ALK-positive ALK-positive Anaplastic Large Cell Lymphoma Research Articles

ALK-positive Anaplastic Large Cell Lymphoma Involving the Spinal Cord

Abstract Introduction/Objective ALK-positive anaplastic large cell lymphoma (ALCL) is a mature T-cell lymphoma that accounts for approximately 3% of non-Hodgkin lymphoma cases in the adult population. Lymph node involvement is most commonly seen, followed by extra-nodal involvement of the skin, soft tissue, and bone. Central nervous system involvement is rare at the time of diagnosis. This case study features a patient with systemic ALK-positive ALCL involving multiple nodal and extra-nodal sites, most notably the spinal cord. Methods/Case Report A 22-year-old female presented to the hospital with worsening back pain accompanied by neurological symptoms. MRI scans of her spine revealed extensive patchy abnormal marrow signal with enhancement of the thoracolumbar vertebrae (T1-L2) and adjacent extension into the spinal canal (T3-T7, T10-L1, L3-S1) and paraspinal soft tissues. A laminectomy of the T5-T7 and T11-T12 levels was performed for surgical decompression, and epidural and paraspinal resection specimens were submitted. These specimens, along with an excisional axillary lymph node biopsy, demonstrated ALK-positive ALCL after comprehensive immunohistochemical studies. In-situ hybridization for EBV was negative. CHOP therapy was initiated, but treatment was complicated by Candidemia and disseminated intravascular coagulation. The patient rapidly declined and passed away less than a month after hospital admission. At autopsy, enlargement of lymph nodes, the left ovary (17.8 g), and spleen (383.6 g) were noted on gross examination. A solid, white-tan lesion (2.1 x 0.9 x 0.8 cm) was found in the left ovary. Necrosis and infiltrates of large, pleomorphic lymphoid cells with irregular nuclear contours and prominent nucleoli were seen on microscopic examination of the spinal cord and leptomeninges, vertebral bone marrow, mediastinal lymph nodes, spleen, and left ovary. Consistent with previously submitted surgical specimens, the viable neoplastic cells stained strongly positive for ALK-1, CD4, and CD30. Results (if a Case Study enter NA) NA Conclusion This case study highlights an atypical presentation of ALCL with a fatal outcome. Given the availability of effective treatment options, prompt diagnosis is crucial to improving survival outcomes. Additional research is necessary to elucidate the clinical and pathological features of ALCL. The differential for back pain is broad, but including this entity for patients with similarly insidious clinical presentations can prevent a delay in diagnosis that allows for rapid progression of disease.

Successful bridging therapy with alectinib prior to allogeneic stem cell transplantation for refractory ALK-positive anaplastic large cell lymphoma

Although alectinib is effective for relapsed or refractory ALK-positive anaplastic large cell lymphoma (ALCL) and has a favorable safety profile, its role as a bridging therapy for allogeneic hematopoietic stem cell transplantation (allo-HSCT) and the role of allo-HSCT itself in this setting are unknown. A 35-year-old man with ALK-positive ALCL experienced relapse after first-line therapy with CHOP. Brentuximab vedotin led to partial response and high-dose chemotherapy combined with autologous HSCT was performed. However, disease progressed 15 months after transplantation, and alectinib was initiated. Complete response (CR) was achieved after three months of treatment, and alectinib was continued for 5 months. After cessation of alectinib, allogeneic bone marrow transplantation from an HLA 1-locus mismatched unrelated donor was performed after conditioning with fludarabine, busulfan, and total body irradiation. GVHD prophylaxis consisted of tacrolimus and short-term methotrexate. The post-transplant course was unremarkable except for grade I acute GVHD. The lymphoma has not recurred for 2 years after allo-HSCT without resuming alectinib. The clinical course of our case suggests that alectinib bridging therapy and allo-HSCT are effective in relapsed/refractory ALK-positive ALCL.

ALK-positive anaplastic large cell lymphoma in adults.

