70 years). Objective responses were documented in eight of 10 patients (80%; 90% confidence interval, 56.2-95.9), with six complete responses. The 1-year progression-free survival, event-free survival, and overall survival rates were 58.3%, 70.0%, and 70.0%, respectively. The median duration of therapy was 340 days. No unexpected adverse events occurred. The most common grade 3 and higher adverse event was a decrease in neutrophil count in two patients. Alectinib showed favorable clinical activity and was well tolerated in patients with ALK-positive ALCL who had progressed on standard chemotherapy. Based on the results of the current study, the Ministry of Health, Labour and Welfare of Japan approved alectinib for the treatment of recurrent or refractory ALK-positive ALCL in February 2020.

Lenalidomide demonstrates clinical activity in anaplastic lymphoma kinase-positive large B-cell lymphoma.

Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK+ LBCL) is known to be a rare and aggressive form of lymphoma that relapses quickly after both conventional chemotherapy and more targeted therapy. Lenalidomide is an immunomodulator that has shown safety and efficacy in multiple myeloma and is also approved for use in several types of lymphoma. In the case described here, the patient had a significant partial response to lenalidomide, which has not previously been described in this type of lymphoma. Given how aggressive and difficult to treat ALK+ LBCL is, further research is warranted to more completely elucidate the mechanism of action of lenalidomide in ALK+ LBCL and its role in treatment.

FDG PET/CT in Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma.

Anaplastic large cell lymphoma (ALCL) is a rare non-Hodgkin's lymphoma, with rearrangements of the anaplastic lymphoma kinase gene in 60%-85% of systemic cases. We report an 11-year-old boy with ALCL in whom serial FDG PET/CT revealed partial response and complete metabolic response at interim and end of treatment, respectively. However, the patient relapsed within 2 weeks, confirmed by cytology. Because of the inherent aggressive nature of ALCL, possibility of an early relapse should always be kept in mind.

Synergistic Effect of Alectinib and Everolimus on ALK-positive Anaplastic Large Cell Lymphoma Growth Inhibition.

BACKGROUND/AIM: The aim of the study was to examine the efficacy of the combination of anaplastic lymphoma kinase (ALK) inhibitors with other inhibitors for the treatment of ALK-positive lymphomas. This approach is predicted to be an alternative way for suppressing ALK-positive anaplastic large cell lymphoma (ALCL). MATERIALS AND METHODS: We treated ALK-positive ALCL cell lines, KARPAS-299 and SU-DHL-1, with the ALK inhibitor alectinib and the mammalian target of rapamycin (mTOR) inhibitor everolimus. RESULTS: The ALK inhibitor alectinib had a selective ALK-dependent inhibitory effect on ALK-positive cancer cell proliferation. Treatment with alectinib or everolimus inhibited target molecules, and their combination augmented their inhibitory effect on the mTOR pathway. Furthermore, the combination treatment significantly inhibited cell proliferation and induced cell cycle arrest at the G0/G1 phase in ALK-positive ALCL cells. CONCLUSION: The combination of both inhibitors synergistically inhibits ALK-positive ALCL cell growth via the ALK/mTOR pathway.

The mechanism of cancer drug addiction in ALK-positive T-Cell lymphoma.

Rational new strategies are needed to treat tumors resistant to kinase inhibitors. Mechanistic studies of resistance provide fertile ground for development of new approaches. Cancer drug addiction is a paradoxical resistance phenomenon, well-described in MEK-ERK-driven solid tumors, in which drug-target overexpression promotes resistance but a toxic overdose of signaling if the inhibitor is withdrawn. This can permit prolonged control of tumors through intermittent dosing. We and others showed previously that cancer drug addiction arises also in the hematologic malignancy ALK-positive anaplastic large-cell lymphoma (ALCL) resistant to ALK-specific tyrosine kinase inhibitors (TKIs). This is driven by the overexpression of the fusion kinase NPM1-ALK, but the mechanism by which ALK overactivity drives toxicity upon TKI withdrawal remained obscure. Here we reveal the mechanism of ALK-TKI addiction in ALCL. We interrogated the well-described mechanism of MEK/ERK pathway inhibitor addiction in solid tumors and found it does not apply to ALCL. Instead, phosphoproteomics and confirmatory functional studies revealed that the STAT1 overactivation is the key mechanism of ALK-TKI addiction in ALCL. The withdrawal of TKI from addicted tumors in vitro and in vivo leads to overwhelming phospho-STAT1 activation, turning on its tumor-suppressive gene-expression program and turning off STAT3's oncogenic program. Moreover, a novel NPM1-ALK-positive ALCL PDX model showed a significant survival benefit from intermittent compared with continuous TKI dosing. In sum, we reveal for the first time the mechanism of cancer drug addiction in ALK-positive ALCL and the benefit of scheduled intermittent dosing in high-risk patient-derived tumors in vivo.