ALK-positive anaplastic large cell lymphoma (ALCL) represents approximately 6-7% of the mature T-cell lymphomas. This subtype contains a translocation between the ALK gene on chromosome 2 and one of several other genes that together form an oncogene. The most frequent translocation is t(2;5) which combines ALK with NPM1. This lymphoma has a median age of 34 years, is more common in males, and is in advanced stage at the time of diagnosis in most patients. ALK-positive ALCL is the most curable of the peripheral T-cell lymphomas. The CHOP regimen has been most frequently used, but results are improved with the substitution of brentuximab vedotin for vincristine (BV-CHP) and the addition of etoposide (CHOEP), with BV-CHP being favored. Salvage therapies include allogeneic or autologous bone marrow transplantation, BV, if not used as part of the primary therapy, and ALK inhibitors. The latter are very active and likely to be incorporated into the primary therapy.

Gastrointestinal ALK-positive anaplastic large cell lymphoma: a clinicopathological analysis of five cases

Objective: To investigate the clinicpathological characteristics of ALK-positive anaplastic large cell lymphoma (ALCL) of the gastrointestinal tract, and to discuss its diagnosis and differential diagnosis. Methods: Five cases of gastrointestinal ALK-positive ALCL diagnosed and treated in Xijing Hospital of the Fourth Military Medical University, between 2011 and 2019 were collected. There were three male and two female patients, aged 5-42 years (mean 25 years). These patients clinically presented with fever and night sweats, weight loss, abdominal pain, abdominal mass, ulcers, bleeding, or intestinal obstruction, and underwent surgical resection of the tumors or endoscopic biopsy. The clinical manifestations, auxiliary examinations, histopathological characteristics, immunophenotypes and genetic alterations were analyzed. Results: In this cohort, one case was common type, two cases were monomorphic variant of common type, and two cases were small cell variant. The tumor cells in all cases expressed ALK, CD30, and one or more T lymphocyte markers, while all the markers of B lymphocyte and plasmacyte were negative. Clonality analysis showed that two cases had clonal T cell receptor (TCR) and immunoglobulin (Ig) gene rearrangement, one case had no clonal TCR but Ig gene rearrangement, and one case had no clonal TCR and Ig gene rearrangements. During the 4 to 67 months' follow-up, two patients died of the disease, two were alive with free of disease and one had a relapse. Conclusions: ALK-positive ALCL of the gastrointestinal tract is extremely rare, and has poor prognosis. Lymphoma originating from this site with CD30 and ALK-positive phenotypes may be considered to be ALCL; however differentiation from other tumors that had anaplastic features, expressed CD30 and or ALK, in particular, ALK positive large B-cell lymphoma is necessary.

Abstract 2181: Treatment-free remission after discontinuation of ALK tyrosine kinase inhibitors (TKIs) in patients with ALK-positive anaplastic large cell lymphoma (ALCL)

Abstract Introduction: ALK-positive ALCL accounts for 0.8% of Korean lymphoma patients. ALK TKIs are effective and durable, but clinical outcomes after treatment discontinuation are unknown. We analyzed outcomes and minimal residual disease (MRD) in adult ALK-positive ALCL patients who discontinued ALK TKIs after achieving treatment-free remission. Methods: Six patients who achieved responses to crizotinib (N=5) and ceritinib (N=1) were analyzed. RNA extracted from the blood or tissues before TKI treatment were subjected to multiplex PCR-based next generation sequencing (NGS) using ARCHER® RNA Fusionplex® to detect NPM-ALK fusion. MRD was assessed by droplet digital PCR (ddPCR) of peripheral blood mononuclear cells (PBMCs) after the treatment discontinuation. Undetectable MRD was defined as less than one lymphocytic cell per 10,000 leukocytes (10-4) in peripheral blood. Results: Patients received ALK TKIs as second line (N=3) or third line and beyond (N=3). All achieved complete metabolic responses after median 28.5 months (range, 1.7-93.9) of treatments (Table 1). Before TKI, LY1 patient had no morphologic evidence of bone marrow involvement, but NPM1-ALK fusions were detected by NGS and ddPCR in blood, bone marrow, and tissue. LY1 patient remained in remission at 27.8 months after crizotinib discontinuation. In all patients, NPM1-ALK fusion were not detected in the PBMCs after discontinuing ALK TKIs for median 38.2 months (range, 0-61.9). No patient re-initiated anti-lymphoma therapies. Conclusion: Out data demonstrated that treatment-free remission was durable for more than two years in ALK-positive ALCL patients following the cessation of ALK TKIs. MRD-guided discontinuation decision may be considered for ALK-positive ALCL patients treated with ALK inhibitors who had undetectable MRD. Table 1. Summary of patient characteristics and outcomes of ALK TKI discontinuation No. Sex Age at diagnosis Ann Arbor stage ALK TKI Lines of prior therapy Treatment duration of ALK TKI (months) Reason for cessation Duration of ALK TKI cessation (months) Best response to ALK TKI Time of MRD assessment after TKI cessation MRD after TKI cessation 1 M 22 IIB Crizotinib 3 93.9 Fertility 27.8 CR 5.6 Negative 2 F 20 IVB Crizotinib 1 90.9 Fertility 25.9 CR 0.0 Negative 3 M 23 IV Ceritinib 1 42.7 Adverse event 61.9 CR 61.9 Negative 4 F 58 IV Crizotinib 3 1.7 Cost 86.6 CR 72.1 Negative 5 F 64 IVB Crizotinib 1 13.6 Cost 31.0 CR 14.4 Negative 6 M 75 IV Crizotinib 2 11.7 Cost 80.2 CR 62.0 Negative Citation Format: Jinyong Kim, Soyeon Kim, Tae Min Kim, Jeonghwan Youk, Miso Kim, Bhumsuk Keam, Dong-Wan Kim, Dae Seog Heo. Treatment-free remission after discontinuation of ALK tyrosine kinase inhibitors (TKIs) in patients with ALK-positive anaplastic large cell lymphoma (ALCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2181.

Clinicopathological features and prognostic significance of programmed death ligand 1 in pediatric ALK-positive anaplastic large cell lymphoma: results of the ALCL99 treatment in Japan

Programmed cell death 1/programmed death ligand 1 (PD-1/PD-L1) blockade is a promising therapy for hematological malignancies. However, the association of PD-L1 expression with the clinicopathological features and prognosis in pediatric ALK-positive anaplastic large cell lymphoma (ALCL) remains unclear. Using PD-L1/ALK immunofluorescence double staining, we evaluated the PD-L1 expression on tumor cells/tumor-infiltrating immune cells (TIICs) and the quantity of TIICs in 54 children with ALK-positive ALCL treated with the ALCL99 protocol. The percentages of PD-L1-positive tumor cells were significantly lower in patients with skin/mediastinum involvement, clinical high-risk group, present minimal disseminated disease (MDD), and a low ALK-antibody titer. The percentages of PD-L1-positive TIICs were significantly higher in patients with absent MDD. The percentages of TIICs were significantly lower in patients with absent MDD and a common morphological pattern. We classified patients according to the PD-L1 expression on tumor cells (Tumor-PD-L1), PD-L1 expression on TIICs (TIIC-PD-L1), and quantity of TIICs (TIIC-quantity). The progression-free survival (PFS) did not differ between Tumor-PD-L1high and Tumor-PD-L1low ALCL; TIIC-PD-L1high and TIIC-PD-L1low ALCL; and TIIC-quantityhigh and TIIC-quantitylow ALCL. According to the combined parameters of Tumor-PD-L1 and TIIC-quantity, Tumor-PD-L1high/TIIC-quantityhigh ALCL had a worse 5-year PFS than other ALCL (50% versus 83%; P=.009). Tumor-PD-L1high/TIIC-quantityhigh ALCL remained a significant prognostic factor in multivariate analysis (P=.044). This is the first study to demonstrate that a high tumoral PD-L1 expression with a high quantity of TIICs was associated with a poor prognosis in pediatric ALK-positive ALCL. The tumor microenvironment of ALK-positive ALCL may be relevant to the clinicopathological features and prognosis.

Low miR-214-5p Expression Correlates With Aggressive Subtypes of Pediatric ALCL With Non-Common Histology.

The unsatisfactory cure rate of relapsing ALK-positive Anaplastic Large-Cell Lymphoma (ALCL) of childhood calls for the identification of new prognostic markers. Here, the small RNA landscape of pediatric ALK-positive ALCL was defined by RNA sequencing. Overall, 121 miRNAs were significantly dysregulated in ALCL compared to non-neoplastic lymph nodes. The most up-regulated miRNA was miR-21-5p, whereas miR-19a-3p and miR-214-5p were reduced in ALCL. Characterization of miRNA expression in cases that relapsed after first line therapy disclosed a significant association between miR-214-5p down-regulation and aggressive non-common histology. Our results suggest that miR-214-5p level may help to refine the prognostic stratification of pediatric ALK-positive ALCL.