PD-L1 expression is associated with ALK positivity and STAT3 activation, but not outcome in patients with systemic anaplastic large cell lymphoma.

The programmed cell death 1 (PD-1) pathway is a recently recognized mechanism of tumor immune evasion. In this study, programmed cell death ligand 1 (PD-L1) expression was evaluated in 95 patients with systemic anaplastic large cell lymphoma: 45 ALK+ and 50 ALK-. ALK+ anaplastic large cell lymphoma was more often positive for PD-L1 than ALK- anaplastic large cell lymphoma (76% vs 42%, p = 0.002). ALK- anaplastic large cell lymphoma showed a strong correlation between PD-L1 expression and STAT3 activation (measured by pSTAT3Tyr705) (r = 0.8, p < 0.0001). In contrast, the PD-L1/pSTAT3 correlation was weaker in ALK+ anaplastic large cell lymphoma (r = 0.4, p = 0.08). In ALK- anaplastic large cell lymphoma, the PD-L1+ subgroup was more often EMA positive (69% vs 20%, p = 0.02) and tended to be less often CD2+ (50% vs 83%, p = 0.059). In ALK+ anaplastic large cell lymphoma, PD-L1 was not associated with pathologic features (all p > 0.05). Negative ALK status and high IPI score (≥3) were associated with shorter overall survival (p = 0.009 and p = 0.0005, respectively). Overall survival was not different between patients with PD-L1+ vs PD-L1- anaplastic large cell lymphoma (p = 0.44), regardless of ALK status and International Prognostic Index (IPI) score. We conclude that PD-L1 expression is more common in ALK+ anaplastic large cell lymphoma than ALK- anaplastic large cell lymphoma. In ALK- anaplastic large cell lymphoma, PD-L1 is strongly correlated with STAT3 activation and is associated with more frequent EMA and less frequent CD2 expression. PD-L1 has no prognostic significance in predicting the outcome of patients with systemic anaplastic large cell lymphoma, regardless of ALK status. PD-L1 expression on the anaplastic large cell lymphoma cells suggests these patients as potential candidates for PD-1 blockade immunotherapy.

Drug-resistance mechanisms of three mutations in anaplastic lymphoma kinase against two inhibitors based on MM/PBSA combined with interaction entropy.

As a promising drug target in the treatment of lung cancer, anaplastic lymphoma kinase (ALK) and its mutations have been studied widely through the development of multiple generations of inhibitors. Experiments have found that compared with the wild-type, the L1198F and C1156Y/L1198F mutations resulted in resistance to 5P8 inhibitors, and the C1156Y mutation resulted in resistance to VGH inhibitors. In this study, the newly developed interaction entropy (IE) method combined with the polarized protein-specific charge (PPC) force field was utilized to explore the origin of the resistance mechanism of the ALK mutant system. The calculated binding free energy was consistent with the experimental results. Per-residue binding free energy decomposition showed that the predicted hot-spot residues (LEU1122, LEU/PHE1198, MET1199, GLY1202 and LEU1256) were almost identical across systems. Especially, the GLU1197 residue played an important role in inducing drug-resistance for both inhibitors. The electrostatic interaction of GLU1197, PHE1198 and MET1199 mainly resulted in the resistances of the L1198F and C1156Y/L1198F mutations to 5P8. And the van der Waals interaction energy of LEU1256 residue, and electrostatic energy and entropy change of GLU1197 resulted in the resistances of the C1156Y mutations to VGH. The indicated origins of the drug-resistance in the ALK systems provide a theoretical foundation for the design of potent inhibitors.