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.

Anaplastic large cell lymphoma as a posttransplant lymphoproliferative disorder in a renal transplant patient: A case report.

We report a case of a 45‐year‐old female who developed an ALK‐positive anaplastic large cell lymphoma (ALCL) 9 years after renal transplant. The patient underwent a cadaveric renal transplant for diabetic nephropathy, and presented 9 years later with fever and multiorgan dysfunction. The initial CT scans showed multiple enlarged supra‐ and infradiaphrgamatic lymph nodes. A CT‐guided core needle biopsy of a retroperitoneal lymph node revealed ALK positive ALCL. She received six cycles of cyclophosphamide, adriamycin, vincristine, etoposide, and prednisone, and has been in remission for over 3 years. Monomorphic T‐cell posttransplant lymphoproliferative disorder (PTLD) is an established but rare entity of PTLD and generally carries poor prognosis. This is a case report of a late PTLD with pathology reporting an aggressive T‐cell lymphoma that has been successfully treated with multiagent chemotherapy.

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

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). 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. 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. 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 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 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 STAT1 overactivation is the key mechanism of ALK-TKI addiction in ALCL. 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 significant survival benefit from intermittent compared to 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.

Prognostic impact of minimal disseminated disease and immune response to NPM-ALK in Japanese children with ALK-positive anaplastic large cell lymphoma.

The prognostic impact of minimal disseminated disease (MDD) and anti-anaplastic lymphoma kinase (ALK) antibody titer in children with ALK-positive anaplastic large cell lymphoma (ALCL) was reported by an Italian/German group. Here, we examine their prognostic value in Japanese children with ALK-positive ALCL. We evaluated nucleophosmin (NPM)-ALK transcripts in 60 patients at diagnosis by RT-PCR and real-time PCR (qPCR). The antibody titer was assessed in 35 patients. Fifty-two percent were MDD positive by RT-PCR and 37% had more than 10 copies of NPM-ALK per 104 copies of ABL (10NCNs) by qPCR. Fifty-one percent of 35 patients had high antibody titer (>1/750). Progression-free survival (PFS) of the patients with >10 NCNs or low antibody titers was significantly poorer than that of patients with ≤10 NCNs or high antibody titers (>1/750) (P=0.016, 0.029), respectively, although we observed no difference in PFS associated with positive MDD on RT-PCR. On stratification using a combination of MDD and antibody titer, PFS for patients with >10 NCNs and low antibody titer was extremely low (30.0%). Combined evaluation of MDD and anti-ALK antibody titer at diagnosis may thus be valuable for stratification of treatment for childhood ALCL.

A Matched-Pair Analysis of EPOCH Versus CHOP As Frontline Therapy for Nodal Peripheral T-Cell Lymphoma, Excluding ALK-Positive Anaplastic T-Cell Lymphoma: A Multicenter Experience in Thailand

BACKGROUNDNodal peripheral T-cell lymphomas (PTCLs), except ALK-positive anaplastic large cell lymphoma (ALCL) have inferior outcomes compared with their B-cell counterparts. CHOP (Cyclophosphamide, Doxorubicin, Vincristine, Prednisolone) is considered the standard therapy despite consistent results showing its ineffectiveness. The addition of etoposide (E) to chemotherapy showed encouraging results in selected subgroups of patients. We performed a matched-pair analysis comparing the efficacy of EPOCH to CHOP for untreated patients with nodal PTCLs, excluding ALK-positive ALCL.PATIENTS AND METHODSAccording to the Thai Lymphoma Study Group uniform treatment project, 58 patients with nodal PTCLs including angioimmunoblastic lymphoma, ALK-negative ALCL and PTCL, not otherwise specified received conventional EPOCH regimen as frontline therapy between January 2009 and December 2015. These patients were matched to 58 patients receiving CHOP (1:1) in multicenter registry of lymphoma in Thailand between 2007-2014 by center, International Prognostic Index (IPI) and age. Patients who underwent upfront autologous stem cell transplantation (ASCT) were censored at the time of transplantation.RESULTSThe baseline characteristics were well balanced between the two groups except a higher proportion of patients with ALK-negative ALCL in EPOCH group (24.1% vs 5.2%) (Table 1). The median cycle of chemotherapy was 6 (range, 1-8) in both groups. Four patients (6.9%) underwent upfront ASCT after EPOCH induction. The overall response rate for evaluable patients were 75.4% (complete response [CR] 57%) and 58% (CR 39%) in EPOCH and CHOP groups, respectively (P<0.0001). With a median follow up of 26 months, 4-year progression free survival (PFS) were 45.8% and 26.7% in EPOCH and CHOP group, respectively (P=0.022) (Figure 1). Projected overall survival (OS) at 4 years were 50.9% and 28.7% for EPOCH- and CHOP-treated patients, respectively (P=0.001) (Figure 2). By multivariate analysis, chemotherapy regimen was the only factor independently predicting PFS (Hazard ratio [HR] 1.82, 95% confidence interval [CI] 1.04-3.19, P=0.034) and OS (HR 2.26, 95% CI 1.28-3.97, P=0.005).CONCLUSIONEPOCH provided superior outcomes than CHOP regimen as frontline therapy for patients with nodal PTCLS, excluding ALK-positive ALCL. The promising efficacy of EPOCH warrants further evaluation in prospective randomized controlled study. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Analysis of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-reactive CD8(+) T cell responses in children with NPM-ALK(+) anaplastic large cell lymphoma.

Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK-positive anaplastic large cell lymphoma (ALCL) have been detected using peptide-based approaches in individuals preselected for human leucocyte antigen (HLA)-A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)-ALK-specific CD8(+) T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK-specific CD8(+) T cells. Autologous dendritic cells (DCs) transfected with in-vitro-transcribed RNA (IVT-RNA) encoding NPM-ALK were used as antigen-presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon-gamma enzyme-linked immunospot (ELISPOT) assays with NPM-ALK-transfected autologous DCs as well as CV-1 in Origin with SV40 genes (COS-7) cells co-transfected with genes encoding the patients' HLA class I alleles and with NPM-ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM-ALK-specific CD8(+) T cell responses were detected in three of five ALK-positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti-ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM-ALK-specific CD8(+) T cell responses were restricted by HLA-C-alleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM-ALK-reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis.

Abstract 3498: Synergistic cytotoxicity of everolimus in combination with crizotinib in ALK-positive anaplastic large cell lymphoma cells

Abstract Everolimus, an mTOR inhibitor, has shown promising anti-tumor activity in a variety of lymphomas, although its clinical efficacy was not satisfactory, due possibly to activation of several pro-surviving signaling pathways. The combined effect of everolimus and crizotinib, an ALK inhibitor, has not yet been investigated in ALK-positive anaplastic large cell lymphoma (ALCL). We treated ALK-positive ALCL cell lines, K-299 and SU-DHL-1, with various concentrations of everolimus and crizotinib at a fixed ratio of 1:40 (0.5 nM, 1.0 nM, and 2.0 nM for everolimus, and 20 nM, 40 nM, and 80 nM for crizotinib, respectively). After 72 hours, the combination index (CI) values were less than 1 (0.596, 0.583, and 0.763 in K-299 cells, and 0.326, 0.616, and 0.271 in SU-DHL-1 cells, respectively) in all tested combinations, suggesting synergistic cytotoxicity of everolimus and crizotinib. Western blot analysis demonstrated that everolimus up-regulated phosphorylation of ERK Thr202/Tyr204 and AKT Thr308 and Ser473. However, this aberrant activation of ERK and AKT was attenuated by the addition of crizotinib. In addition, while everolimus selectively inhibited mTOR Ser2448 phosphorylation, a marker for mTORC1 activity, the combination treatment more potently inhibited mTOR Ser2448 phosphorylation and decreased mTOR Ser2481 phosphorylation, a marker for mTORC2, as well. Cell-cycle analysis showed that the combination treatment induced G1 arrest. PARP cleavage was also increased after the combination treatment. To test the hypothesis that our findings could be applyed to other ALK-positive malignancies, we treated NCI-H2228, a lung adenocarcinoma cell line that harbors an EML4-ALK fusion gene, with everolimus and crizotinib for 72 hours. The CI values were less than 1 in all tested combinations: 0.228 in 1 nM everolimus plus 80 nM crizotinib, 0.216 in 2 nM everolimus plus 160 nM crizotinib, and 0.349 in 4 nM everolimus and 320 nM crizotinib. In summary, everolimus in combination with crizotinib synergistically inhibited the growth of ALK-positive ALCL cells. Crizotinib abrogated aberrant ERK and AKT activation induced by everolimus and more potently inhibited both mTORC1 and mTORC2 activity when combined with everolimus, resulting in increased G1 cell-cycle arrest and apoptosis. Our findings may provide an evidence for future research using everolimus and crizotinib combination in ALK-positive ALCL and could be used to improve the therapeutic outcome in patients with ALK-positive ALCL. Citation Format: Ji-Won Kim, Wendan Xu, Sung-Soo Yoon. Synergistic cytotoxicity of everolimus in combination with crizotinib in ALK-positive anaplastic large cell lymphoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3498. doi:10.1158/1538-7445.AM2015-3498

RNASET2 — An autoantigen in anaplastic large cell lymphoma identified by protein array analysis

Characterising tumour-associated antigens (TAAs) not only represents an important approach to the identification of new diagnostic/prognostic markers, but can also provide information on disease processes and additional potential therapeutic targets. Preliminary screening of a protein macroarray, containing more than 12,000 different proteins, with sera from anaplastic lymphoma kinase (ALK)-negative and ALK-positive anaplastic large cell lymphoma (ALCL) patients identified ribonuclease and tumour suppressor protein Ribonuclease T2 (RNASET2), phosphatase lipid phosphate phosphatase-related protein type 3 (LPPR3) and apoptotic adaptor molecule Fas-associating protein (FADD) as ALK-negative ALCL-associated TAAs. Further validation of these observations was confirmed using the ALCL sera in reverse ELISAs. The circulating anti-RNASET2 autoantibodies present in ALCL patients' sera also recognised eukaryotically expressed RNASET2 protein. RNASET2 expression was then investigated in normal tissues and in lymphomas to explore its clinical potential. RNASET2 protein and mRNA levels showed highest expression in the spleen, leucocytes and pancreas. RNASET2 protein expression was not restricted to ALK-negative ALCL (81%), being expressed in ALK-positive ALCL (65%) as well as in a number of other lymphomas. The immunological recognition of RNASET2, its expression in ALCL and other lymphomas together with its known tumourigenic properties suggest that further studies on this autoantigen are warranted.

CEP-28122, a Highly Potent and Selective Orally Active Inhibitor of Anaplastic Lymphoma Kinase with Antitumor Activity in Experimental Models of Human Cancers

Anaplastic lymphoma kinase (ALK) is constitutively activated in a number of human cancer types due to chromosomal translocations, point mutations, and gene amplification and has emerged as an excellent molecular target for cancer therapy. Here we report the identification and preclinical characterization of CEP-28122, a highly potent and selective orally active ALK inhibitor. CEP-28122 is a potent inhibitor of recombinant ALK activity and cellular ALK tyrosine phosphorylation. It induced concentration-dependent growth inhibition/cytotoxicity of ALK-positive anaplastic large-cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma cells, and displayed dose-dependent inhibition of ALK tyrosine phosphorylation in tumor xenografts in mice, with substantial target inhibition (>90%) for more than 12 hours following single oral dosing at 30 mg/kg. Dose-dependent antitumor activity was observed in ALK-positive ALCL, NSCLC, and neuroblastoma tumor xenografts in mice administered CEP-28122 orally, with complete/near complete tumor regressions observed following treatment at doses of 30 mg/kg twice daily or higher. Treatment of mice bearing Sup-M2 tumor xenografts for 4 weeks and primary human ALCL tumor grafts for 2 weeks at 55 or 100 mg/kg twice daily led to sustained tumor regression in all mice, with no tumor reemergence for more than 60 days postcessation of treatment. Conversely, CEP-28122 displayed marginal antitumor activity against ALK-negative human tumor xenografts under the same dosing regimens. Administration of CEP-28122 was well tolerated in mice and rats. In summary, CEP-28122 is a highly potent and selective orally active ALK inhibitor with a favorable pharmaceutical and pharmacokinetic profile and robust and selective pharmacologic efficacy against ALK-positive human cancer cells and tumor xenograft models in mice.

The Prognosis of Limited Stage Peripheral T-Cell Lymphoma (PTCL): A Population-Based Analysis and Comparison to Diffuse Large B-Cell Lymphoma (DLBCL)

AbstractAbstract 4129Multiple prospective and retrospective studies have demonstrated an inferior prognosis of peripheral T-cell lymphoma (PTCL), with the exception of ALK-positive anaplastic large cell lymphoma (ALCL). However, there is very little information regarding the prognosis of patients with limited stage PTCL and how it compares to diffuse large B-cell lymphoma (DLBCL). Given disease rarity, patients with limited stage PTCL from two institutions, the British Columbia Cancer Agency (BCCA) and the University of Nebraska Medical Center (UNMC) were evaluated. Methods:Patients >15 y of age diagnosed with limited stage PTCL between 1981 and 2008, excluding NK/T-cell lymphoma, nasal type and cutaneous ALCL, were identified in the BCCA Lymphoid Cancer Database and from the UNMC. Limited stage PTCL was defined as stage I/IIA, non-bulky (< 10cm) and considered ‘minimal’ extent ie. potentially encompassable in a radiotherapy field. Results:62 patients with limited stage PTCL were identified (n=35 BCCA and n=27 UNMC). The main histologic subtype was PTCL-NOS (n= 50, 81%) and the remaining patients had ALCL (ALK-negative (neg) ALCL n=7; ALK-positive (pos) ALCL n=5). 14 cases of PTCL-NOS had cutaneous-only disease. Patients had the following clinical characteristics: M:F 1.2:1, median age 52y (18-98y), stage I 68%, 52% extranodal involvement. The majority of patients were treated with anthracycline-based chemotherapy (81%) and over half (53%) received combined modality therapy. The 5-y time to progression (TTP) was similar in irradiated and non-irradiated patients (p=0.52). The outcome of patients with cutaneous-only limited stage PTCL-NOS was excellent with a 5-y TTP and OS of 86% and 92%, respectively. Similarly, patients with ALK-pos ALCL had a very favourable outcome with only 1/5 patients developing relapsed disease and no deaths to date. The 5-y TTP and OS for the remaining cases of PTCL-NOS and ALK-neg ALCL (n=43) were 61% and 67%, respectively. For the whole cohort, there was a more favourable prognosis in patients with a low risk disease by the stage adjusted International Prognostic Index (L-IPI) score at diagnosis (5-y TTP 0 factor 83%, 1 factor 66%, 2 factors 50%, 3 factors 25%, p=.013 and 5-y OS 0 factor 91%, 1 factor 73%, 2 factors 50%, 3 factors 25%, p<.0001). Late relapses (> 5 y) were uncommon, occurring in 2 patients with PTCL-NOS presenting with skin relapses and 1 patient with ALK-neg ALCL with a nodal relapse. The outcome of limited stage PTCL patients was compared to a cohort of BCCA patients diagnosed with limited stage DLBCL who were treated with CHOP-like chemotherapy (without rituximab) and radiotherapy (n=245) (Campbell BA ASCO 2010). In multivariate analysis, excluding cutaneous-only PTCL-NOS and ALK-pos ALCL, there was an inferior TTP in cases with a T-cell phenotype (HR 2.28, p=.003) and a high L-IPI score (HR 1.57, p<.00001). Similarly, the OS was inferior in patients with a T-cell phenotype (HR 1.90, p=.011) and high L-IPI score (HR 1.78, p<.0001). Surprisingly, all of the patients with relapsed (n=4) or refractory (n=3) disease who were able to receive salvage treatment which included stem cell transplant (SCT), (n=7 allogeneic (n=1), autologous (n=6)) are alive and free of disease a median of 7.2 y from the date of transplant (range 1.4 – 16 years). This is in stark contrast to DLBCL patients (n=6), where all but one patient relapsed and died of lymphoma post-transplant. Conclusion:Limited stage cutaneous-only PTCL-NOS and ALK-pos ALCL as well as selected patients with PTCL-NOS and ALK-neg ALCL with low risk disease have a favourable prognosis. However, the prognosis remains inferior to limited stage DLBCL. Despite an overall higher risk of relapse, the cure rate is high in limited stage PTCL patients who are able to receive a stem cell transplant as part of their salvage therapy for relapsed or refractory disease. Disclosures:No relevant conflicts of interest to declare.

Primary Esophageal CD30-Positive ALK-Positive Anaplastic Large Cell Lymphoma: A Case Report and Literature Review

To introduce a case of primary esophageal CD30-positive ALK-positive anaplastic large cell lymphoma (ALCL) and discuss its diagnosis and treatment. Esophagectomy was done for a 37-year-old male with a submucosal lesion after a frozen section failed to give a definite diagnosis. Samples were sent for hematoxylin-eosin staining and immunohistochemical analysis. Six cycles of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy were given and the patient was followed up. The operation was uneventful. Postoperative pathologic and immunohistochemical examination yielded a diagnosis of primary CD30-positive and ALK-positive ALCL of the esophagus. The patient was in complete remission at the 14-month follow-up. ALCL of the esophagus should be considered in the differential diagnosis of esophageal submucosal lesions. Biopsy through either esophagoscopy or surgical exploration, chemotherapy, and radiotherapy can be chosen for long-term survival.

Circulating Antibodies to ALK Inversely Correlat with Relapse Risk and Circulating Tumor Cells in Children and Adolescents with ALK-Positive Anaplastic Large Cell Lymphoma

Tumour-associated antigens (TAAs) recognised by patients' immune systems may be of prognostic relevance and also represent new immunotherapeutic targets. Circulating antibodies and T-cell responses to the TAA anaplastic lymphoma kinase (ALK) have been detected in patients with ALK-positive anaplastic large cell lymphoma (ALCL) (Pulford et al. Blood 2000, 96:1605–7; Ait-Tahar et al. Int J Cancer 2006, 118:688–95; Cancer Res 2007, 65:1891–901). Their exact significance with regard to prognosis is, however, unclear at present. In contrast, and unusually for non-Hodgkin's lymphoma, low numbers of circulating tumor cells (CTCs) expressing nucleophosmin (NPM)-ALK-transcripts at diagnosis in bone marrow or blood confers an unfavourable prognosis (Damm-Welk et al. Blood 2007, 110:670–7). The current study was performed to address whether there was any correlation between the magnitude of the autoantibody response to ALK withprognosis andthe levels of CTCs at diagnosis in a cohort of uniformly treated children and adolescents with ALK-positive ALCL for whom full clinical data was available.The anti-ALK-antibody titers were analyzed in initial serum or plasma samples from 86 patients and copies of NPM-ALK in initial bone marrow (n=61) and/or blood samples (n=53). The children were treated according to the NHL-BFM95 and ALCL99 trials carried out between 1996 and 2006. Antibody titers against ALK and copies of NPM-ALK were measured using ALK transfectants and quantitative RT-PCR as previously described (Pulford et al. Blood 2000, 96:1605–7; Damm-Welk et al. Blood 2007, 110:670–7). Circulating antibodies to ALK were detected in 80/86 (93%) of patients studied. The titers ranged between 1/50 and ≥1/60750. The median follow-up of the patients was 4.6 years (range 1.4–10.4 years). Antibody titers <1/60750, detected in 61 patients (71%), significantly correlated with advanced stage, mediastinal or visceral involvement. The 5 year event free survival rate (pEFS) of 25 patients with antibody titres ≥1/60750 was 88+/− 0.06% compared to 51+/−0.07% for 61 patients with titres less than 1/60750 (p<.003). Among those patients mounting an antibody response, higher antibody titers were significantly associated with lower cumulative incidence of relapses (CI-R): 17 patients with a titer between 1/50 and <1/2025 had a CI-R of 74±12% compared to a CI-R of 32±8% for 38 patients with titers between 1/2025 and <1/60750 and a CI-R of 12±7% for 25 patients with titers ≥1/60750 (p<.001). There was a significant inverse correlation between the magnitude of antibody titers and CTCs in 61 patients studied for the presence of both autoantibodies and CTC in bone marrow. None of the 20 patients with a high antibody titer (≥1/60750) had more than 10 copies NPM-ALK/104 copies ABL in bone marrow compared to 13 of 41 patients with lower antibody titers (p=.003). Comparison of antibody titers with CTCs in peripheral blood led to a similar grouping. In conclusion, the current study shows, for the first time, that the magnitude of the autoantibody response to ALK is inversely correlated with the relapse risk and disease dissemination in ALK-positive ALCL. Our results, therefore, provide clinical evidence that a strong immune response to ALK may control the progression of ALCL supporting the possibility that CTCs are a secondary phenomenon due to a limited anti-tumor immune response. These results also support the use of ALK as an immunotherapeutic target in ALCL.